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Subcellular Transcriptomics and Proteomics: A Comparative Methods Review

Open AccessPublished:December 15, 2021DOI:https://doi.org/10.1016/j.mcpro.2021.100186

      Highlights

      • Subcellular information of protein and RNA give insights into molecular function.
      • This review discusses strategies available to measure subcellular information.
      • Hybridization of methods shows promise for exploring the composition of organelles.
      • Advances are aiding understanding of the organisation and dynamics of cells.

      Abstract

      The internal environment of cells is molecularly crowded, which requires spatial organization via subcellular compartmentalization. These compartments harbor specific conditions for molecules to perform their biological functions, such as coordination of the cell cycle, cell survival, and growth. This compartmentalization is also not static, with molecules trafficking between these subcellular neighborhoods to carry out their functions. For example, some biomolecules are multifunctional, requiring an environment with differing conditions or interacting partners, and others traffic to export such molecules. Aberrant localization of proteins or RNA species has been linked to many pathological conditions, such as neurological, cancer, and pulmonary diseases. Differential expression studies in transcriptomics and proteomics are relatively common, but the majority have overlooked the importance of subcellular information. In addition, subcellular transcriptomics and proteomics data do not always colocate because of the biochemical processes that occur during and after translation, highlighting the complementary nature of these fields. In this review, we discuss and directly compare the current methods in spatial proteomics and transcriptomics, which include sequencing- and imaging-based strategies, to give the reader an overview of the current tools available. We also discuss current limitations of these strategies as well as future developments in the field of spatial -omics.

      Graphical Abstract

      Keywords

      Abbreviations:

      BAP (biotin acceptor peptide), bDNA (branched DNA), cyTOF (cytometry by time of flight), DFHBI (3,5-difluoro-4-hydroxybenzylideneimidazolidinone), ER (endoplasmic reticulum), EV (extracellular vesicle), FFE (free-flow electrophoresis), FIFFF (flow field-flow fractionation), FISSEQ (fluorescent in situ sequencing), FP (fluorescent protein), HPA (Human Protein Atlas), IFC (imaging flow cytometry), IMC (imaging mass cytometry), LOPIT (localization of organelle proteins by isotope tagging), MCP (bacteriophage MS2 coat protein), MERFISH (multiplexed error-robust FISH), MS (mass spectrometry), MSI (MS imaging), PAINT (point accumulation in nanoscale topography), PCP (P77 bacteriophage coat protein), PM (plasma membrane), PTM (post-translational modification), RBP (RNA-binding protein), RIP (RNA-co-immunoprecipitation), scRNA-Seq (single-cell RNA-Seq), seqFISH (sequential barcoding FISH), SINC-Seq (single-cell integrated nucRNA and cytRNA sequencing), TREAT (3(three)′-RNA end accumulation during turnover), TRICK (translating RNA imaging by coat protein knockoff)
      Molecular biology is the study of cellular functions via processes such as molecular synthesis, modification, and interactions. RNA and proteins can have multiple roles and interacting partners that require close physical proximity to each other within the cell to function. Therefore, precise control of localization or colocalization by selective congregation and isolation of biochemical processes is integral and intrinsically linked to cellular functions. For instance, in context of transcription and translation, mRNA is shuttled out of the nucleus, where it docks at ribosomes within the cytosol, at the endoplasmic reticulum (ER) or near the mitochondria, dependent on the coded protein and cellular conditions (
      • Kloc M.
      • Zearfoss N.R.
      • Etkin L.D.
      Mechanisms of subcellular mRNA localization.
      ,
      • Dennerlein S.
      • Wang C.
      • Rehling P.
      Plasticity of mitochondrial translation.
      ,
      • Fazal F.M.
      • Han S.
      • Parker K.R.
      • Kaewsapsak P.
      • Xu J.
      • Boettiger A.N.
      • Chang H.Y.
      • Ting A.Y.
      Atlas of subcellular RNA localization revealed by APEX-seq.
      ). Translation of mRNA at the coded protein's functional site, rather than at a singular canonical and/or punctate location, is clearly demonstrated within polarized cells, such as neurons or intestinal epithelial cells (
      • Cajigas I.J.
      • Tushev G.
      • Will T.J.
      • tom Dieck S.
      • Fuerst N.
      • Schuman E.M.
      The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging.
      ,
      • Moor A.E.
      • Golan M.
      • Massasa E.E.
      • Lemze D.
      • Weizman T.
      • Shenhav R.
      • Baydatch S.
      • Mizrahi O.
      • Winkler R.
      • Golani O.
      • Stern-Ginossar N.
      • Itzkovitz S.
      Global mRNA polarization regulates translation efficiency in the intestinal epithelium.
      ). Hence, studying subcellular localization not only gives insights into the organization of cellular compartments but how cells function; so techniques that provide spatial context are important tools in molecular biology.
      The relationship between DNA, RNA and proteins does not represent a linear dogma. Interactions, or “interactomes,” between nucleic acids and proteins are fundamental for cellular function. RNA-binding proteins (RBPs), originally thought to exclusively function in gene regulation via ribonucleoprotein complex formation, have now been shown to have more extensive interplay between protein and RNA interactomes (
      • Hentze M.W.
      • Castello A.
      • Schwarzl T.
      • Preiss T.
      A brave new world of RNA-binding proteins.
      ). A prime example of RNA-mediated and RBP-mediated regulation via subcellular relocalization is the short noncoding RNA transcript Y3 RNA, which orchestrates translocation of the RBP Rho 60-kDa autoantigen between the cytosol and nucleus as part of a UV-induced survival mechanism (
      • Sim S.
      • Weinberg D.E.
      • Fuchs G.
      • Choi K.
      • Chung J.
      • Wolin S.L.
      The subcellular distribution of an RNA quality control protein, the Ro autoantigen, is regulated by noncoding y RNA binding.
      ,
      • Sim S.
      • Yao J.
      • Weinberg D.E.
      • Niessen S.
      • Yates J.R.
      • Wolin S.L.
      The zipcode-binding protein ZBP1 influences the subcellular location of the Ro 60-kDa autoantigen and the noncoding Y3 RNA.
      ). A more classic example of subcellular control is during the cell cycle, where cyclins and cyclin-dependent kinases traffic between nuclei and cytosol (
      • Yang J.
      • Kornbluth S.
      All aboard the cyclin train: Subcellular trafficking of cyclins and their CDK partners.
      ). An in-depth immunofluorescence study has recently captured single-cell variability of subcellular composition during the cell cycle (
      • Mahdessian D.
      • Cesnik A.J.
      • Gnann C.
      • Danielsson F.
      • Stenström L.
      • Arif M.
      • Zhang C.
      • Le T.
      • Johansson F.
      • Shutten R.
      • Bäckström A.
      • Axelsson U.
      • Thul P.
      • Cho N.H.
      • Carja O.
      • et al.
      Spatiotemporal dissection of the cell cycle with single-cell proteogenomics.
      ).
      Aberrant trafficking of RNA and protein has been implicated in several pathological conditions, including amyotrophic lateral sclerosis and pulmonary atrial hypertension, respectively (
      • Guo L.
      • Shorter J.
      Biology and pathobiology of TDP-43 and emergent therapeutic strategies.
      ,
      • Sehgal P.B.
      • Lee J.E.
      Protein trafficking dysfunctions: Role in the pathogenesis of pulmonary arterial hypertension.
      ). A well-documented example of mislocalization causing severe disease is the most common mutation in cystic fibrosis, F508del. Immunofluorescence and subcellular fractionation strategies have shown that this mutation causes the cystic fibrosis transmembrane regulator ion channel to misfold and accumulate at the ER, preventing cystic fibrosis transmembrane regulator expression at the plasma membrane (PM) and, consequently, impairing mucus clearance in the lungs (
      • Kopito R.R.
      Biosynthesis and degradation of CFTR.
      ,
      • Cheng S.H.
      • Gregory R.J.
      • Marshall J.
      • Paul S.
      • Souza D.W.
      • White G.A.
      • O'Riordan C.R.
      • Smith A.E.
      Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.
      ,
      • Lukacs G.L.
      • Mohamed A.
      • Kartner N.
      • Chang X.B.
      • Riordan J.R.
      • Grinstein S.
      Conformational maturation of CFTR but not its mutant counterpart (delta F508) occurs in the endoplasmic reticulum and requires ATP.
      ). This has aided the design of pharmacological intervention to correct this misfolding and subsequent mislocalization (
      • Ren H.Y.
      • Grove D.E.
      • De La Rosa O.
      • Houck S.A.
      • Sopha P.
      • Van Goor F.
      • Hoffman B.J.
      • Cyr D.M.
      VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.
      ). In many of these cases, early stages of disease can be identified by translocation events, which can precede or be independent to detectable changes in gene expression and, therefore, can only be studied at the subcellular level (
      • Lu A.X.
      • Chong Y.T.
      • Hsu I.S.
      • Strome B.
      • Handfield L.F.
      • Kraus O.
      • Andrews B.J.
      • Moses A.M.
      Integrating images from multiple microscopy screens reveals diverse patterns of change in the subcellular localization of proteins.
      ). Despite this, temporal or differential expression is more commonly studied because it is more straightforward, though novel tools to study the spatial dimension on an -omics scale are opening new opportunities for a better understanding of cellular function.
      Spatial proteomics and transcriptomics have often been reviewed independently with technical details covered in previous articles (
      • Fazal F.M.
      • Chang H.Y.
      Subcellular spatial transcriptomes: Emerging frontier for understanding gene regulation.
      ,
      • Christopher J.A.
      • Stadler C.
      • Martin C.E.
      • Morgenstern M.
      • Pan Y.
      • Betsinger C.N.
      • Rattray D.G.
      • Mahdessian D.
      • Gingras A.C.
      • Warscheid B.
      • Lehtiö J.
      • Cristea I.M.
      • Foster L.J.
      • Emili A.
      • Lilley K.S.
      Subcellular proteomics.
      ,
      • Lundberg E.
      • Borner G.H.H.
      Spatial proteomics: A powerful discovery tool for cell biology.
      ). Here, we outline and directly compare methods (summarized in Table 1) that interrogate the spatial transcriptomic and proteomic within subcellular compartments of cells, rather than spatial information at the tissue level, and suggest gaps in technology in need of further advancement. We aim to provide a resource for newcomers to spatial -omics who wish to unpick the busy, yet spatially organized, environment within cells.
      Table 1Summary of each method covered within this review
      MethodPrincipleExamples of biological insightsLive, fixed, or lysed samples?In situ?Targeted?
      Imaging
       Affinity reagentsExogenous dyes or probes (e.g., antibodies or oligonucleotides) designed to target specific molecules of interest (MOIs)The largest database of human protein subcellular localizations using stringently validated antibodies, giving insights into cell variability and mapping subcellular localization of SARS-coronavirus 2 interactors (
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ). smFISH aided the understanding of how liquid–liquid phase separation aids formation of rotavirus replication factories (considered virus-made membraneless organelles) (
      • Geiger F.
      • Acker J.
      • Papa G.
      • Wang X.
      • Arter W.E.
      • Saar K.L.
      • Erkamp N.A.
      • Qi R.
      • Bravo J.P.
      • Strauss S.
      • Krainer G.
      • Burrone O.R.
      • Jungmann R.
      • Knowles T.P.
      • Engelke H.
      • et al.
      Liquid–liquid phase separation underpins the formation of replication factories in rotaviruses.
      )
      Primarily fixed samples (exception of live FISH)Targeted, label MOI
       Fluorescently tagged proteinsFluorescent proteins (typically) genetically fused to MOI and, therefore coexpressed with the MOIGenetically fused fluorescent proteins were used to gain insight into the pH- and receptor-dependent endocytic entry of severe acute respiratory syndrome virus into the host cell (
      • Wang H.
      • Yang P.
      • Liu K.
      • Guo F.
      • Zhang Y.
      • Zhang G.
      • Jiang C.
      SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway.
      )
      Live/fixedTargeted, label MOI
       IFCA combination of flow cytometry and microscopy to capture spatial information using fluorescent probesAn IFC method was developed to provide a more informative diagnostic tool for types of acute leukaemia (
      • Grimwade L.F.
      • Fuller K.A.
      • Erber W.N.
      Applications of imaging flow cytometry in the diagnostic assessment of acute leukaemia.
      )
      Live/fixedTargeted, label MOI
       IMCUses heavy-metal probes conjugated to antibodies, which ablated pixel by pixel and measured using MS. This improved multiplexing of probes because of the reduced spectral overlap compared with fluorescent strategiesUsed for cellular phenotyping of breast cancer and lesions in multiple sclerosis and lymphoid organs (
      • Giesen C.
      • Wang H.A.
      • Schapiro D.
      • Zivanovic N.
      • Jacobs A.
      • Hattendorf B.
      • Schüffler P.J.
      • Grolimund D.
      • Buhmann J.M.
      • Brandt S.
      • Varga Z.
      • Wild P.J.
      • Günther D.
      • Bodenmiller B.
      Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.
      ,
      • Park C.
      • Ponath G.
      • Levine-Ritterman M.
      • Bull E.
      • Swanson E.C.
      • De Jager P.L.
      • Segal B.M.
      • Pitt D.
      The landscape of myeloid and astrocyte phenotypes in acute multiple sclerosis lesions.
      ,
      • Durand M.
      • Walter T.
      • Pirnay T.
      • Naessens T.
      • Gueguen P.
      • Goudot C.
      • Lameiras S.
      • Chang Q.
      • Talaei N.
      • Ornatsky O.
      • Vassilevskaia T.
      • Baulande S.
      • Amigorena S.
      • Segura E.
      Human lymphoid organ cDC2 and macrophages play complementary roles in T follicular helper responses.
      ). Primarily used for tissue-level insights, rather than subcellular, though some subcellular information is achievable with the method
      FixedTargeted, label MOI
       MSISimilar to IMC, but ablation leads to ionization of all molecules within the pixel, producing a separate spectra per pixel of the samplePrimarily, still tissue-level resolution, rather than subcellular resolution. Has been used for intraoperative imaging of pituitary adenomas for biomarkers that are usually difficult to detect efficiently (
      • Huang K.T.
      • Ludy S.
      • Calligaris D.
      • Dunn I.F.
      • Laws E.
      • Santagata S.
      • Agar N.Y.
      Rapid mass spectrometry imaging to assess the biochemical profile of pituitary tissue for potential intraoperative usage.
      )
      Typically fixedUntargeted, cell-wide
      Biochemical separation
       Basic centrifugation/detergent basedUses targeted centrifugation or detergent step(s) to achieve enrichment of a specific cellular component or organelle of interestUsed in the study of mitochondrial transport in Trypanosoma brucei to aid understanding of parasitic physiology (
      • Peikert C.D.
      • Mani J.
      • Morgenstern M.
      • Käser S.
      • Knapp B.
      • Wenger C.
      • Harsman A.
      • Oeljeklaus S.
      • Schneider A.
      • Warscheid B.
      Charting organellar importomes by quantitative mass spectrometry.
      ) and gain insights into proinflammatory gene regulation in context of subcellular dynamics of macrophages from mice (
      • Bhatt D.M.
      • Pandya-Jones A.
      • Tong A.J.
      • Barozzi I.
      • Lissner M.M.
      • Natoli G.
      • Black D.L.
      • Smale S.T.
      Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.
      )
      Lysed, in vitroUntargeted, enrich organelle(s) of interest
       Correlation profilingUses multiple centrifugation or detergent steps of increasing spin speed/time or solubility, respectively, to collect an abundance profile of one or multiple subcellular components. Can be used for cell-wide analysis of moleculesUsed to track the subcellular proteome of host cells over the course of human cytomegalovirus infection in a spatial and temporal context (
      • Jean Beltran P.M.
      • Mathias R.A.
      • Cristea I.M.
      • Beltran J.
      A Portrait of the human organelle proteome in space and time during cytomegalovirus infection.
      ). Also, used to identify that lysosomal trapping is important for the efficacy of drugs that aid antigen presentation (
      • Kozik P.
      • Gros M.
      • Itzhak D.N.
      • Joannas L.
      • Heurtebise-Chrétien S.
      • Krawczyk P.A.
      • Rodríguez-Silvestre P.
      • Alloatti A.
      • Magalhaes J.G.
      • Del Nery E.
      • Borner G.H.H.
      • Amigorena S.
      Small molecule enhancers of endosome-to-cytosol import augment anti-tumor immunity.
      )
      Lysed, in vitroUntargeted, cell-wide
       Electrophoresis basedSeparates subcellular components via their charge state using modified electrophoresis techniquesUsed to assess the protein composition of the secretory pathway in plants that are otherwise difficult to resolve with centrifugation because of their similar density (
      • De Michele R.
      • McFarlane H.E.
      • Parsons H.T.
      • Meents M.J.
      • Lao J.
      • González Fernández-Niño S.M.
      • Petzold C.J.
      • Frommer W.B.
      • Samuels A.L.
      • Heazlewood J.L.
      Free-flow electrophoresis of plasma membrane vesicles enriched by two-phase partitioning enhances the quality of the proteome from Arabidopsis seedlings.
      )
      Lysed, in vitroUntargeted, cell-wide
      Proximity labeling
       BioID and APEXFusion of bait protein(s) to either a biotin ligase (e.g., BioID) or peroxidase (e.g., APEX) that covalently labels molecules in immediate proximity of the bait with a small and exogenous substrate. The substrate can then be purified along with the labeled moleculesBioID has revealed novel organellar components of the Trypanosoma brucei, flies, and worms (
      • Branon T.C.
      • Bosch J.A.
      • Sanchez A.D.
      • Udeshi N.D.
      • Svinkina T.
      • Carr S.A.
      • Feldman J.L.
      • Perrimon N.
      • Ting A.Y.
      Efficient proximity labeling in living cells and organisms with TurboID.
      ,
      • Dang H.Q.
      • Zhou Q.
      • Rowlett V.W.
      • Hu H.
      • Lee K.J.
      • Margolin W.
      • Li Z.
      Proximity interactions among basal body components in trypanosoma brucei identify novel regulators of basal body biogenesis and inheritance.
      ,
      • Morriswood B.
      • Havlicek K.
      • Demmel L.
      • Yavuz S.
      • Sealey-Cardona M.
      • Vidilaseris K.
      • Anrather D.
      • Kostan J.
      • Djinovic-Carugo K.
      • Roux K.J.
      • Warren G.
      Novel bilobe components in Trypanosoma brucei identified using proximity-dependent biotinylation.
      ,
      • Ramanathan M.
      • Majzoub K.
      • Rao D.S.
      • Neela P.H.
      • Zarnegar B.J.
      • Mondal S.
      • Roth J.G.
      • Gai H.
      • Kovalski J.R.
      • Siprashvili Z.
      • Palmer T.D.
      • Carette J.E.
      • Khavari P.A.
      RNA-protein interaction detection in living cells.
      ) and identifying novel proteins involved in hyperpolarization that are linked to neurodegenerative diseases (
      • Uezu A.
      • Kanak D.J.
      • Bradshaw T.W.
      • Soderblom E.J.
      • Catavero C.M.
      • Burette A.C.
      • Weinberg R.J.
      • Soderling S.H.
      Identification of an elaborate complex mediating postsynaptic inhibition.
      ,
      • Spence E.F.
      • Dube S.
      • Uezu A.
      • Locke M.
      • Soderblom E.J.
      • Soderling S.H.
      In vivo proximity proteomics of nascent synapses reveals a novel regulator of cytoskeleton-mediated synaptic maturation.
      )

