Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening *S

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of α-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant α-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Fö rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new allsolid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of ␣-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant ␣-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.

Molecular & Cellular Proteomics 6:1446 -1454, 2007.
Above and beyond the isolation and identification of proteins, the field of proteomics faces the challenges of detecting protein cellular localization and quantifying molecular states such as protein conformations, protein-protein interactions, and post-translational modifications. In the past decade, Fö rster resonance energy transfer (FRET) 1 and fluorescence lifetime imaging microscopy (FLIM) have proven to be instrumental for the quantitative imaging of these biochemical states in single cells (1). Similarly the analysis of different cellular populations (cellomes) will also benefit from these imaging methods. Quantitative multiparametric microscopy is a very young field in which advances in liquid/sample handling robotics and information technology are gradually being integrated into automated microscopes (2,3). These automated imaging systems merge the high content image information with the high throughput volumes provided by their automation and unsupervised operation.
Screening techniques have now reached (ultra)high throughput levels, i.e. they are capable of performing more than 10 5 assays/day in microliter volumes. Such a high throughput is necessary for applications where (bio)chemical libraries are tested, e.g. for drug discovery and interactomics research (4). Although the advance in throughput scale is necessary, it is often accompanied by low information content. Evidently multiparametric detection at high numbers would present a powerful tool. Moreover the screening reproducibility and estimators need to respect comparatively high quality standards, e.g. coefficient of variations (CVs) and zscores should not exceed 5% and should be higher than 0.5, respectively.
High content applications typically involve the quantitative and multiparametric analysis of the effect of analytes or other perturbing conditions on cellular behavior (5). The understanding of molecular mechanisms underlying disease, for instance, requires high resolution information because the screens target the cellular and/or subcellular level. Such applications aim at the monitoring of molecular pathways: the localization and interactions of biomolecules and their altered behavior in response to drugs or pathogens. An automated fluorescence lifetime imaging microscope capable of unsupervised operation was developed to provide the basis for a scalable screening platform that combines high throughput levels and high content information gained from quantitative multiparametric imaging.
Fluorescent protein engineering offers a wide variety of genetically expressible fluorescent biosensors, e.g. for the detection of ion concentration, pH, molecular oxygen, proteolytic and chaperone activity, and ubiquitination, many of which can be quantitatively detected by fluorescence lifetime sensing (6). Their exquisite selectivity is derived from the fact that these biosensors can be targeted to specific proteins of interest, organelles, and other subcompartments of the cell. In addition, a wide variety of site-specific orthogonal protein labeling strategies using synthetic dyes is available nowadays, e.g. FlAsH (fluorescein arsenical hairpin binder), ReAsH (resorufin arsenical hairpin binder), SnapTag, HaloTag (Promega), and CoA binding (6). The availability of commercial systems for automated fluorescence imaging is constantly growing (3,8,9). Moreover recent works demonstrate the usefulness of time-resolved fluorescence assays in screening (10,11).
In this work, an automated FLIM that is based on state-ofthe-art technology, i.e. intensified charge-coupled devices (ICCDs), is described. We recently introduced new all-solidstate technologies (12,13) that will enable the construction of cost-effective and turnkey systems that do not require specialized knowledge for their maintenance and operation.
In light of the presented results and novel technologies, we envisage comparatively inexpensive and simple high throughput and high content quantitative screening platforms to become available in the near future. These systems would provide a substantial impulse to the recent and actively expanding fields of drug discovery, interactomics, cellomics, and proteomics.