      APEX-Seq identified stress type–dependent RNA interactions with stress granules (
      • Fazal F.M.
      • Chang H.Y.
      Subcellular spatial transcriptomes: Emerging frontier for understanding gene regulation.
      ,
      • Padrón A.
      • Iwasaki S.
      • Ingolia N.T.
      Proximity RNA labeling by APEX-seq reveals the organization of translation initiation complexes and repressive RNA granules.
      )
      Lysed, in vivo labelingUntargeted, label organelle(s) of interest
      The table includes a short description about the principle of each method, examples of their biological insights or applications, and basic comparisons of the characteristics of the methods.

      Imaging the Spatial Transcriptome and Proteome

      Microscopy-Based Imaging

      Microscopy is the most well-established and largest branch of imaging with a variety of labeling strategies for targeting proteins and transcripts, often at a single-cell level. Conducting microscopy studies on a global spatial scale can be challenging and laborious because of costly generation of antibodies or recombinant organisms and limited multiplexing capacity. In addition, sample preparation is rarely a one-size-fits-all process. For example, fixing is usually dependent on the subcellular compartment of interest, and phototoxicity is a limiting factor in live-cell imaging. Fixing cells can disrupt molecular organization and macroorganization and structures, causing artificial localization of molecules (
      • Stadler C.
      • Skogs M.
      • Brismar H.
      • Uhlén M.
      • Lundberg E.
      A single fixation protocol for proteome-wide immunofluorescence localization studies.
      ) but does not suffer from issues of phototoxicity and can capture snapshots of transcripts and proteins, which rapidly fluctuate or have low copy number. Some applications are limited to fixed samples only, such as immunofluorescence or FISH, whereas others have the capacity for live-cell imaging, such as genetically fusing fluorescent tags. Recent emergence of high-throughput and super-resolution microscopy has allowed mid-scale to large-scale spatial studies of transcripts and proteins, permitting quantitative measurements alongside the “seeing is believing” aspect at which imaging excels. Furthermore, while simultaneous genome-wide live-cell imaging is not yet possible, recent advancements in the field of high-content imaging are enabling faster image acquisition at higher resolution, though often with a trade-off between the two (
      • Mattiazzi Usaj M.
      • Styles E.B.
      • Verster A.J.
      • Friesen H.
      • Boone C.
      • Andrews B.J.
      High-content screening for quantitative cell biology.
      ). Both the technological advancements of the instrumentation and bioimaging informatics have been extensively reviewed (
      • Wollman R.
      • Stuurman N.
      High throughput microscopy: From raw images to discoveries.
      ,
      • Eliceiri K.W.
      • Berthold M.R.
      • Goldberg I.G.
      • Ibáñez L.
      • Manjunath B.S.
      • Martone M.E.
      • Murphy R.F.
      • Peng H.
      • Plant A.L.
      • Roysam B.
      • Stuurman N.
      • Stuurmann N.
      • Swedlow J.R.
      • Tomancak P.
      • Carpenter A.E.
      Biological imaging software tools.
      ,
      • Aspelmeier T.
      • Egner A.
      • Munk A.
      Modern statistical challenges in high-resolution fluorescence microscopy.
      ,
      • Khater I.M.
      • Nabi I.R.
      • Hamarneh G.
      A review of super-resolution single-molecule localization microscopy cluster analysis and quantification methods.
      ,
      • Wu Y.L.
      • Tschanz A.
      • Krupnik L.
      • Ries J.
      Quantitative data analysis in single-molecule localization microscopy.
      ).
      Here, we briefly discuss the main labeling options and some alternative imaging approaches, whilst outlining the advantages and disadvantages, and giving representative examples of their use in subcellular research, specifically in the context of large-scale spatial studies. The following labeling strategies are not necessarily exclusive to each other, and combinational labeling protocols have been documented (
      • Pineau I.
      • Barrette B.
      • Vallières N.
      • Lacroix S.
      A novel method for multiple labeling combining in situ hybridization with immunofluorescence.
      ,
      • Chaudhuri A.D.
      • Yelamanchili S.V.
      • Fox H.S.
      Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers.
      ,
      • VanZomeren-Dohm A.
      • Flannery E.
      • Duman-Scheel M.
      Whole-mount in situ hybridization detection of mRNA in GFP-marked drosophila imaginal disc mosaic clones.
      ,
      • Zaglia T.
      • Di Bona A.
      • Chioato T.
      • Basso C.
      • Ausoni S.
      • Mongillo M.
      Optimized protocol for immunostaining of experimental GFP-expressing and human hearts.
      ,
      • Oliva A.A.
      • Swann J.W.
      Fluorescence in situ hybridization method for co-localization of mRNA and GEP.
      ).

      Visualization of Using Affinity Reagents

      Antibodies and Organelle-Specific Dyes

      In the case of proteins, the use of antibodies against specific endogenous proteins of interest is often known as immunofluorescence or immunocytometry. Immunofluorescence can be highly sensitive when using signal-amplifying reagents, such as secondary antibodies conjugated to various fluorophores. Readily available commercial antibodies make comparative studies of protein localization in different cell or tissue samples easy and fast, particularly in commonly used model organisms, such as humans and mice. Finding commercial antibodies for some less well-studied species and proteins can be more difficult. This can be overcome by genetically fusing an epitope, such as FLAG, to the protein of interest and then using an antibody against this epitope to indirectly label the protein. However, in this case, a fluorescent protein (FP), such as GFP, genetically fused to the protein is often favored as it negates the need for the antibody-labeling step. Chemical organelle–specific dyes, such as 4′,6-diamidino-2-phenylindole for nuclei staining, can also be used alongside antibodies. Reviews are available detailing such dyes (
      • Kilgore J.A.
      • Dolman N.J.
      • Davidson M.W.
      A review of reagents for fluorescence microscopy of cellular compartments and structures, part II: Reagents for non-vesicular organelles.
      ,
      • Zhu H.
      • Fan J.
      • Du J.
      • Peng X.
      Fluorescent probes for sensing and imaging within specific cellular organelles.
      ). It should be noted that antibodies are prone to batch-to-batch variability and poor specificity that can yield false results from nonspecific and variable binding. These drawbacks have caused major reproducibility crises amongst the scientific community (
      • Baker M.
      Reproducibility crisis: Blame it on the antibodies.
      ). In recent years, however, there has been a huge drive to address this key issue with commercial suppliers providing extensive validation and moving toward recombinant products with less batch variability. In addition, with the increasing accessibility of CRISPR technology, validating specificity of antibodies using CRISPR knockouts is becoming common practice. Immunofluorescence-based methods are also restricted to static end-point measurements since such experiments require cell fixation and permeabilization prior to intracellular staining (Fig. 1A). Sample preparation can be very context specific, and inappropriate selection of fixation and permeabilization approaches can affect protein localization by introducing artifacts or causing loss of soluble proteins (
      • Lundberg E.
      • Borner G.H.H.
      Spatial proteomics: A powerful discovery tool for cell biology.
      • Schnell U.
      • Dijk F.
      • Sjollema K.A.
      • Giepmans B.N.
      Immunolabeling artifacts and the need for live-cell imaging.
      ). However, standardization of sample preparation and developments in automation has allowed multiplexing of off-the-shelf antibodies to improve throughput (
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Gut G.
      • Herrmann M.D.
      • Pelkmans L.
      Multiplexed protein maps link subcellular organization to cellular states.
      ).
      Figure thumbnail gr1
      Fig. 1Microscopy-based imaging approaches for subcellular proteomics or transcriptomics, focusing on the probing strategies. A, traditional antibody staining involves probing subcellular targets (such as the mitochondrial substructure) using monoclonal antibodies. These may be directly conjugated to a fluorescent label (direct immunofluorescence) or with a fluorescently labeled secondary antibody (indirect immunofluorescence). To determine subcellular location of proteins, an antibody against an organelle marker or a dye must be used alongside an antibody against the protein of interest. Then analysis can be performed to determine and quantify the colocalization of these antibodies/dyes. B, fluorescent protein reporters, such as GFP, can be genetically engineered to be fused and expressed with a target gene/protein of interest. Therefore, allowing confocal imaging of molecules that have no antibody or require live-cell imaging. In MS2 labeling systems for RNA, fluorescent reporter proteins can be genetically fused to MCP. C, RNA aptamers are an alternative to MS2 systems for labeling RNA, which allow for fusion of an RNA structure that binds and stabilizes an exogenous fluorescent molecule (e.g., DFHBI). RNA aptamers can be used either as affinity reagents or as reporters. D, in situ hybridization (ISH) employs a variety of antisense nucleic acid probes for the detection of RNA of interest in permeabilized and fixed cellular material. Recent ISH strategies have allowed for highly multiplexed experimental designs using molecular barcoding (e.g., seqFISH and MERFISH). DFHBI, 3,5-difluoro-4-hydroxybenzylideneimidazolidinone; MCP, bacteriophage MS2 coat protein; MERFISH, multiplexed error-robust FISH; seqFISH, sequential barcoding FISH.
      Limited global spatial proteomics experiments have been conducted because of the aforementioned restrictions. The largest immunofluorescence-based subcellular proteomics study performed to date is the work of the Cell Atlas database. This work is part of the wider Human Protein Atlas (HPA) initiative, aiming to document the entirety of the human subcellular proteome in different human cell and tissue types to elucidate protein function and create a comprehensive biological resource for human proteins in health and disease (
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
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      • Sivertsson Å.
      • Kampf C.
      • Sjöstedt E.
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      • Olsson I.
      • Edlund K.
      • Lundberg E.
      • Navani S.
      • Szigyarto C.A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ,
      • Thul P.J.
      • Åkesson L.
      • Wiking M.
      • Mahdessian D.
      • Geladaki A.
      • Ait Blal H.
      • Alm T.
      • Asplund A.
      • Björk L.
      • Breckels L.M.
      • Bäckström A.
      • Danielsson F.
      • Fagerberg L.
      • Fall J.
      • Gatto L.
      • et al.
      A subcellular map of the human proteome.
      ,
      • Uhlen M.
      • Zhang C.
      • Lee S.
      • Sjöstedt E.
      • Fagerberg L.
      • Bidkhori G.
      • Benfeitas R.
      • Arif M.
      • Liu Z.
      • Edfors F.
      • Sanli K.
      • von Feilitzen K.
      • Oksvold P.
      • Lundberg E.
      • Hober S.
      • et al.
      A pathology atlas of the human cancer transcriptome.
      ). HPA has collaborated with other international-scale projects, such as UniProt, NextProt, GO, ELIXIR, to provide publicly available databases of subcellular information for the wider scientific community (
      • Bateman A.
      • Martin M.J.
      • Orchard S.
      • Magrane M.
      • Agivetova R.
      • Ahmad S.
      • Alpi E.
      • Bowler-Barnett E.H.
      • Britto R.
      • Bursteinas B.
      • Bye-A-Jee H.
      • Coetzee R.
      • Cukura A.
      • Da Silva A.
      • Denny P.
      • et al.
      The UniProt Consortium
      UniProt: The universal protein knowledgebase in 2021.
      ,
      • Zahn-Zabal M.
      • Michel P.A.
      • Gateau A.
      • Nikitin F.
      • Schaeffer M.
      • Audot E.
      • Gaudet P.
      • Duek P.D.
      • Teixeira D.
      • Rech de Laval V.
      • Samarasinghe K.
      • Bairoch A.
      • Lane L.
      The neXtProt knowledgebase in 2020: Data, tools and usability improvements.
      ,
      • The Gene Ontology Consortium
      The Gene Ontology resource: Enriching a GOld mine.
      ,
      • Vizcaíno J.A.
      • Walzer M.
      • Jiménez R.C.
      • Bittremieux W.
      • Bouyssié D.
      • Carapito C.
      • Corrales F.
      • Ferro M.
      • Heck A.J.R.
      • Horvatovich P.
      • Hubalek M.
      • Lane L.
      • Laukens K.
      • Levander F.
      • Lisacek F.
      • et al.
      A community proposal to integrate proteomics activities in ELIXIR.
      ). During the past two decades, a near proteome-wide collection of antibodies has been created and validated for the purpose of this initiative (
      • Nilsson P.
      • Paavilainen L.
      • Larsson K.
      • Odling J.
      • Sundberg M.
      • Andersson A.C.
      • Kampf C.
      • Persson A.
      • Al-Khalili Szigyarto C.
      • Ottosson J.
      • Björling E.
      • Hober S.
      • Wernérus H.
      • Wester K.
      • Pontén F.
      • et al.
      Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling.
      ,
      • Uhlén M.
      • Björling E.
      • Agaton C.
      • Szigyarto C.A.
      • Amini B.
      • Andersen E.
      • Andersson A.C.
      • Angelidou P.
      • Asplund A.
      • Asplund C.
      • Berglund L.
      • Bergström K.
      • Brumer H.
      • Cerjan D.
      • Ekström M.
      • et al.
      A human protein atlas for normal and cancer tissues based on antibody proteomics.
      ,
      • Algenäs C.
      • Agaton C.
      • Fagerberg L.
      • Asplund A.
      • Björling L.
      • Björling E.
      • Kampf C.
      • Lundberg E.
      • Nilsson P.
      • Persson A.
      • Wester K.
      • Pontén F.
      • Wernérus H.
      • Uhlén M.
      • Ottosson Takanen J.
      • et al.
      Antibody performance in western blot applications is context-dependent.
      ,
      • Skogs M.
      • Stadler C.
      • Schutten R.
      • Hjelmare M.
      • Gnann C.
      • Björk L.
      • Poser I.
      • Hyman A.
      • Uhlén M.
      • Lundberg E.
      Antibody validation in bioimaging applications based on endogenous expression of tagged proteins.
      ,
      • Stadler C.
      • Hjelmare M.
      • Neumann B.
      • Jonasson K.
      • Pepperkok R.
      • Uhlén M.
      • Lundberg E.
      Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy.
      ). This work used 14,000 antibodies to systematically map the spatial distribution of 12,003 proteins at single-cell resolution to one or more of 30 different subcellular niches. Of those proteins, 5,662 lacked subcellular localization information in the literature prior to this study. This classification was performed using a combination of manual and computational image analysis approaches (
      • Thul P.J.
      • Åkesson L.
      • Wiking M.
      • Mahdessian D.
      • Geladaki A.
      • Ait Blal H.
      • Alm T.
      • Asplund A.
      • Björk L.
      • Breckels L.M.
      • Bäckström A.
      • Danielsson F.
      • Fagerberg L.
      • Fall J.
      • Gatto L.
      • et al.
      A subcellular map of the human proteome.
      ,
      • Sullivan D.P.
      • Winsnes C.F.
      • Åkesson L.
      • Hjelmare M.
      • Wiking M.
      • Schutten R.
      • Campbell L.
      • Leifsson H.
      • Rhodes S.
      • Nordgren A.
      • Smith K.
      • Revaz B.
      • Finnbogason B.
      • Szantner A.
      • Lundberg E.
      Deep learning is combined with massive-scale citizen science to improve large-scale image classification.
      ). Notably, the images were obtained using high-resolution confocal microscopy, enabling assignment of proteins to fine and less-well characterized cellular structures, such as microtubule ends, cytokinetic bridge subcompartments, and the nucleolar fibrillar center, as well as to functionally uncharacterized subcellular niches, such as rods and rings. Moreover, this work showed that approximately half of all human proteins (6,163 out of 12,003 proteins in this dataset) localize to multiple (two or more) subcellular niches. This dataset also revealed that more than one-sixth of the human proteome displays variability in terms of expression levels or subcellular distribution at the level of single cells (
      • Thul P.J.
      • Åkesson L.
      • Wiking M.
      • Mahdessian D.
      • Geladaki A.
      • Ait Blal H.
      • Alm T.
      • Asplund A.
      • Björk L.
      • Breckels L.M.
      • Bäckström A.
      • Danielsson F.
      • Fagerberg L.
      • Fall J.
      • Gatto L.
      • et al.
      A subcellular map of the human proteome.
      ). During the coronavirus disease 2019 pandemic, with collaborators, HPA turned to mapping the distribution of the virus' key host interactor, ACE2, across >150 human tissues, as well as the human interactome of coronavirus disease 2019 with the aim to determine whether readily available drugs can be repurposed in the fight against the virus (
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ).
      In immunofluorescence, the number of proteins of interest that can be probed in one sample is largely limited to the number of fluorochromes that can be used without causing signal interference by spectral overlap or fluorescent bleed-through into other channels. Therefore, traditionally, only around 4 to 6 fluorochromes could be used at a time, where each primary antibody is labeled with its unique fluorochrome (e.g., conjugated to a secondary antibody). Recent developments in using either cyclical probing with antibodies, such as cyclic immunofluorescence, or using them in combination with other types of probes, such as oligonucleotides as molecular barcodes in co-detection by indexing, has allowed for improved multiplexing (
      • Gut G.
      • Herrmann M.D.
      • Pelkmans L.
      Multiplexed protein maps link subcellular organization to cellular states.
      ,
      • Lin J.R.
      • Fallahi-Sichani M.
      • Sorger P.K.
      Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.
      ,
      • Goltsev Y.
      • Samusik N.
      • Kennedy-Darling J.
      • Bhate S.
      • Hale M.
      • Vazquez G.
      • Black S.
      • Nolan G.P.
      Deep profiling of mouse splenic architecture with CODEX multiplexed imaging.
      ,
      • Lin J.R.
      • Izar B.
      • Wang S.
      • Yapp C.
      • Mei S.
      • Shah P.M.
      • Santagata S.
      • Sorger P.K.
      Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes.
      ). Another example of overcoming fluorescent signal overlap was the use of unique and fidentifiable DNA origami structures with the blinking kinetics of DNA-point accumulation in nanoscale topography (DNA-PAINT) that allowed multiplexing antibody probes in a single channel with super-resolution microscopy (
      • Jungmann R.
      • Avendaño M.S.
      • Woehrstein J.B.
      • Dai M.
      • Shih W.M.
      • Yin P.
      Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and exchange-PAINT.
      ,
      • Schnitzbauer J.
      • Strauss M.T.
      • Schlichthaerle T.
      • Schueder F.
      • Jungmann R.
      Super-resolution microscopy with DNA-PAINT.
      ).
      The targeting of RNA transcripts directly via antibodies is far less prevalent than proteins. Antibodies against RNA antigens do exist; however, these are limited to global RNA applications. For example, antibodies against a subtype of RNA, such as rRNA, or for epigenetic applications, such as certain global modifications of RNA (e.g., methylation or acetylation groups). This restricts their primary use to immunoblotting or immunopurifications and is not typically applicable to imaging (
      • Doerr A.
      RNA antibodies: Upping the ante.
      • Ye J.D.
      • Tereshko V.
      • Frederiksen J.K.
      • Koide A.
      • Fellouse F.A.
      • Sidhu S.S.
      • Koide S.
      • Kossiakoff A.A.
      • Piccirilli J.A.
      Synthetic antibodies for specific recognition and crystallization of structured RNA.
      ).