EXPERIMENTAL PROCEDURES
Microscopy-The automated microscope used in this work is based on a frequency-domain FLIM setup that is described elsewhere (14) in more detail (see also Supplemental Figs. 1 and 2). The core of the system consists of a motorized Axiovert200M (Carl Zeiss Jena GmbH, Jena, Germany) and an ICCD (PicoStar by LaVision GmbH, Gö ttingen, Germany). Additionally a high resolution CCD camera (Imager Compact by LaVision GmbH) and a SwissRanger-2 time-offlight imager (Centre Suisse d'Electronique et de Microtechnique SA, Zü rich, Switzerland) can be mounted on the binocular phototube output port. The samples were scanned by translating the computerassisted microscope stage (LSTEP by Mä rzhä user GmbH and Co. KG, Wetzlar-Steindorf, Germany). Focus, optical port selection, shutters, objective revolver, filter turret, and filter wheel are also motorized. The microscope is equipped with HBO (ATTO-Arc 100 by Zeiss) and XBO (HAL 100 by Zeiss) lamps, an argon ion laser (Innova 300C argon laser, Coherent Inc., Santa Clara CA), a solid-state laser (Compass 405 nm by Coherent Inc.), and a light-emitting diode illumination module (NSPB500S, Nichia Corp.). The excitation source can be freely chosen and switched. Specific exciter and emitter filter cubes are used to select different fluorophores in a sample. In the present work, rhodamine 6G, enhanced green fluorescence protein (EGFP), and EYFP were excited by the 488 nm laser line of the argon ion laser. In the case of the screening of REACh ubiquitination of GFP-␣synuclein, GFP was excited with the 458 nm line of the argon ion laser. The filter turret hosts a beam splitter, a low efficiency reflector, and two filter cubes whose emitter, dichroic, and exciter filters were as follows: (i) band pass, 440 -460 nm; long pass, 470 nm, and band pass, 480 -500 nm; (ii) band pass, 490 -510 nm; long pass, 515 nm; and band pass, 520 -550 nm; (iii) band pass, 460 -480 nm; long pass, 493 nm; and band pass, 505-530 nm (AHF Analysentechnik AG, Tü bingen, Germany). These filter cubes were used for the experiments show in Fig. 5, Fig. 2, and Figs. 1, 3, and 4, respectively. All above mentioned features were integrated in a virtual microscope environment that allows the automation of the entire imaging process. This environment was controlled by in-house developed software programmed in the DaVis suite (LaVision GmbH). Schematics are available in the supplemental material.
Screening Protocol-Initially the user defines the type of screening and calibrates the microscope with a fluorescence lifetime standard positioned at the sample plane. At regular intervals, the microscope compares the calibration parameters with the phase and demodulation of the light source by a low efficiency reflector positioned in the filter turret to correct for possible drifts in the relative phase of the system over time. The dynamic calibration offered by this internal reference does not require the sample to be removed or the interaction of the user. The user can define an arbitrary number of virtual acquisition channels. The microscope is not equipped with a single detector with spectral and lifetime capabilities, but acquisition channels are defined by (i) the light source (laser, HBO, XBO, or lightemitting diode module), (ii) the filter set (the turret hosts four different filter cubes and a filter wheel in front of the HBO lamp that is fitted with eight excitation filters), and (iii) the detectors (ICCD for FLIM or a high resolution CCD) and are selected via software. With a profile chosen, the microscope selects and presents a series of fields of view on which the user may manually focus. These focus landmarks are used in the autofocusing routines by interpolation over the sample. Subsequently the system scans the sample and computes the exposure time of the detector to avoid its saturation. For this, two images are acquired at opposite phases (0°and 180°) with a low exposure time (typically 20 ms), and a pixel-by-pixel average intensity and fluorescence lifetime are computed using the rapid lifetime determination algorithm (15). Based on these parameters, the platform decides whether to image the current field of view, i.e. whether a fluorescent object is present, and computes the optimal exposure time. If requested, the microscope can refine the focus position by the use of an iterative "staircase" procedure (16) with a focus score based on sampling at half the Nyquist frequency as described previously (17). As this process is rather time-consuming, it is ideally limited to a small number of fields of view, e.g. those containing objects identified by certain search criteria. This system could be equipped with autofocus hardware (18) for improved acquisition throughput. The microscope then switches to the next virtual channel to acquire the images and stores the data in the memory. The images of each field of view are stored on mass storage devices during the movement of the stage between fields. In the case of time lapse screening, this procedure is repeated after a user-defined time lag to follow a process on a large number of cells over time. The recorded object time and spatial coordinates allow time-dependent measurements to be made for each individual object.