      In Situ Hybridization

      In situ hybridization was first discovered as a useful nucleic acid labeling tool in 1969 using radioactive tritium-labeled antisense sequences to image the nuclei of frog eggs (
      • Gall J.G.
      • Pardue M.L.
      Formation and detection of RNA-DNA hybrid molecules in cytological preparations.
      ). FISH was soon adopted as a safer and more stable alternative (
      • Rudkin G.T.
      • Stollar B.D.
      High resolution detection of DNA-RNA hybrids in situ by indirect immunofluorescence.
      ). The oligonucleotides used in FISH are designed to hybridize on the RNA target by sequence complementarity. These oligonucleotides are labeled either directly or indirectly, via a secondary probe (such as an antibody) conjugated to a fluorophore (Fig. 1D). Single-molecule resolution was enabled by “tiling” multiple antisense probes along a sequence of interest to boost signal and has been a powerful tool in understanding the role of RNA localization in biology, such as in meiosis and neuromuscular junctions (
      • Chen J.
      • McSwiggen D.
      • Ünal E.
      Single molecule fluorescence in situ hybridization (SmFISH) analysis in budding yeast vegetative growth and meiosis.
      ,
      • Ding D.Q.
      • Okamasa K.
      • Katou Y.
      • Oya E.
      • Nakayama J.I.
      • Chikashige Y.
      • Shirahige K.
      • Haraguchi T.
      • Hiraoka Y.
      Chromosome-associated RNA–protein complexes promote pairing of homologous chromosomes during meiosis in Schizosaccharomyces pombe.
      ,
      • Titlow J.S.
      • Yang L.
      • Parton R.M.
      • Palanca A.
      • Davis I.
      Super-resolution single molecule FISH at the Drosophila neuromuscular junction.
      ). The main restriction of FISH has been its low throughput and need to be performed in fixed cells to prevent RNase and DNase degradation of nucleic acids, limiting its use for temporal applications. However, FISH imaging in live cells, known as “live FISH,” has been achieved with the caveats of using toxic permeabilization techniques and rapid sequestering of the molecular beacons in the nucleus (
      • Simon B.
      • Sandhu M.
      • Myhr K.L.
      Live FISH: Imaging mRNA in living neurons.
      ,
      • Oomoto I.
      • Suzuki-Hirano A.
      • Umeshima H.
      • Han Y.W.
      • Yanagisawa H.
      • Carlton P.
      • Harada Y.
      • Kengaku M.
      • Okamoto A.
      • Shimogori T.
      • Wang D.O.
      ECHO-liveFISH: In vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.
      ). It has only been with the recent developments within CRISPR–Cas9 technology that live FISH has been possible without such drawbacks (
      • Wang H.
      • Nakamura M.
      • Abbott T.R.
      • Zhao D.
      • Luo K.
      • Yu C.
      • Nguyen C.M.
      • Lo A.
      • Daley T.P.
      • La Russa M.
      • Liu Y.
      • Qi L.S.
      CRISPR-mediated live imaging of genome editing and transcription.
      ). Despite live-imaging alternatives, such as aptamers, RNA FISH is still a gold-standard technique for RNA localization, and recent advancements, such as CRISPR–Cas9, have kept it current and pervasive.
      FISH has a multitude of available signal-amplifying probes to choose from, which are particularly useful for overcoming hurdles commonly found in difficult targets and samples, such as short noncoding RNA and tissues (
      • Pichon X.
      • Lagha M.
      • Mueller F.
      • Bertrand E.
      A growing toolbox to image gene expression in single cells: Sensitive approaches for demanding challenges.
      ). Generally, these probes have branched structures that increase the molecular surface area for multiple fluorophores to bind to the molecule of interest, which form the basis for single-molecule inexpensive FISH, FISH with sequential tethered and intertwined oligodeoxynucleotide complexes, branched DNA (bDNA) FISH, and hybridization chain reaction FISH (
      • Sinnamon J.R.
      • Czaplinski K.
      RNA detection in situ with FISH-STICs.
      ,
      • Wang F.
      • Flanagan J.
      • Su N.
      • Wang L.C.
      • Bui S.
      • Nielson A.
      • Wu X.
      • Vo H.T.
      • Ma X.J.
      • Luo Y.
      RNAscope: A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.
      ,
      • Choi H.M.
      • Chang J.Y.
      • Trinh le A.
      • Padilla J.E.
      • Fraser S.E.
      • Pierce N.A.
      Programmable in situ amplification for multiplexed imaging of mRNA expression.
      ,
      • Choi H.M.T.
      • Schwarzkopf M.
      • Fornace M.E.
      • Acharya A.
      • Artavanis G.
      • Stegmaier J.
      • Cunha A.
      • Pierce N.A.
      Third-generation in situ hybridization chain reaction: Multiplexed, quantitative, sensitive, versatile, robust.
      ). For targets that require particularly high specificity, such as short noncoding RNAs, padlock probes can covalently “lock” and amplify the signal using a rolling circle mechanism (
      • Banér J.
      • Nilsson M.
      • Mendel-Hartvig M.
      • Landegren U.
      Signal amplification of padlock probes by rolling circle replication.
      ,
      • Deng R.
      • Zhang K.
      • Sun Y.
      • Ren X.
      • Li J.
      Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification.
      ). bDNA probes were used for a large-scale imaging study, which targeted 928 genes involved in cancer, endocytosis, and metabolism at a single-cell level (
      • Battich N.
      • Stoeger T.
      • Pelkmans L.
      Image-based transcriptomics in thousands of single human cells at single-molecule resolution.
      ). The use of enzyme amplification of in situ hybridization probes and 96-well plates enabled mapping of mRNA dynamics in embryogenesis of Drosophila, achieving analysis of 3,370 transcripts and demonstrated a correlation between mRNA localization and subsequent protein localization and function (
      • Lécuyer E.
      • Yoshida H.
      • Parthasarathy N.
      • Alm C.
      • Babak T.
      • Cerovina T.
      • Hughes T.R.
      • Tomancak P.
      • Krause H.M.
      Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.
      ). In addition, super-resolution FISH was used alongside RNA-Seq methods to track the dynamics of proteins in dendritic cells (
      • Cajigas I.J.
      • Tushev G.
      • Will T.J.
      • tom Dieck S.
      • Fuerst N.
      • Schuman E.M.
      The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging.
      ).
      Multiplexing is also a powerful feature of FISH with easy to perform probe generation and sequential rehybridization of the sample, allowing for multiple rounds of reprobing and fluorescent barcoding of thousands of molecules, with minimal loss of signal (
      • Lubeck E.
      • Coskun A.F.
      • Zhiyentayev T.
      • Ahmad M.
      • Cai L.
      Single-cell in situ RNA profiling by sequential hybridization.
      ). Novel methods, such as multiplexed error-robust FISH (MERFISH) and sequential barcoding FISH (seqFISH), exploit such characteristics and, in theory, have the capability of generating spatial information of the entire known transcriptome in just eight rounds of hybridization and four dyes (48 = 65,536) (
      • Lubeck E.
      • Coskun A.F.
      • Zhiyentayev T.
      • Ahmad M.
      • Cai L.
      Single-cell in situ RNA profiling by sequential hybridization.
      ,
      • Chen K.H.
      • Boettiger A.N.
      • Moffitt J.R.
      • Wang S.
      • Zhuang X.
      RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells.
      ,
      • Xia C.
      • Babcock H.P.
      • Moffitt J.R.
      • Zhuang X.
      Multiplexed detection of RNA using MERFISH and branched DNA amplification.
      ). Realistically, this level of coverage is not achievable with the exponential increase in error rates per round of hybridization. MERFISH employs an error-detection barcoding scheme to account for a proportion of this error and when used in conjunction with bDNA probes to amplify the signal across ∼10,000 transcripts by 10.5-fold (
      • Xia C.
      • Babcock H.P.
      • Moffitt J.R.
      • Zhuang X.
      Multiplexed detection of RNA using MERFISH and branched DNA amplification.
      ). Optical overcrowding of transcripts is also a limiting factor for such techniques. seqFISH+ was developed to circumvent this optical overcrowding by expanding the fluorophore palette from 4 to 5 colors to 60 “pseudocolors” using molecular barcoding, allowing analysis of 24,000 genes in four rounds with one round of error correction (
      • Eng C.L.
      • Lawson M.
      • Zhu Q.
      • Dries R.
      • Koulena N.
      • Takei Y.
      • Yun J.
      • Cronin C.
      • Karp C.
      • Yuan G.C.
      • Cai L.
      Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+.
      ). MERFISH and seqFISH have provided insight into the spatial organization of the cell cycle, mouse hippocampus, and tissue development and homeostasis, as well as capturing nascent transcription active sites of genes (
      • Xia C.
      • Fan J.
      • Emanuel G.
      • Hao J.
      • Zhuang X.
      Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression.
      ,
      • Shah S.
      • Lubeck E.
      • Zhou W.
      • Cai L.
      seqFISH accurately detects transcripts in single cells and reveals robust spatial organization in the Hippocampus.
      ,
      • Mayr U.
      • Serra D.
      • Liberali P.
      Exploring single cells in space and time during tissue development, homeostasis and regeneration.
      ,
      • Shah S.
      • Takei Y.
      • Zhou W.
      • Lubeck E.
      • Yun J.
      • Eng C.L.
      • Koulena N.
      • Cronin C.
      • Karp C.
      • Liaw E.J.
      • Amin M.
      • Cai L.
      Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH.
      ). Amplification is very powerful, though it only provides a global increase of intensity across targets, which cannot distinguish real RNA spots/signals from nonspecifically bound probes, which affects the resolution. To overcome this, experimentation with different split probes was conducted to achieve impressively punctate transcript spots, which can only fluoresce when two probes dock within immediate proximity on a highly specific and shared bridge sequence (
      • Goh J.J.L.
      • Chou N.
      • Seow W.Y.
      • Ha N.
      • Cheng C.P.P.
      • Chang Y.C.
      • Zhao Z.W.
      • Chen K.H.
      Highly specific multiplexed RNA imaging in tissues with split-FISH.
      ). An untargeted alternative to the aforementioned, fluorescent in situ sequencing (FISSEQ) used crosslinking and reverse transcription of RNA in situ to perform RNA-Seq with cyclic fluorescent probe ligations directly on the sample, which was measured via confocal microscopy (
      • Lee J.H.
      • Daugharthy E.R.
      • Scheiman J.
      • Kalhor R.
      • Ferrante T.C.
      • Terry R.
      • Turczyk B.M.
      • Yang J.L.
      • Lee H.S.
      • Aach J.
      • Zhang K.
      • Church G.M.
      Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.
      ). This method was demonstrated in a variety of sample types, such as primary fibroblasts, tissues, and whole embryos, and could be powerful in applications such as cellular phenotyping and gene regulation. The original FISSEQ publication uncovered that long noncoding RNAs preferentially localize in the nucleus. The premise of this method is powerful, but FISSEQ struggles to attain read counts comparable with standard single-cell RNA-Seq (scRNA-Seq), is difficult to perform in tissues, and is limited to short reads (
      • Lee J.H.
      • Daugharthy E.R.
      • Scheiman J.
      • Kalhor R.
      • Ferrante T.C.
      • Terry R.
      • Turczyk B.M.
      • Yang J.L.
      • Lee H.S.
      • Aach J.
      • Zhang K.
      • Church G.M.
      Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.
      ). A new variation of FISSEQ, known as In Situ Transcriptome Accessibility sequencing, has recently been developed for longer reads (
      • Fürth D.
      • Hatini V.
      • Lee J.H.
      In situ transcriptome accessibility sequencing (INSTA-seq).
      ). To determine the precise subcellular localization of transcripts, it is recommended that organelle-specific dyes or immunofluorescence or organellar proteins are used as counterstains in these approaches (
      • Lee J.H.
      • Daugharthy E.R.
      • Scheiman J.
      • Kalhor R.
      • Ferrante T.C.
      • Terry R.
      • Turczyk B.M.
      • Yang J.L.
      • Lee H.S.
      • Aach J.
      • Zhang K.
      • Church G.M.
      Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.
      ). The versatility of FISH shows that it still has untapped potential in the transcriptomics world, and some of the newer methods have been recently reviewed (
      • Liao J.
      • Lu X.
      • Shao X.
      • Zhu L.
      • Fan X.
      Uncovering an organ’s molecular architecture at single-cell resolution by spatially resolved transcriptomics.
      ).