Data Analysis-A second computer analyzes the images that are acquired by the platform. The data are transferred in a local area network and are processed on line. The results are typically available on screen within a few seconds after the acquisition. A stand alone tool for remote monitoring of the screening activities was developed in Microsoft Visual Basic, allowing the fully unsupervised acquisition/ analysis process to be monitored by local area network access or an internet connection (see supplemental material).
This process returns the ensemble of original images, the processed intensity and lifetime maps, a low resolution global map of the sample, and an array of estimators for every imaged object. Every object is flagged with its relative position in space and time. The features of single objects that are analyzed comprise intensity (in different spectral ranges), homogeneity of the intensity (coefficient of variation), fluorescence lifetime and the lifetime moments analysis heterogeneity estimator (14), and morphological estimators, e.g. area, perimeter, elongation, and roundness. Other analyses that can be performed on the data include invariant moment analysis and the analysis of intensity/lifetime in specific cellular compartments that are identified by morphological estimators or fluorescent labeling. Userassisted statistical software provides access to the unsupervised readout. These routines allow object counting of the imaged sample and the extraction of subpopulations from the ensemble by inspection of data clusters in combinatorial bidimensional histograms. Both the unsupervised batch processing and the supervised statistical analysis software were developed in Matlab (Mathwork, Natick, MA). Part of the Matlab code and further information are available upon request.
Sample Preparation-EGFP was purified from a liquid culture of transformed BL21DE3 Escherichia coli bacteria by immobilized metal chromatography using the His 6 tag of the protein. Rhodamine 6G was prepared at a concentration of 200 M in distilled water from a 10 mM methanol stock solution. A 1536-multiwell plate (SensoPlate by Greiner Bio-One GmbH, Frickenhausen, Germany) was loaded using an automated liquid handling station (Freedom EVO by TECAN). Potassium iodine was added at the indicated concentrations (see Fig.  1) to the wells prior to plate loading.
For the imaging of bacterial colonies, BL21DE3 E. coli bacteria were transformed with the pRSET(B)::YFP vector and plated on an agar layer that was cast in a custom-built plate with removable Teflon walls to facilitate their removal before imaging. This allows the entire cultured surface to be exposed to the objective when the plate is mounted on the stage.
CHO cells were transfected with pEYFP and/or pEGFP vectors using the Effectene 2000 lipid formulation according to the protocol provided by the supplier (Qiagen GmbH, Hilden, Germany). Liposomes containing either pEGFP vector, pEYFP vector, or pEGFP and pEYFP vectors were prepared by incubating the respective DNA or a 1:1 mixture of both DNAs with the Effectene reagent. The 4 wells of a Labtek chamber slide with glass bottom were transfected with the two individual DNA-lipid solutions, the mixed DNA-lipid solution, and a mixture of both individual DNA-lipid solutions. Rat striatal CSM14.1 cells were transfected with pcDNA3.1 vectors encoding the genes for wild type ␣-synuclein, the A30P mutant, or the A53T mutant in a 4-well Labtek chamber slide. These constructs were co-transfected with the recently described REACh2-ubiquitin fusion construct (19). The cells were allowed to express the fluorescent proteins for 48 h upon which the cells were fixed in 4% (w/v) formaldehyde in PBS, washed, and mounted in Mowiol.

RESULTS
Unsupervised FLIM for High Throughput-The ultrahigh throughput standard (uHTS), requiring more than 10 5 assays/ day in microliter volume, defines the highest current throughput level of screening platforms. The 1536-multiwell plate is a format that allows these assays to be performed under the given criteria when read in ϳ20 min. Fig. 1 shows the intensity and lifetime maps of a 1536multiwell plate whose wells were loaded with different fluorescent solutions. Pairs of rows, i.e. sets of 96 wells, were loaded with purified EGFP, rhodamine 6G (R6G), and potassium iodine by liquid handling robotics. The EGFP and R6G solutions exhibited fluorescence lifetimes of ϳ2.6 and ϳ4.2 ns, respectively. Under these experimental conditions, EGFP was brighter than R6G (Fig. 1A, first and last rows, respectively). Additionally quenching of R6G with decreasing concentrations of potassium iodine (63, 50, 38, 25, 13, and 0 mM) was used to generate a gradient of intensities and lifetimes. The comparison between Fig. 1, A and B, shows that lifetime detection provides excellent contrast and that the readout is independent of the probe concentration. In fact, differences in brightness due to pipetting errors or illumination inhomogeneity are not present in the lifetime map. Fig. 1C shows the averages and the S.E. of the eight different groups of wells that contained the same solutions. The R6G quenching curve FIG. 1. High throughput screening and reproducibility. Eight groups of 96 wells in a 1536-multiwell plate were loaded with 1) EGFP, 2) a mixture of R6G and EGFP, and 3-8) a gradient of R6G quenched with potassium iodine at 63, 50, 38, 25, 13, and 0 mM concentration, respectively. Two empty wells served as a control for plate autofluorescence. The plate was screened in less than 20 min (5ϫ objective) with high sensitivity and reproducibility, providing maps of the sample for brightness (A) and lifetime (B). Note that the S.E. and coefficients of variation of the fluorescence lifetimes (table in C) are in the range of picoseconds and 1-2%, respectively. a.u., arbitrary units; MAX, maximum. Error bars are standard error of the mean.
is resolved with high accuracy. The coefficients of variation are in the range of only 1-2%, and the S.E. values are in the picosecond range.
The parallel imaging of 4 wells per field of view with a low magnification objective (5ϫ) allowed the complete multiwell plate to be imaged in ϳ20 min (ϳ0.7 s/well). Therefore, these instruments could, in principle, perform at uHTS levels when equipped with sample handling robotics and rapid autofocusing hardware.
Scalability-One of the advantages offered by an automated microscopy platform is its straightforward scalability. Light sources, filters, detectors, and objectives can be selected to match the sample requirements. Fig. 2 shows images of a custom-built 14 ϫ 9-cm bacterial plate. E. coli bacteria transformed with pRSETB-EYFP were plated at the maximal achievable density that is compatible with imaging, ϳ20,000 colonies/plate. The agar plate can be screened in ϳ90 min by imaging ϳ1900 fields of view (corresponding to a total of ϳ7600 exposures) using a 5ϫ objective. The segmentation of single objects (Fig. 2, B, D, and E) allows the retrieval of fluorescence intensity ( Fig. 2A), lifetime (Fig. 2C), and morphological estimators for each bacterial colony.
The images shown in Figs. 1 and 2 were acquired using the rapid lifetime determination algorithm. This algorithm allows higher throughputs than the common multipoint phase acquisition used in frequency-domain FLIM because it requires the acquisition of only two opposite-phase images. Lifetime heterogeneity and photostability can be obtained upon acquisition of more phase-dependent images but at the cost of (approximately half the) acquisition speed (20).