      Visualization Using Fluorescently Tagged Proteins

      FPs

      Genetically fused FPs are the next most prolific method of fluorescently labeling molecules, with the work that allowed scientists to harness FPs for research winning the Nobel Prize in chemistry in 2008 (
      • Shimomura O.
      Structure of the chromophore of Aequorea green fluorescent protein.
      ,
      • Chalfie M.
      • Tu Y.
      • Euskirchen G.
      • Ward W.W.
      • Prasher D.C.
      Green fluorescent protein as a marker for gene expression.
      ,
      • Heim R.
      • Tsien R.Y.
      Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer.
      ). Since the discovery and enhanced engineering of FPs, their use has provided immense biological insights into multiple processes, including demonstrating pH- and receptor-dependent endocytic viral entry during severe acute respiratory syndrome infection (
      • Wang H.
      • Yang P.
      • Liu K.
      • Guo F.
      • Zhang Y.
      • Zhang G.
      • Jiang C.
      SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway.
      ). This strategy involves fusing a reporter protein gene, usually an FP or a sequence that can be fluorescently labeled downstream, to a protein of interest using transfection. When the protein of interest is expressed, so is the fused reporter protein or sequence, which can then either be directly excited at the appropriate wavelength or labeled with a fluorophore (e.g., a fluorescent antibody) (Fig. 1B). In contrast to strategies with affinity reagents, FPs allow for live-cell imaging, capturing temporal protein dynamics. An innovative and multicolored system called fluorescent ubiquitination-based cell cycle indicator utilizes fused FP monomers to two proteins, Cdt1 and geminin, that are specifically degraded in different parts of the cell cycle, at S/G2 and M/G1 phases, respectively. This strategy allows for cell cycle–dependent multicolored labeling of the nuclei (
      • Sakaue-Sawano A.
      • Kurokawa H.
      • Morimura T.
      • Hanyu A.
      • Hama H.
      • Osawa H.
      • Kashiwagi S.
      • Fukami K.
      • Miyata T.
      • Miyoshi H.
      • Imamura T.
      • Ogawa M.
      • Masai H.
      • Miyawaki A.
      Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.
      ). The strategy has allowed for deconvolution of cell cycle states and cellular processes that are otherwise difficult to distinguish. For example, it has been used to determine the relationship between the progression of double-stranded break repair and cell cycle status in living cells with the aim to help development and assessment of cancer therapies (
      • Otsuka K.
      • Tomita M.
      Concurrent live imaging of DNA double-strand break repair and cell-cycle progression by CRISPR/Cas9-mediated knock-in of a tricistronic vector.
      ). However, sensitivity can be an issue, as it has been shown that only a third of the most abundant proteins in mammalian cells can be detected using the most widely used FP, GFP, although this can be mitigated via using more photostable or/and brighter tags (
      • Kamiyama D.
      • Sekine S.
      • Barsi-Rhyne B.
      • Hu J.
      • Chen B.
      • Gilbert L.A.
      • Ishikawa H.
      • Leonetti M.D.
      • Marshall W.F.
      • Weissman J.S.
      • Huang B.
      Versatile protein tagging in cells with split fluorescent protein.
      ). Furthermore, it has been shown that in certain cases, tagging endogenous proteins can interfere with specific properties of native molecules, including its subcellular localization. For instance, FPs have been found to erroneously locate at the endomembrane system of mammalian cells (
      • Stadler C.
      • Rexhepaj E.
      • Singan V.R.
      • Murphy R.F.
      • Pepperkok R.
      • Uhlén M.
      • Simpson J.C.
      • Lundberg E.
      Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells.
      ,
      • Simpson J.C.
      • Wellenreuther R.
      • Poustka A.
      • Pepperkok R.
      • Wiemann S.
      Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.
      ). This localization artifact can be influenced by where the FP has been genetically encoded on the target protein (e.g., on the N or C terminus). This effect was extensively examined in budding yeast (
      • Weill U.
      • Krieger G.
      • Avihou Z.
      • Milo R.
      • Schuldiner M.
      • Davidi D.
      Assessment of GFP tag position on protein localization and growth fitness in yeast.
      ). As well as this, protein fusion can also impair the normal expression, function, or degradation patterns of the native protein. Therefore, verification is required to ensure that endogenous localization and expression of the target molecule is unaffected by genetic fusion.
      Saccharomyces cerevisiae has highly efficient homologous recombination processes compared with mammalian cells, making it relatively easy to generate FP-fused libraries, while generally preserving the normal expression patterns of the endogenous genes. Therefore, this species was used to conduct the first genome-wide library of a eukaryote for live-cell imaging using GFP tagging, achieving systematic localization of 75% of the yeast proteome to 22 distinct subcellular niches under normal culture conditions. This study provided novel localization information on 1630 proteins (
      • Huh W.K.
      • Falvo J.V.
      • Gerke L.C.
      • Carroll A.S.
      • Howson R.W.
      • Weissman J.S.
      • O'Shea E.K.
      Global analysis of protein localization in budding yeast.
      ). Subsequent studies have used this yeast library under multiple conditions of environmental stress to uncover yeast protein localization dynamics as well as providing a quantitative dimension (
      • Chong Y.T.
      • Koh J.L.
      • Friesen H.
      • Duffy S.K.
      • Duffy K.
      • Cox M.J.
      • Moses A.
      • Moffat J.
      • Boone C.
      • Andrews B.J.
      Yeast proteome dynamics from single cell imaging and automated analysis.
      ,
      • Breker M.
      • Gymrek M.
      • Schuldiner M.
      A novel single-cell screening platform reveals proteome plasticity during yeast stress responses.
      ,
      • Dénervaud N.
      • Becker J.
      • Delgado-Gonzalo R.
      • Damay P.
      • Rajkumar A.S.
      • Unser M.
      • Shore D.
      • Naef F.
      • Maerkl S.J.
      A chemostat array enables the spatio-temporal analysis of the yeast proteome.
      ,
      • Tkach J.M.
      • Yimit A.
      • Lee A.Y.
      • Riffle M.
      • Costanzo M.
      • Jaschob D.
      • Hendry J.A.
      • Ou J.
      • Moffat J.
      • Boone C.
      • Davis T.N.
      • Nislow C.
      • Brown G.W.
      Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress.
      ,
      • Newman J.R.
      • Ghaemmaghami S.
      • Ihmels J.
      • Breslow D.K.
      • Noble M.
      • DeRisi J.L.
      • Weissman J.S.
      Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise.
      ) (reviewed in Ref. (
      • Torres N.P.
      • Ho B.
      • Brown G.W.
      High-throughput fluorescence microscopic analysis of protein abundance and localization in budding yeast.
      )). Improved technology has led to further ease with creating genome-wide fluorescent fusion libraries. For example, the SWAp-Tag method, which allows efficient modification of a parental library and was employed for generating both an N-terminally–tagged and C-terminally–tagged yeast proteomes (
      • Yofe I.
      • Weill U.
      • Meurer M.
      • Chuartzman S.
      • Zalckvar E.
      • Goldman O.
      • Ben-Dor S.
      • Schütze C.
      • Wiedemann N.
      • Knop M.
      • Khmelinskii A.
      • Schuldiner M.
      One library to make them all: Streamlining the creation of yeast libraries via a SWAp-Tag strategy.
      ,
      • Meurer M.
      • Duan Y.
      • Sass E.
      • Kats I.
      • Herbst K.
      • Buchmuller B.C.
      • Dederer V.
      • Huber F.
      • Kirrmaier D.
      • Štefl M.
      • Van Laer K.
      • Dick T.P.
      • Lemberg M.K.
      • Khmelinskii A.
      • Levy E.D.
      • et al.
      Genome-wide C-SWAT library for high-throughput yeast genome tagging.
      ,
      • Weill U.
      • Yofe I.
      • Sass E.
      • Stynen B.
      • Davidi D.
      • Natarajan J.
      • Ben-Menachem R.
      • Avihou Z.
      • Goldman O.
      • Harpaz N.
      • Chuartzman S.
      • Kniazev K.
      • Knoblach B.
      • Laborenz J.
      • Boos F.
      • et al.
      Genome-wide SWAp-Tag yeast libraries for proteome exploration.
      ). Such extensive and numerous libraries enabled meta-analysis of protein localization dynamics in a quantitative manner with an unsupervised computational method (
      • Lu A.X.
      • Moses A.M.
      An unsupervised knn method to systematically detect changes in protein localization in high-throughput microscopy images.
      ). Such approaches have been able to differentiate perturbation-specific relocalization events from more generalized stress responses, concluding that protein subcellular localization provides an important layer of cellular regulation, independent from modulation of protein expression levels (
      • Lu A.X.
      • Chong Y.T.
      • Hsu I.S.
      • Strome B.
      • Handfield L.F.
      • Kraus O.
      • Andrews B.J.
      • Moses A.M.
      Integrating images from multiple microscopy screens reveals diverse patterns of change in the subcellular localization of proteins.
      ,
      • Lu A.X.
      • Moses A.M.
      An unsupervised knn method to systematically detect changes in protein localization in high-throughput microscopy images.
      ). Because of the efforts mentioned previously, several databases containing imaging data on the spatial organization of the S. cerevisiae proteome are now publicly available (
      • Breker M.
      • Gymrek M.
      • Moldavski O.
      • Schuldiner M.
      LoQAtE-Localization and Quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast.
      ,
      • Riffle M.
      • Davis T.N.
      The yeast resource center public image repository: A large database of fluorescence microscopy images.
      ,
      • Chuartzman S.G.
      • Schuldiner M.
      Database for high throughput screening hits (dHITS): A simple tool to retrieve gene specific phenotypes from systematic screens done in yeast.
      ,
      • Cherry J.M.
      • Hong E.L.
      • Amundsen C.
      • Balakrishnan R.
      • Binkley G.
      • Chan E.T.
      • Christie K.R.
      • Costanzo M.C.
      • Dwight S.S.
      • Engel S.R.
      • Fisk D.G.
      • Hirschman J.E.
      • Hitz B.C.
      • Karra K.
      • Krieger C.J.
      • et al.
      Saccharomyces Genome Database: The genomics resource of budding yeast.
      ,
      • Koh J.L.
      • Chong Y.T.
      • Friesen H.
      • Moses A.
      • Boone C.
      • Andrews B.J.
      • Moffat J.
      CYCLoPs: A comprehensive database constructed from automated analysis of protein abundance and subcellular localization patterns in Saccharomyces cerevisiae.
      ,
      • Dubreuil B.
      • Sass E.
      • Nadav Y.
      • Heidenreich M.
      • Georgeson J.M.
      • Weill U.
      • Duan Y.
      • Meurer M.
      • Schuldiner M.
      • Knop M.
      • Levy E.D.
      YeastRGB: Comparing the abundance and localization of yeast proteins across cells and libraries.
      ).
      Similar efforts to systematically probe human protein subcellular localization using fluorescent reporter fusions have also been published but so far have only covered a small proportion of the proteome. For example, a collection of N-terminal and C-terminal GFP fusions to complementary DNA was generated to study protein localization in living human cells, resulting in localization assignment for 1600 human proteins (
      • Simpson J.C.
      • Wellenreuther R.
      • Poustka A.
      • Pepperkok R.
      • Wiemann S.
      Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.
      ). Similarly, an annotated reporter clone collection was built via exon tagging using retroviral particle–mediated delivery in 2006 (
      • Sigal A.
      • Milo R.
      • Cohen A.
      • Geva-Zatorsky N.
      • Klein Y.
      • Alaluf I.
      • Swerdlin N.
      • Perzov N.
      • Danon T.
      • Liron Y.
      • Raveh T.
      • Carpenter A.E.
      • Lahav G.
      • Alon U.
      Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins.
      ). This collection has been used in combination with time-lapse fluorescence microscopy to track the abundance and localization dynamics of more than 1,000 endogenous proteins in living human cells under different conditions (
      • Sigal A.
      • Milo R.
      • Cohen A.
      • Geva-Zatorsky N.
      • Klein Y.
      • Alaluf I.
      • Swerdlin N.
      • Perzov N.
      • Danon T.
      • Liron Y.
      • Raveh T.
      • Carpenter A.E.
      • Lahav G.
      • Alon U.
      Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins.
      ,
      • Sigal A.
      • Danon T.
      • Cohen A.
      • Milo R.
      • Geva-Zatorsky N.
      • Lustig G.
      • Liron Y.
      • Alon U.
      • Perzov N.
      Generation of a fluorescently labeled endogenous protein library in living human cells.
      ,
      • Cohen A.A.
      • Geva-Zatorsky N.
      • Eden E.
      • Frenkel-Morgenstern M.
      • Issaeva I.
      • Sigal A.
      • Milo R.
      • Cohen-Saidon C.
      • Liron Y.
      • Kam Z.
      • Cohen L.
      • Danon T.
      • Perzov N.
      • Alon U.
      Dynamic proteomics of individual cancer cells in response to a drug.
      ,
      • Frenkel-Morgenstern M.
      • Cohen A.A.
      • Geva-Zatorsky N.
      • Eden E.
      • Prilusky J.
      • Issaeva I.
      • Sigal A.
      • Cohen-Saidon C.
      • Liron Y.
      • Cohen L.
      • Danon T.
      • Perzov N.
      • Alon U.
      Dynamic proteomics: A database for dynamics and localizations of endogenous fluorescently-tagged proteins in living human cells.
      ). More recently, 1,311 proteins were fluorescently tagged using CRISPR-based fusion in multiple cell lines to achieve deep profiling of these proteins using 3D confocal microscopy, immunoprecipitation–mass spectrometry (MS), and next-generation sequencing (
      • Cho N.H.
      • Cheveralls K.C.
      • Brunner A.-D.
      • Kim K.
      • Michaelis A.C.
      • Raghavan P.
      • Kobayashi H.
      • Savy L.
      • Li J.Y.
      • Canaj H.
      • Kim J.Y.S.
      • Stewart E.M.
      • Gnann C.
      • McCarthy F.
      • Cabrera J.P.
      • et al.
      OpenCell: Proteome-scale endogenous tagging enables the cartography of human cellular organization.
      ).
      The fluorescent tagging methods described previously center on protein labeling, but variations of these approaches have also allowed probing of RNA localization. Typically, this has been possible by encoding RNA hairpins into the gene of interest, which when transcribed can then be targeted by a corresponding RBP that is coexpressed and fused with FPs (
      • Lampasona A.A.
      • Czaplinski K.
      RNA voyeurism: A coming of age story.
      ). The first and most used system of this kind is the MS2 system, which uses bacteriophage MS2 coat proteins (MCPs), which are RBPs, to target genetically inserted MS2 loops (
      • Bertrand E.
      • Chartrand P.
      • Schaefer M.
      • Shenoy S.M.
      • Singer R.H.
      • Long R.M.
      Localization of ASH1 mRNA particles in living yeast.
      ). Similar systems exploiting FPs have been added to the RNA localization repertoire, such as the P77 bacteriophage coat protein (PCP) system (
      • Daigle N.
      • Ellenberg J.
      LambdaN-GFP: An RNA reporter system for live-cell imaging.
      ,
      • Chen J.
      • Nikolaitchik O.
      • Singh J.
      • Wright A.
      • Bencsics C.E.
      • Coffin J.M.
      • Ni N.
      • Lockett S.
      • Pathak V.K.
      • Hu W.S.
      High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis.
      ,
      • Yiu H.-W.
      • Demidov V.V.
      • Toran P.
      • Cantor C.R.
      • Broude N.E.
      RNA detection in live bacterial cells using fluorescent protein complementation triggered by interaction of two RNA aptamers with two RNA-binding peptides.
      ,
      • Yin J.
      • Zhu D.
      • Zhang Z.
      • Wang W.
      • Fan J.
      • Men D.
      • Deng J.
      • Wei H.
      • Zhang X.E.
      • Cui Z.
      Imaging of mRNA-protein interactions in live cells using novel mCherry trimolecular fluorescence complementation systems.
      ,
      • Valencia-Burton M.
      • McCullough R.M.
      • Cantor C.R.
      • Broude N.E.
      RNA visualization in live bacterial cells using fluorescent protein complementation.
      ,
      • Wu B.
      • Chen J.
      • Singer R.H.
      Background free imaging of single mRNAs in live cells using split fluorescent proteins.
      ). tdTomato-labeled PCP was used to successfully track individual mRNA molecules during translation at polysomes in different subcellular locations in dendrites (
      • Wang C.
      • Han B.
      • Zhou R.
      • Zhuang X.
      Real-time imaging of translation on single mRNA transcripts in live cells.
      ). This study also utilized SunTag molecules, which provide protein scaffolds for multimerization of fluorescent tags to boost poor signal and to study translation in real time (
      • Wang C.
      • Han B.
      • Zhou R.
      • Zhuang X.
      Real-time imaging of translation on single mRNA transcripts in live cells.
      ,
      • Tanenbaum M.E.
      • Gilbert L.A.
      • Qi L.S.
      • Weissman J.S.
      • Vale R.D.
      A protein-tagging system for signal amplification in gene expression and fluorescence imaging.
      ,
      • Tanenbaum M.E.
      • Gilbert L.A.
      • Qi L.S.
      • Weissman J.S.
      • Vale R.D.
      A protein-tagging system for signal amplification in gene expression and fluorescence imaging.
      ). Several other methods have been developed for studying translation in both fixed and live-cell applications, which are reviewed in detail (
      • Biswas J.
      • Liu Y.
      • Singer R.H.
      • Wu B.
      Fluorescence imaging methods to investigate translation in single cells.
      ). Single-molecule imaging of both translation and degradation in live cells can be achieved using the entertainingly named translating RNA imaging by coat protein knockoff (TRICK) and 3(three)′-RNA end accumulation during turnover (TREAT) methods, both of which use dual-color MCP and PCP reporter systems. TRICK can distinguish untranslated from translated transcripts by incorporating loops for MCP and PCP at different locations in the sequence of the mRNA of interest (
      • Russo J.
      • Wilusz J.
      Trick or TREAT: A scary-good new approach for single-molecule mRNA decay analysis.
      ). During translation, the ribosome knocks off PCP in the coding region of the transcript leaving MCP behind (
      • Halstead J.M.
      • Wilbertz J.H.
      • Wippich F.
      • Lionnet T.
      • Ephrussi A.
      • Chao J.A.
      TRICK: A single-molecule method for imaging the first round of translation in living cells and animals.
      ). TREAT uses a similar concept, where PCP is used to label the 3′ end of the transcript, which is lost during degradation (
      • Horvathova I.
      • Voigt F.
      • Kotrys A.V.
      • Zhan Y.
      • Artus-Revel C.G.
      • Eglinger J.
      • Stadler M.B.
      • Giorgetti L.
      • Chao J.A.
      The dynamics of mRNA turnover revealed by single-molecule imaging in single cells.
      ). Both TRICK and TREAT have been used independently in HeLa cells under arsenite stress to show reporter mRNAs retained in P-bodies are suspended, neither being translated nor degraded (
      • Horvathova I.
      • Voigt F.
      • Kotrys A.V.
      • Zhan Y.
      • Artus-Revel C.G.
      • Eglinger J.
      • Stadler M.B.
      • Giorgetti L.
      • Chao J.A.
      The dynamics of mRNA turnover revealed by single-molecule imaging in single cells.
      ,
      • Halstead J.M.
      • Lionnet T.
      • Wilbertz J.H.
      • Wippich F.
      • Ephrussi A.
      • Singer R.H.
      • Chao J.A.
      Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals.
      ,
      • Wilbertz J.H.
      • Voigt F.
      • Horvathova I.
      • Roth G.
      • Zhan Y.
      • Chao J.A.
      Single-molecule imaging of mRNA localization and regulation during the integrated stress response.
      ).
      MS2-based and MS2-like systems tended to suffer from low signal-to-noise ratios. Constitutive fusion of coat proteins to FPs means that the fluorescence is independent of being bound to the sequence of interest. The signal-to-noise ratio can be significantly improved by including a nuclear localization sequence, so unbound protein is sequestered in the nucleus to improve the background of cytoplasmic transcripts (
      • Lampasona A.A.
      • Czaplinski K.
      RNA voyeurism: A coming of age story.
      ,
      • Wu B.
      • Chao J.A.
      • Singer R.H.
      Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells.
      ). Also, much like FP tagging, there is no clear rule as to where to genetically encode the RNA–stem loops within the endogenous transcript (
      • Weil T.T.
      • Parton R.M.
      • Davis I.
      Making the message clear: Visualizing mRNA localization.
      ). There has been evidence that introduction of MS2-coated stem loops in yeast causes inhibition of mRNA decay, leading to RNA fragments that can continue to fluoresce and consequent aberrant localization measurements (
      • Garcia J.F.
      • Parker R.
      MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: Implications for the localization of mRNAs by MS2-MCP system.
      ,
      • Heinrich S.
      • Sidler C.L.
      • Azzalin C.M.
      • Weis K.
      Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing.
      ). However, there is debate whether this evidence was an artifact of gene expression and/or the methods used to assess this degradation (
      • Haimovich G.
      • Zabezhinsky D.
      • Haas B.
      • Slobodin B.
      • Purushothaman P.
      • Fan L.
      • Levin J.Z.
      • Nusbaum C.
      • Gerst J.E.
      Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to ‘MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: Implications for the localization of mRNAs by MS2-MCP system’.
      ). To address these concerns, a modified coat-protein reporter system allowing for efficient RNA degradation was established in both yeast and mammalian cells (
      • Tutucci E.
      • Vera M.
      • Biswas J.
      • Garcia J.
      • Parker R.
      • Singer R.H.
      An improved MS2 system for accurate reporting of the mRNA life cycle.
      ).