High Content Screening- Fig. 3 shows images of a 4-well chamber microscope slide on which CHO cells were cultured that express EGFP and EYFP. The first two wells were transfected with liposome preparations containing only pEGFP or pEYFP, respectively. The second two wells were co-transfected by mixing pEGFP and pEYFP vectors in the same liposomes (EGFP ϩ EYFP) or by mixing the two separately prepared homogeneous liposome solutions containing pEGFP and pEYFP (EGFP/EYFP). 15,000 cells were imaged in 90 min using a 20ϫ objective. The cells were segmented and analyzed for intensity and lifetimes by the unsupervised software. Here six phase images were acquired for the computation of the lifetime heterogeneity by lifetime moments analysis (14). Therefore, the "EGFP," "EYFP," and EGFP ϩ EYFP samples represent homogeneous cell populations that exhibit single Gaussian distributions (solid lines). The EGFP/EYFP can be fitted by three Gaussian distribution components (black solid line) whose averages and S.D. values were constrained to those retrieved from the homogeneous conditions (see Supplemental Fig. 7 for further information). Therefore, it follows that only about 30% of the cells received the two different liposomes that were present in the preparation. Fig. 3, C and D, summarizes the average lifetime and relative brightness in each well in a representative region of 5 ϫ 25 fields of view. Because of differences in protein expression levels, the brightness (Fig. 3, B and D) does not provide a robust estimator for the comparison of the samples. On the other hand, the fluorescence lifetimes (Fig. 3, A and C) clearly distinguish between the different transfection conditions. The cells expressing EGFP or EYFP alone exhibited a fluorescence lifetime of 2.13 Ϯ 0.10 ns (mean Ϯ S.D., n ϭ 2488) and 2.43 Ϯ 0.11 ns (n ϭ 3472), respectively. The relative brightness of the two samples shows a bimodal distribution dem-FIG. 2. High throughput and system scalability. The relative brightness (A) and fluorescence lifetimes (C) of a large agar surface (9 ϫ 14 cm) plated with ϳ20,000 E. coli bacterial colonies expressing EYFP was imaged in ϳ1 h with ϳ1900 fields of view (5ϫ objective). A higher magnification (B) shows the single colonies. Each field of view (D, E, and squares in B) is segmented (D), and the fluorescence lifetime of single colonies is assigned (E). The spatial coordinates of each segmented colony is stored to allow subsequent higher resolution imaging or sample retrieval. a.u., arbitrary units; MAX, maximum.
onstrating that cells express more EGFP than EYFP under the conditions used.
The lifetime heterogeneity estimator also shows differences between the four samples: 81 Ϯ 12, 92 Ϯ 12, 86 Ϯ 11, and 85 Ϯ 12% for EGFP, EYFP, EGFP ϩ EYFP, and EGFP/EYFP, respectively. Bidimensional histograms of the fluorescence lifetime heterogeneity versus the average fluorescence lifetime of each cell (Fig. 3E) show the correlation between these two estimators. In fact, EYFP has a higher lifetime and a higher heterogeneity than EGFP. Such analysis can be extended to other pairs of estimators for the analysis of subpopulations in a manner comparable to fluorescence-activated cell sorting analysis (see bidimensional histograms in the supplemental material). Fig. 4 shows the individual cells in this field of view that are marked with a circle in Fig. 3C. Each cell was identified by an image processing routine that consists of automatic background subtraction and automatic threshold detection followed by a watershed algorithm and a morphological mask operation. Segmented objects with fluorescence intensities below 5% of the CCD dynamic range were masked out and ignored. Object classification was performed off line by supervised software. Fig. 4A shows the result of this process; segmented cells are color-coded, and rejected objects are presented in a nonlinear gray level map to highlight their low fluorescence levels. Fig. 4, B and C, shows the lifetime map of the successfully segmented cells.
Although the cells that were transfected with EGFP and EYFP alone exhibited average lifetimes that differ only by 300 ps, up to 95% of cells can be correctly classified as either EGFP or EYFP by a linear separation of the two populations. The two co-transfections EGFP ϩ EYFP and EGFP/EYFP exhibited average lifetimes and a distribution broadness that differ by only 30 and 50 ps, respectively. However, the high number of analyzed cells permits the retrieval of the weight of the three underlying distributions.