      RNA Aptamers

      RNA aptamers have been used in both in vitro and in vivo imaging as affinity reagents and reporter tags, respectively (
      • Bai J.
      • Luo Y.
      • Wang X.
      • Li S.
      • Luo M.
      • Yin M.
      • Zuo Y.
      • Li G.
      • Yao J.
      • Yang H.
      • Zhang M.
      • Wei W.
      • Wang M.
      • Wang R.
      • Fan C.
      • et al.
      A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering.
      ,
      • Yan Q.
      • Cai M.
      • Zhou L.
      • Xu H.
      • Shi Y.
      • Sun J.
      • Jiang J.
      • Gao J.
      • Wang H.
      Using an RNA aptamer probe for super-resolution imaging of native EGFR.
      ). They are short RNA oligonucleotides that can be conjugated to fluorescent dyes or designed to bind and induce the fluorescence of exogenous small molecules such as 3,5-difluoro-4-hydroxybenzylideneimidazolidinone (DFHBI), which is structurally related to the GFP chromophore (
      • Paige J.S.
      • Wu K.Y.
      • Jaffrey S.R.
      RNA mimics of green fluorescent protein.
      ) (Fig. 1C). DFHBI is structurally unstable, preventing its fluorescent activity until it is bound to the complementary active site of the fluorogenic RNA aptamer, bypassing the constitutive fluorescence that is caused by the persistent RBP–FP interaction in MS2-style systems.
      The original DFHBI-binding RNA aptamer, Spinach, demonstrated excellent brightness with minimal background fluorescence and resistance to photobleaching. Typically, fluorogenic RNA aptamers are expressed fused to an RNA of interest for subcellular RNA imaging in live cells (
      • Bai J.
      • Luo Y.
      • Wang X.
      • Li S.
      • Luo M.
      • Yin M.
      • Zuo Y.
      • Li G.
      • Yao J.
      • Yang H.
      • Zhang M.
      • Wei W.
      • Wang M.
      • Wang R.
      • Fan C.
      • et al.
      A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering.
      ,
      • Paige J.S.
      • Wu K.Y.
      • Jaffrey S.R.
      RNA mimics of green fluorescent protein.
      ). Guet et al. used spinach to show nuclear relocalization of STL1 and CTT1 transcripts in S. cerevisiae upon osmotic stress (
      • Guet D.
      • Burns L.T.
      • Maji S.
      • Boulanger J.
      • Hersen P.
      • Wente S.R.
      • Salamero J.
      • Dargemont C.
      Combining spinach-tagged RNA and gene localization to image gene expression in live yeast.
      ). Conversely, cyanine-conjugated RNA aptamers have been used as affinity reagents for live-cell imaging of proteins including epidermal growth factor receptor, human retinoblastoma protein, and transferrin (
      • Yan Q.
      • Cai M.
      • Zhou L.
      • Xu H.
      • Shi Y.
      • Sun J.
      • Jiang J.
      • Gao J.
      • Wang H.
      Using an RNA aptamer probe for super-resolution imaging of native EGFR.
      ,
      • Tan X.
      • Constantin T.P.
      • Sloane K.L.
      • Waggoner A.S.
      • Bruchez M.P.
      • Armitage B.A.
      Fluoromodules consisting of a promiscuous RNA aptamer and red or blue fluorogenic cyanine dyes: Selection, characterization, and bioimaging.
      ,
      • Le T.T.
      • Bruckbauer A.
      • Tahirbegi B.
      • Magness A.J.
      • Ying L.
      • Ellington A.D.
      • Cass A.E.G.
      A highly stable RNA aptamer probe for the retinoblastoma protein in live cells.
      ,
      • Yoon S.
      • Huang K.W.
      • Andrikakou P.
      • Vasconcelos D.
      • Swiderski P.
      • Reebye V.
      • Sodergren M.
      • Habib N.
      • Rossi J.J.
      Targeted delivery of C/EBPα-saRNA by RNA aptamers shows anti-tumor effects in a mouse model of advanced PDAC.
      ).
      In comparison to antibodies, aptamers have improved versatility with flexible modifications, less batch-to-batch variability, less steric hindrance, and are capable of labeling both nucleic acids and proteins (
      • Yan Q.
      • Cai M.
      • Zhou L.
      • Xu H.
      • Shi Y.
      • Sun J.
      • Jiang J.
      • Gao J.
      • Wang H.
      Using an RNA aptamer probe for super-resolution imaging of native EGFR.
      ). However, spinach, plus other RNA aptamers, have had issues with RNA degradation, intracellular folding, and thermal stability. Further aptamers, such as spinach2 and broccoli, have been designed to overcome these complications (
      • Strack R.L.
      • Disney M.D.
      • Jaffrey S.R.
      A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA.
      ,
      • Filonov G.S.
      • Moon J.D.
      • Svensen N.
      • Jaffrey S.R.
      Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.
      ). Additional fluorophores with corresponding fluorogenic aptamers have been designed to cover more of the visible and near-infrared spectra (
      • Song W.
      • Filonov G.S.
      • Kim H.
      • Hirsch M.
      • Li X.
      • Moon J.D.
      • Jaffrey S.R.
      Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex.
      ,
      • Filonov G.S.
      • Song W.
      • Jaffrey S.R.
      Spectral tuning by a single nucleotide controls the fluorescence properties of a fluorogenic aptamer.
      ,
      • Wirth R.
      • Gao P.
      • Nienhaus G.U.
      • Sunbul M.
      • Jäschke A.
      SiRA: A silicon rhodamine-binding aptamer for live-cell super-resolution RNA imaging.
      ). Indeed, near-IR aptamers were the first to be adapted for live-cell super-resolution RNA imaging and have been used to detect subnuclear RNA structures in mammalian cells (
      • Wirth R.
      • Gao P.
      • Nienhaus G.U.
      • Sunbul M.
      • Jäschke A.
      SiRA: A silicon rhodamine-binding aptamer for live-cell super-resolution RNA imaging.
      ,
      • Sunbul M.
      • Lackner J.
      • Martin A.
      • Englert D.
      • Hacene B.
      • Grün F.
      • Nienhaus K.
      • Nienhaus G.U.
      • Jäschke A.
      Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics.
      ). For further reviews of RNA aptamers, see Refs. (
      • Trachman R.J.
      • Truong L.
      • Ferré-D'Amaré A.R.
      Structural principles of fluorescent RNA aptamers.
      ,
      • Swetha P.
      • Fan Z.
      • Wang F.
      • Jiang J.H.
      Genetically encoded light-up RNA aptamers and their applications for imaging and biosensing.
      ,
      • Gao T.
      • Luo Y.
      • Li W.
      • Cao Y.
      • Pei R.
      Progress in the isolation of aptamers to light-up the dyes and the applications.
      ).

      Imaging Flow Cytometry

      Imaging flow cytometry (IFC) could be considered an alternative microscopy-based technique and can achieve up to 20 nm resolution (Fig. 2A) (
      • Headland S.E.
      • Jones H.R.
      • D'Sa A.S.
      • Perretti M.
      • Norling L.V.
      Cutting-edge analysis of extracellular microparticles using imagestreamx imaging flow cytometry.
      ). IFC combines the multiparameter capabilities of flow cytometry and the morphological and subcellular spatial capabilities of microscopy (including dark field, light field, and fluorescence). However, in IFC, there tends to be a trade-off between throughput, sensitivity, and spatial resolution. To compensate for this, a technique to control the flow of cells in the microfluidics system was used to virtually “freeze” cells on the image sensor enabling longer exposure times in image acquisition (
      • Headland S.E.
      • Jones H.R.
      • D'Sa A.S.
      • Perretti M.
      • Norling L.V.
      Cutting-edge analysis of extracellular microparticles using imagestreamx imaging flow cytometry.
      ). This improved signal-to-noise, throughput, sensitivity, and resolution. Whilst IFC cannot perform super high-resolution imaging and capture more intricate subcellular features, its application has been particularly useful for rare cell events and in diagnostic contexts (
      • Doan M.
      • Vorobjev I.
      • Rees P.
      • Filby A.
      • Wolkenhauer O.
      • Goldfeld A.E.
      • Lieberman J.
      • Barteneva N.
      • Carpenter A.E.
      • Hennig H.
      Diagnostic potential of imaging flow cytometry.
      ). For example, it has been used as a diagnostic tool in acute leukemia to assess promyelocytic leukemia protein bodies and the cytoplasmic versus nuclear localization of a characteristic antigen (
      • Grimwade L.F.
      • Fuller K.A.
      • Erber W.N.
      Applications of imaging flow cytometry in the diagnostic assessment of acute leukaemia.
      ). Another major consideration is that the approach requires cells to be in suspension, and dissociation of adherent cells or tissues may cause aberrant localization of molecules. Whilst performed less frequently than protein analysis, RNA transcripts can be visualized using IFC (
      • Pekle E.
      • Smith A.
      • Rosignoli G.
      • Sellick C.
      • Smales C.M.
      • Pearce C.
      Application of imaging flow cytometry for the characterization of intracellular attributes in Chinese hamster ovary cell lines at the single-cell level.
      ,
      • Lalmansingh A.S.
      • Arora K.
      • DeMarco R.A.
      • Hager G.L.
      • Nagaich A.K.
      High-throughput RNA FISH analysis by imaging flow cytometry reveals that pioneer factor Foxa1 reduces transcriptional stochasticity.
      ).
      Figure thumbnail gr2
      Fig. 2Alternative imaging for subcellular proteomics and/or transcriptomics, which couple technologies in MS, microfluidics, and/or microdissection. A, instrumentation coupling flow cytometry and microscopy allows for multiplexing of several protein–RNA targets using fluorescent labels, gaining both spatial and single-cell information. B, microlaser ablation and ionization of molecules, such as peptides, lipids, or metabolites, directly from tissue or cell culture sample enables label-free acquisition of mass spectra across each “pixel” of sample. Very rich datasets but still have poor resolution because of current technical limitations. C, similar to MSI, microlaser ablation allows for acquisition of spectra per “pixel” of a sample. Though, this method has improved subcellular resolution and uses labeling of antibodies conjugated to non-naturally occurring metal isotopes to quantify ∼40 target proteins/RNAs of interest. The metal isotope signals have less signal overlap than fluorescent methods allowing improved multiplexing than traditional antibody probing. MSI, MS imaging.