A High Throughput, High Content Cellular Assay for Ubiquitination by FRET-Ubiquitination of ␣-synuclein in the rat striatal CSM14.1 cell line was investigated using a FRETbased assay (19). A total of 871 cells were imaged with ϳ1140 fields of view using a 40ϫ objective (Fig. 5). GFP fusion proteins of ␣-synuclein and its familial mutations A30P and A53T were co-expressed with ubiquitin fused to a non-fluorescent YFP mutant (REACh2). The transfer of energy from the GFP donor to the REACh acceptor is highly efficient due to their optimized spectral overlap. Ubiquitination is quantified by the occurrence of FRET between the GFP-tagged ␣-synuclein protein and the REACh2-labeled ubiquitin that causes a reduction in the GFP lifetime. Four samples (control, wild type, and A30P and A53T mutants) were imaged in 45 min each, including the focusing procedure. The GFP lifetime in the absence of FRET was determined in a control sample that expresses GFP-␣-synuclein alone and amounted to 2255 Ϯ 9 ps (average Ϯ S.E., n ϭ 191). The stability of the measurement was verified by rescreening of this sample at the end of the measurement in a smaller number of cells. The retrieved lifetime was statistically identical to the control (2248 Ϯ 8 ps, n ϭ 35). The lifetime of wild type GFP-␣synuclein in the presence of ubiquitin-REACh2 was reduced to 1817 Ϯ 13 ps (n ϭ 219) and significantly lower than for the two ␣-synuclein mutants whose lifetimes were not statistically different from each other (A30P: 1928 Ϯ 13 ps, n ϭ 270; A53T: 1914 Ϯ 14 ps, n ϭ 156). FRET efficiencies were computed by the reduction in lifetime relative to the control sample. The distribution of FRET efficiencies computed on a cell-by- cell   FIG. 3. High content screening. A 4-well Labtek chamber was seeded with CHO cells, transfected with EGFP and/or EYFP, and imaged in ϳ90 min with ϳ600 fields of view (20ϫ objective). The wells were transfected with a vector encoding EGFP or EYFP, co-transfected with liposomes containing a mixture of EGFP and EYFP vectors (EGFP ϩ EYFP), and co-transfected with a mixture of EGFP liposomes and EYFP liposomes that were produced separately (EGFP/EYFP). A shows the distributions of lifetimes computed over the single cells (ϳ15,000 in total; circles) and the data fitted with Gaussian functions (solid lines); the EGFP/EYFP sample is best represented by a sum of the other three populations. B shows the distribution of brightness. C and D show the synthetic representation of the average lifetime and brightness, respectively, in each field of view of a 5 ϫ 25 field of view region of interest. The circle in the EGFP well indicates the field of view that is shown in more detail in Fig. 4. E shows the bidimensional distribution of lifetime heterogeneity plotted versus the average lifetime of the four samples. The black arrows indicate the averages of the EGFP and the EYFP samples. a.u., arbitrary units; MIN, minimum; MAX, maximum; ADU, analog-to-digital units.
basis for all ␣-synuclein constructs is shown in Fig. 5A. An increase in FRET efficiencies was observed when ubiquitin-REACh was co-expressed with the respective GFP-labeled ␣-synuclein construct. Higher FRET efficiencies were accom-panied by the broadening of their distribution due to the presence of multiple interaction modes and/or ubiquitination levels. Additionally the average distributions of FRET efficiencies were broader than the distributions of the single cells (Supplemental Fig. 8), indicating the presence of separate ubiquitination levels also between individual cells. Gaussian fitting of the composite FRET efficiency (ubiquitination) distribution showed the presence of three populations for wild type ␣-synuclein (a fraction of 0.33 at 12% FRET, 0.39 at 21%, and 0.28 at 27%). The mutant ␣-synuclein forms lost the highest FRET efficiency population, and the remaining populations were redistributed in favor of the lowest population (A30P: 0.6 at 12% and 0.4 at 16%; A53T: 0.56 at 11% and 0.47 at 19%). In contrast to the GFP-YFP co-mixing experiment shown in Figs. 3 and 4, a reversed correlation of the coefficient of variation (14) with the lifetime exists. This is caused by the increase of heterogeneity with increasing FRET and presents an additional quality criterion for FRET. DISCUSSION FRET operates at intermolecular distances on the scale of protein dimensions (Ͻ10 nm) and exhibits sensitivity to changes in the Ångstrom range. FLIM provides a non-invasive, fast, and quantitative FRET measurement, thus giving access to molecular information like protein-protein interactions and conformational changes. Furthermore lifetime sensing was used for the quantification of oxygen content, ion concentration, and pH and can be used to map biochemical events in living cells (6), proving its value for molecular proteomics studies. The diversity of available synthetic dyes with sensing capabilities for different small molecules and conditions can be exploited by FLIM to create new sensitive and reproducible assays for a variety of cellular functions. This holds particularly true for those dyes that respond with otherwise difficult to calibrate quantum yield changes and that are now avoided in favor of ratiometric dyes.