      Non-microscopy-Based Imaging Methods

      Imaging techniques that do not rely on microscopy are also available to map subcellular localization. These typically consist of hybridizing flow cytometry and/or MS to imaging. Whilst exciting, their use is still limited. Therefore, we only briefly provide an overview but direct to relevant sources of further reading.

      Imaging Mass Cytometry

      Imaging mass cytometry (IMC) uses a similar instrumental setup to mass cytometry, which hybridizes flow cytometry and MS using a cytometry by time of flight (cyTOF). This technology does not suffer from the same degree of signal overlap compared with fluorescent tagging systems (
      • Spitzer M.H.
      • Nolan G.P.
      Mass cytometry: Single cells, many features.
      ,
      • Baharlou H.
      • Canete N.P.
      • Cunningham A.L.
      • Harman A.N.
      • Patrick E.
      Mass cytometry imaging for the study of human diseases—applications and data analysis strategies.
      ). The MS element allows discrimination between targets at an isotopic scale. This is achieved by coupling probes, commonly antibodies, to discrete heavy-metal isotope tags (
      • Spitzer M.H.
      • Nolan G.P.
      Mass cytometry: Single cells, many features.
      ,
      • Giesen C.
      • Wang H.A.
      • Schapiro D.
      • Zivanovic N.
      • Jacobs A.
      • Hattendorf B.
      • Schüffler P.J.
      • Grolimund D.
      • Buhmann J.M.
      • Brandt S.
      • Varga Z.
      • Wild P.J.
      • Günther D.
      • Bodenmiller B.
      Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.
      ). Currently, this tagging system allows around 40 targets of interest to be measured per single cell. In traditional mass cytometry, cells are passed through a microfluidics-style droplet system and through argon plasma at a high temperature when entering the instrument where covalent bonds within molecules are broken, releasing free atomic-level ions. The ions enter a quadrupole where the heavy-metal isotope tags are selected. These tags go on to be separated by mass-to-charge in the cyTOF component of the instrument (
      • Spitzer M.H.
      • Nolan G.P.
      Mass cytometry: Single cells, many features.
      ,
      • Giesen C.
      • Wang H.A.
      • Schapiro D.
      • Zivanovic N.
      • Jacobs A.
      • Hattendorf B.
      • Schüffler P.J.
      • Grolimund D.
      • Buhmann J.M.
      • Brandt S.
      • Varga Z.
      • Wild P.J.
      • Günther D.
      • Bodenmiller B.
      Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.
      ). This is a destructive process, so cells cannot be sorted via this technique, unlike flow cytometry sorting methods (e.g., fluorescence-activated cell sorting), and spatial information is lost. IMC overcame this loss of spatial information by coupling laser ablation of tissue slide or cell culture a pixel at a time into a cyTOF (Fig. 2C). In the first publication of this method, the ability to untangle the heterogeneity of breast cancer samples was demonstrated (
      • Giesen C.
      • Wang H.A.
      • Schapiro D.
      • Zivanovic N.
      • Jacobs A.
      • Hattendorf B.
      • Schüffler P.J.
      • Grolimund D.
      • Buhmann J.M.
      • Brandt S.
      • Varga Z.
      • Wild P.J.
      • Günther D.
      • Bodenmiller B.
      Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.
      ). A similar study recently claimed to achieve subcellular resolution using IMC for 37 proteins in 483 breast cancer tumors to assess the phenogenomic correlation with protein expression (
      • Ali H.R.
      • Jackson H.W.
      • Zanotelli V.R.T.
      • Danenberg E.
      • Fischer J.R.
      • Bardwell H.
      • Provenzano E.
      • CRUK IMAXT Grand Challenge Team
      • Rueda O.M.
      • Chin S.-F.
      • Aparicio S.
      • Caldas C.
      • Bodenmiller B.
      Imaging mass cytometry and multiplatform genomics define the phenogenomic landscape of breast cancer.
      ). Breast cancer samples were also used to simultaneously image 16 proteins and three mRNA targets using a combination of antibodies and oligonucleotide probes, respectively (
      • Schulz D.
      • Zanotelli V.R.T.
      • Fischer J.R.
      • Schapiro D.
      • Engler S.
      • Lun X.K.
      • Jackson H.W.
      • Bodenmiller B.
      Simultaneous multiplexed imaging of mRNA and proteins with subcellular resolution in breast cancer tissue samples by mass cytometry.
      ). A variant of IMC was developed, which employed an ion beam to liberate metal ion reporters, known as multiplexed ion beam imaging, which increased speed, sensitivity, and resolution, and has been reported to give “super-resolution” images of 5 to 30 nm (
      • Angelo M.
      • Bendall S.C.
      • Finck R.
      • Hale M.B.
      • Hitzman C.
      • Borowsky A.D.
      • Levenson R.M.
      • Lowe J.B.
      • Liu S.D.
      • Zhao S.
      • Natkunam Y.
      • Nolan G.P.
      Multiplexed ion beam imaging of human breast tumors.
      ,
      • Keren L.
      • Bosse M.
      • Marquez D.
      • Angoshtari R.
      • Jain S.
      • Varma S.
      • Yang S.R.
      • Kurian A.
      • Van Valen D.
      • West R.
      • Bendall S.C.
      • Angelo M.
      A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging.
      ). Currently, IMC-like strategies have been used successfully for cellular phenotyping of lesions in multiple sclerosis and lymphoid organs, primarily at a tissue level rather than subcellular level (
      • Park C.
      • Ponath G.
      • Levine-Ritterman M.
      • Bull E.
      • Swanson E.C.
      • De Jager P.L.
      • Segal B.M.
      • Pitt D.
      The landscape of myeloid and astrocyte phenotypes in acute multiple sclerosis lesions.
      ,
      • Durand M.
      • Walter T.
      • Pirnay T.
      • Naessens T.
      • Gueguen P.
      • Goudot C.
      • Lameiras S.
      • Chang Q.
      • Talaei N.
      • Ornatsky O.
      • Vassilevskaia T.
      • Baulande S.
      • Amigorena S.
      • Segura E.
      Human lymphoid organ cDC2 and macrophages play complementary roles in T follicular helper responses.
      ). Yet their capabilities for providing such resolution are coming into fruition.

      MS Imaging

      IMC may be confused with MS imaging (MSI), though MSI differs in instrumentation and does not require heavy isotope–derivatized antibody labeling. As with IMC, laser ablation is used to ionize individual “pixels” of a sample, with each pixel having a corresponding label-free spectrum, which allows deeper coverage of molecules than IMC (Fig. 2B). However, the technique suffers from poor sensitivity and resolution (commercial instruments ranging from 5 to 20 μm), so is predominantly only useful for macroscopic imaging where subcellular resolution is not in the scope of the experiment (
      • Buchberger A.R.
      • DeLaney K.
      • Johnson J.
      • Li L.
      Mass spectrometry imaging: A review of emerging advancements and future insights.
      ,
      • Niehaus M.
      • Soltwisch J.
      • Belov M.E.
      • Dreisewerd K.
      Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution.
      ,
      • Swales J.G.
      • Hamm G.
      • Clench M.R.
      • Goodwin R.J.A.
      Mass spectrometry imaging and its application in pharmaceutical research and development: A concise review.
      ), although hybrid MS setups have allowed this technology to improve its resolution. For example, researchers mixed and matched ion sources, such as atmospheric pressure and laser-induced postionization (MALDI-2) sources, coupled to orbitrap analyzers to achieve 1.4 and <1 μm resolution, respectively (
      • Niehaus M.
      • Soltwisch J.
      • Belov M.E.
      • Dreisewerd K.
      Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution.
      ,
      • Kompauer M.
      • Heiles S.
      • Spengler B.
      Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-μm lateral resolution.
      ). Because of MS vulnerability to contaminants, a lot of sample preparation methods, such as fixatives, are incompatible with this method and often flash-freezing is preferential, but further MS-friendly methods are under investigation (
      • Buchberger A.R.
      • DeLaney K.
      • Johnson J.
      • Li L.
      Mass spectrometry imaging: A review of emerging advancements and future insights.
      ). Currently, MSI still suffers from shortfalls in achieving subcellular resolution; so there is limited discussion in this review, and more comprehensive details of MSI can be found (
      • Buchberger A.R.
      • DeLaney K.
      • Johnson J.
      • Li L.
      Mass spectrometry imaging: A review of emerging advancements and future insights.
      ,
      • Niehaus M.
      • Soltwisch J.
      • Belov M.E.
      • Dreisewerd K.
      Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution.
      ,
      • Swales J.G.
      • Hamm G.
      • Clench M.R.
      • Goodwin R.J.A.
      Mass spectrometry imaging and its application in pharmaceutical research and development: A concise review.
      ). Arguably, MSI has yet to be fully integrated within subcellular -omics workflows because of limited resolution, but advances in the technologies associated with the approach promise to improve the general utility of the MSI. Currently, MSI has been considered for tissue-level intraoperative imaging, particularly on difficult to image and difficult to measure biomarkers in pancreatic adenoma (
      • Huang K.T.
      • Ludy S.
      • Calligaris D.
      • Dunn I.F.
      • Laws E.
      • Santagata S.
      • Agar N.Y.
      Rapid mass spectrometry imaging to assess the biochemical profile of pituitary tissue for potential intraoperative usage.
      ,
      • St John E.R.
      • Rossi M.
      • Pruski P.
      • Darzi A.
      • Takats Z.
      Intraoperative tissue identification by mass spectrometric technologies.
      ). What makes this approach particularly exciting is that it can be applied to any molecules that can be ionized, which include proteins, metabolites, or lipids.

      Sequencing-Based Methods in Spatial Transcriptomics and Proteomics

      In the postgenomic era, advances in MS-based and RNA-Seq-based technology have allowed researchers to simultaneously quantify thousands of proteins and RNA species in whole cells and tissues. Along with the concurrent advancement of computational tools, powerful spatial -omics workflows can analyze the structure and molecular composition of specific or several subcellular compartments in one experiment. The methods in this section provide spatially enriched samples of proteins or RNA on a subcellular level that are measured downstream using MS or RNA-Seq. Generally, these methods eliminate in situ spatial information during sample preparation and capture “bulk” information of all cells within a given sample. Therefore, achieving single-cell information using the following methods is still challenging, particularly in proteomics because of the inability to amplify proteins (
      • Macosko E.Z.
      • Basu A.
      • Satija R.
      • Nemesh J.
      • Shekhar K.
      • Goldman M.
      • Tirosh I.
      • Bialas A.R.
      • Kamitaki N.
      • Martersteck E.M.
      • Trombetta J.J.
      • Weitz D.A.
      • Sanes J.R.
      • Shalek A.K.
      • Regev A.
      • et al.
      Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets.
      ,
      • Rich-Griffin C.
      • Stechemesser A.
      • Finch J.
      • Lucas E.
      • Ott S.
      • Schäfer P.
      Single-cell transcriptomics: A high-resolution avenue for plant functional genomics.
      ,
      • Hwang B.
      • Lee J.H.
      • Bang D.
      Single-cell RNA sequencing technologies and bioinformatics pipelines.
      ,
      • Birnbaum K.D.
      Power in numbers: Single-cell RNA-seq strategies to dissect complex tissues.
      ,
      • Budnik B.
      • Levy E.
      • Harmange G.
      • Slavov N.
      SCoPE-MS: Mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation.
      ,
      • Minakshi P.
      • Kumar R.
      • Ghosh M.
      • Saini H.M.
      • Ranjan K.
      • Brar B.
      • Prasad G.
      Single-cell proteomics: Technology and applications.
      ,
      • Zhu Y.
      • Scheibinger M.
      • Ellwanger D.C.
      • Krey J.F.
      • Choi D.
      • Kelly R.T.
      • Heller S.
      • Barr-Gillespie P.G.
      Single-cell proteomics reveals changes in expression during hair-cell development.
      ). Details on the type of MS and RNA-Seq approaches that can be coupled with the methods in this section are reviewed (
      • Zhang Y.
      • Fonslow B.R.
      • Shan B.
      • Baek M.C.
      • Yates J.R.
      Protein analysis by shotgun/bottom-up proteomics.
      ,
      • Stark R.
      • Grzelak M.
      • Hadfield J.
      RNA sequencing: The teenage years.
      ,
      • Zhang F.
      • Ge W.
      • Ruan G.
      • Cai X.
      • Guo T.
      Data-independent acquisition mass spectrometry-based proteomics and software tools: A glimpse in 2020.
      ).

      Biochemical Separation

      Established techniques that enrich or isolate cellular structures by their physicochemical properties have been in use for decades. Typically, subcellular distribution of molecules was assessed using target-specific enzymatic assays (
      • de Duve C.
      Tissue fraction-past and present.
      ), whereas modern techniques employ robust quantitative sequencing using RNA-Seq and MS (
      • Zhang Y.
      • Fonslow B.R.
      • Shan B.
      • Baek M.C.
      • Yates J.R.
      Protein analysis by shotgun/bottom-up proteomics.
      ,
      • Stark R.
      • Grzelak M.
      • Hadfield J.
      RNA sequencing: The teenage years.
      ,
      • Zhang F.
      • Ge W.
      • Ruan G.
      • Cai X.
      • Guo T.
      Data-independent acquisition mass spectrometry-based proteomics and software tools: A glimpse in 2020.
      ).

      Basic Centrifugation-Based and Detergent-Based Fractionation

      Centrifugation is one of the simplest methods to separate organelles based on their size, density, and shape. Organellar preparations using centrifugation date back to the late 1800s, initially to isolate nuclei (
      • Miescher F.
      ,
      • Behrens M.
      Untersuchungen an isolierten Zell- und Gewebsbestandteilen. I. Mitteilung: Isolierung von Zellkernen des Kalbsherzmuskels.
      ,
      • Bensley R.R.
      • Hoerr N.L.
      Studies on cell structure by the freezing-drying method VI. The preparation and properties of mitochondria.
      ). Today, there are two generalized categories of centrifugal organellar fractionation, sedimentation, and equilibrium density centrifugation. These result in either an enriched pellet at the base of the tube or at the organelle's equivalent density within a sucrose (or equivalent) gradient, respectively. When coupled with current sequencing technologies, these enrichment strategies are powerful for exploring subcellular composition.
      Early spatial proteomics studies focused on purification of a singular organelle of interest, giving insights into the molecular composition of many cellular compartments, such as the nucleolus, nucleus, nuclear pore, and mitochondria, across many cell/tissue types and models (
      • Andersen J.S.
      • Lyon C.E.
      • Fox A.H.
      • Leung A.K.
      • Lam Y.W.
      • Steen H.
      • Mann M.
      • Lamond A.I.
      Directed proteomic analysis of the human nucleolus.
      ,
      • De Castro Moreira Dos Santos A.
      • Eluan Kalume D.
      • Camargo R.
      • Paola Gómez-Mendoza D.
      • Raimundo Correa J.
      • Charneau S.
      • Valle de Sousa M.
      • Dolabela de Lima B.
      • André Ornelas Ricart C.
      Unveiling the Trypanosoma cruzi nuclear proteome.
      ,
      • Cronshaw J.M.
      • Krutchinsky A.N.
      • Zhang W.
      • Chait B.T.
      • Matunis M.J.
      Proteomic analysis of the mammalian nuclear pore complex.
      ,
      • Taylor S.W.
      • Fahy E.
      • Zhang B.
      • Glenn G.M.
      • Warnock D.E.
      • Wiley S.
      • Murphy A.N.
      • Gaucher S.P.
      • Capaldi R.A.
      • Gibson B.W.
      • Ghosh S.S.
      Characterization of the human heart mitochondrial proteome.
      ). However, purifying subcellular compartments is challenging because of cofractionation with other components of the cell, because of organelles having overlapping biochemical and biophysical properties, and their constant interaction with one another. “Subtractive” or “differential” approaches account for this “contamination” or interactions. These methods involve quantitative comparisons of technically equivalent non-enriched fractions against organelle-enriched fractions (Fig. 3A). Proteins only detected or highly enriched in the organelle-enriched fractions are assigned to that organelle of interest. This strategy has provided valuable information on the subcellular proteomes of the human spliceosome (
      • Zhou Z.
      • Licklider L.J.
      • Gygi S.P.
      • Reed R.
      Comprehensive proteomic analysis of the human spliceosome.
      ,
      • Neubauer G.
      • King A.
      • Rappsilber J.
      • Calvio C.
      • Watson M.
      • Ajuh P.
      • Sleeman J.
      • Lamond A.
      • Mann M.
      Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex.
      ), rodent liver nuclear envelope (
      • Schirmer E.C.
      • Florens L.
      • Guan T.
      • Yates J.R.
      • Gerace L.
      Nuclear membrane proteins with potential disease links found by subtractive proteomics.
      ), rat lung endothelial cell PM, and caveolae (
      • Oh P.
      • Li Y.
      • Yu J.
      • Durr E.
      • Krasinska K.M.
      • Carver L.A.
      • Testa J.E.
      • Schnitzer J.E.
      Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy.
      ,
      • Yates J.R.
      • Gilchrist A.
      • Howell K.E.
      • Bergeron J.J.
      Proteomics of organelles and large cellular structures.
      ), plus multiple subcellular niches in S. cerevisiae and other yeasts using diverse enrichment approaches (
      • Wiederhold E.
      • Gandhi T.
      • Permentier H.P.
      • Breitling R.
      • Poolman B.
      • Slotboom D.J.
      The yeast vacuolar membrane proteome.
      ,
      • Valli M.
      • Grillitsch K.
      • Grünwald-Gruber C.
      • Tatto N.E.
      • Hrobath B.
      • Klug L.
      • Ivashov V.
      • Hauzmayer S.
      • Koller M.
      • Tir N.
      • Leisch F.
      • Gasser B.
      • Graf A.B.
      • Altmann F.
      • Daum G.
      • et al.
      A subcellular proteome atlas of the yeast Komagataella phaffii.
      ,
      • Delom F.
      • Szponarski W.
      • Sommerer N.
      • Boyer J.C.
      • Bruneau J.M.
      • Rossignol M.
      • Gibrat R.
      The plasma membrane proteome of Saccharomyces cerevisiae and its response to the antifungal calcofluor.
      ,
      • Boersema P.J.
      • Raijmakers R.
      • Lemeer S.
      • Mohammed S.
      • Heck A.J.
      Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.
      ,
      • Wang X.
      • Li S.
      • Wang H.
      • Shui W.
      • Hu J.
      Quantitative proteomics reveal proteins enriched in tubular endoplasmic reticulum of Saccharomyces cerevisiae.
      ,
      • Sickmann A.
      • Reinders J.
      • Wagner Y.
      • Joppich C.
      • Zahedi R.
      • Meyer H.E.
      • Schönfisch B.
      • Perschil I.
      • Chacinska A.
      • Guiard B.
      • Rehling P.
      • Pfanner N.
      • Meisinger C.
      The proteome of Saccharomyces cerevisiae mitochondria.
      ,
      • Vögtle F.N.
      • Burkhart J.M.
      • Gonczarowska-Jorge H.
      • Kücükköse C.
      • Taskin A.A.
      • Kopczynski D.
      • Ahrends R.
      • Mossmann D.
      • Sickmann A.
      • Zahedi R.P.
      • Meisinger C.
      Landscape of submitochondrial protein distribution.
      ,
      • Morgenstern M.
      • Stiller S.B.
      • Lübbert P.
      • Peikert C.D.
      • Dannenmaier S.
      • Drepper F.
      • Weill U.
      • Höß P.
      • Feuerstein R.
      • Gebert M.
      • Bohnert M.
      • van der Laan M.
      • Schuldiner M.
      • Schütze C.
      • Oeljeklaus S.
      • et al.
      Definition of a high-confidence mitochondrial proteome at quantitative scale.
      ). However, despite accounting for contaminants, it is still difficult to confidently identify organellar proteins, as the composition of any cofractionating organelle will be erroneously assigned to the organelle of interest. In addition, this technique is not always appropriate for multilocalized molecules or dynamic studies (
      • Gatto L.
      • Vizcaíno J.A.
      • Hermjakob H.
      • Huber W.
      • Lilley K.S.
      Organelle proteomics experimental designs and analysis.
      ). Coupling of subtractive proteomics with machine learning has improved classification of organellar proteomes (
      • Ohta S.
      • Bukowski-Wills J.C.
      • Sanchez-Pulido L.
      • Alves Fde L.
      • Wood L.
      • Chen Z.A.
      • Platani M.
      • Fischer L.
      • Hudson D.F.
      • Ponting C.P.
      • Fukagawa T.
      • Earnshaw W.C.
      • Rappsilber J.
      The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics.
      ,
      • Kustatscher G.
      • Hégarat N.
      • Wills K.L.
      • Furlan C.
      • Bukowski-Wills J.C.
      • Hochegger H.
      • Rappsilber J.
      Proteomics of a fuzzy organelle: Interphase chromatin.
      ), which somewhat mitigated this issue by providing more robust statistical comparison between enriched and non-enriched fractions. These strategies have been used to establish biological functions and confident inventories of organellar proteomes, such as the mitochondria, peroxisome, and lysosome (
      • Calvo S.E.
      • Clauser K.R.
      • Mootha V.K.
      MitoCarta2.0: An updated inventory of mammalian mitochondrial proteins.
      ,
      • Güther M.L.
      • Urbaniak M.D.
      • Tavendale A.
      • Prescott A.
      • Ferguson M.A.
      High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics.
      ,
      • Islinger M.
      • Lüers G.H.
      • Li K.W.
      • Loos M.
      • Völkl A.
      Rat liver peroxisomes after fibrate treatment. A survey using quantitative mass spectrometry.
      ,
      • Marelli M.
      • Smith J.J.
      • Jung S.
      • Yi E.
      • Nesvizhskii A.I.
      • Christmas R.H.
      • Saleem R.A.
      • Tam Y.Y.
      • Fagarasanu A.
      • Goodlett D.R.
      • Aebersold R.
      • Rachubinski R.A.
      • Aitchison J.D.
      Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.
      ,
      • Ray G.J.
      • Boydston E.A.
      • Shortt E.
      • Wyant G.A.
      • Lourido S.
      • Chen W.W.
      • Sabatini D.M.
      A PEROXO-tag enables rapid isolation of peroxisomes from human cells.
      ,
      • Schmidtke C.
      • Tiede S.
      • Thelen M.
      • Käkelä R.
      • Jabs S.
      • Makrypidi G.
      • Sylvester M.
      • Schweizer M.
      • Braren I.
      • Brocke-Ahmadinejad N.
      • Cotman S.L.
      • Schulz A.
      • Gieselmann V.
      • Braulke T.
      Lysosomal proteome analysis reveals that CLN3-defective cells have multiple enzyme deficiencies associated with changes in intracellular trafficking.
      ). Such studies can be particularly useful for poorly characterized species, such as eukaryotic parasites, with the intention to aid biological understanding and pharmacological developments. For example, this strategy was used to assess the proteins involved in the mitochondrial “importome” of Trypanosoma brucei by coupling with RNA interference of a key translocase (
      • Peikert C.D.
      • Mani J.
      • Morgenstern M.
      • Käser S.
      • Knapp B.
      • Wenger C.
      • Harsman A.
      • Oeljeklaus S.
      • Schneider A.
      • Warscheid B.
      Charting organellar importomes by quantitative mass spectrometry.
      ).
      Figure thumbnail gr3
      Fig. 3Sequencing-based approaches to subcellular proteomics and transcriptomics. The approaches consist of biochemical organellar separation (A and B) or biotinylation of proximal molecules to a bait protein (C). A, quantifying proteins/RNAs in a targeted organelle-enrichment preparation (via centrifugation or detergents) against crude contaminant samples can infer resident proteins/RNAs of the organelle of interest. Quantification of enriched samples can be performed using MS or RNA-Seq. B, more extensive sequential centrifugation or detergent strategies can determine cell-wide residence of proteins/RNAs. The quantitative profiles of proteins/RNAs across the fractions aid identification of their localization by using organellar markers and machine learning techniques. C, a bait protein of interest (e.g., associated with a particular subcellular localization) is fused to an enzyme that catalyzes the biotinylation of proximal proteins/RNAs in the cell once the substrate (e.g., biotin) is added to the cells in vivo. The biotinylated molecules can be purified and analyzed using either MS or RNA-Seq.
      Organelles can also be enriched using different detergent-containing buffers with increasing solubilization capacity to sequentially extract molecules from distinct parts of the cell (
      • McCarthy F.M.
      • Cooksey A.M.
      • Burgess S.C.
      Sequential detergent extraction prior to mass spectrometry analysis.
      ). For instance, the use of digitonin to permeabilize the PM or NP-40 to release contents of double-membrane organelles. The most popular workflow in proteomics achieves subcellular separation of the cytosol, nucleus, cytoskeleton, and membranous compartments (such as those found in the secretory pathway) (
      • Lee Y.H.
      • Tan H.T.
      • Chung M.C.
      Subcellular fractionation methods and strategies for proteomics.
      ). Modified protocols can further distinguish between DNA-associated and soluble nuclear proteins or insoluble proteins in the cytosolic, nuclear, and membrane-bound components (
      • Stasyk T.
      • Huber L.A.
      Zooming in: Fractionation strategies in proteomics.
      ). This approach was implemented in a phosphoproteomics study to resolve three crude subcellular compartments with a very limited amount of starting material (
      • Masuda T.
      • Sugiyama N.
      • Tomita M.
      • Ohtsuki S.
      • Ishihama Y.
      Mass spectrometry-compatible subcellular fractionation for proteomics.
      ). Notably, detergent enrichment workflows have the advantage of preserving the cytoskeletal network, which is prone to fragmentation in centrifugal fractionation (
      • Lee Y.H.
      • Tan H.T.
      • Chung M.C.
      Subcellular fractionation methods and strategies for proteomics.
      ). Differential detergent extraction is primarily reserved for proteomic studies. However, it has been used for studying polysomal RNA and in a two-step detergent protocol to investigate cotranslational trafficking of mRNA from cytosolic polysomes to ER-bound polysomes (
      • Jagannathan S.
      • Nwosu C.
      • Nicchitta C.V.
      Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation.
      ,
      • Jagannathan S.
      • Reid D.W.
      • Cox A.H.
      • Nicchitta C.V.
      De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum.
      ).
      The development of equivalent biochemical fractionation methods to determine subcellular RNA localization is, in comparison, limited. Several studies use basic cell fractionation via centrifugation and detergent lysis followed by RNA-Seq to infer transcript subcellular enrichment (
      • Bhatt D.M.
      • Pandya-Jones A.
      • Tong A.J.
      • Barozzi I.
      • Lissner M.M.
      • Natoli G.
      • Black D.L.
      • Smale S.T.
      Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.
      ,
      • Werner M.S.
      • Ruthenburg A.J.
      • Werner M.S.
      • Ruthenburg A.J.
      Nuclear fractionation reveals thousands of chromatin-tethered noncoding RNAs adjacent to active genes.
      ,
      • Tilgner H.
      • Knowles D.G.
      • Johnson R.
      • Davis C.A.
      • Chakrabortty S.
      • Djebali S.
      • Curado J.
      • Snyder M.
      • Gingeras T.R.
      • Guigó R.
      Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs.
      ). A sequential detergent strategy was employed to map spatial dynamics of RNA between the cytosol, nucleoplasm, and chromatin in inflammatory-stimulated macrophages by assessing the relative enrichment of transcripts in the different fractions to gain insights into proinflammatory gene regulation (
      • Bhatt D.M.
      • Pandya-Jones A.
      • Tong A.J.
      • Barozzi I.
      • Lissner M.M.
      • Natoli G.
      • Black D.L.
      • Smale S.T.
      Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.