Such detailed and quantitative information is equally important for the life sciences and the screening industry. It was shown (see Fig. 1) that an automated FLIM, capable of unsupervised operation, provides very high throughput with good reproducibility (CV Ͻ 5%) and sensitivity (high z-score). An assay is considered robust when its statistical z-score exceeds 0.5 (21). With the coefficient of variation in our studies, this stringent statistical requirement can be fulfilled with 20% lifetime difference detected in a single well. High sensitivity and reliability are of crucial importance for the FRET-based detection of protein-protein interactions and protein conformational changes. Furthermore assays can be performed in a variable environment, e.g. in cells and in "homogeneous" assay formats that do not require washing steps, by the virtue of the independence of the fluorescence lifetime from fluorophore concentration. FLIM screening platforms could be used for the validation of protein-protein interaction found by other (u)HTS approaches. One such application example is shown for the screening of ubiquitination levels of ␣-synuclein and its familial mutations that are causative for Parkinson disease. With its high throughput, automated FLIM systems could be directly used for the screening of fluorescently labeled genomics banks or drug libraries.
Our experiments also exemplify that the scalability of an automated microscope allows the analysis of samples that do not respect a standardized format: we showed the unsupervised imaging of microtiter plates (Fig. 1), bacterial plates (Fig.  2), and microscope slides (Figs. [3][4][5]. Other samples like tissue slices, electrophoresis gels, DNA or protein arrays, and nanotiter plates could also be easily accommodated. Fig. 2 shows the screening of bacterial colonies. Besides screening for optimization of fluorescent proteins and fluorescent biosensors by random mutagenesis, fluorescence lifetime-based assays could be performed in bacteria as a biological model system that carries the advantage of the simplicity of sample handling, biochemistry, and retrieval of genetic/proteomic compositions. The microscope stores the relative position of each imaged object. The sample can therefore be revisited iteratively for real time data analysis. In addition to the "inventory" use of the platform in cell screening, the platform can therefore also be used to "hunt" for rare events with the aim of sample retrieval. Single colonies, cells, or cellular subpopulations could be isolated, for instance, by photogelation procedures (22) or laser microdissection and pressure catapulting (23) techniques. The protein or genetic content of the objects with specific lifetime properties can then be analyzed by the relevant techniques.
These two modes of operation are generally known as image cytometry for analysis and sorting (ICAS) (22). ICAS is suitable for adherent cells and tissues where flow cytometric techniques cannot be used. Our work shows that the highly informative and sensitive fluorescence lifetime parameter can be used for the selection of cells for ICAS. Fig. 3 demonstrates the unsupervised cellular imaging and data analysis of extended surfaces. Data acquisition with six phase images was performed here to analyze the lifetime heterogeneity (14) and to compensate for photobleaching (24). In the case of FRET imaging, the quantification of lifetime heterogeneity by lifetime moment analysis can provide a measure of the molecular fraction that undergoes FRET, e.g. the relative concentration of interacting proteins and their average intermolecular distance. When photobleaching and lifetime heterogeneity of the fluorophores can be neglected, the rapid lifetime determination algorithm that requires only two phase-dependent images can be used. Under these conditions, the screening of an entire 4-well Labtek chamber would take a third of the current time, i.e. 30 min. The maximal cell density and transfection efficiency that allow single cells to be distinguished amount to ϳ100,000 cells in this format. Therefore, a maximum of 200,000 cells/h can be screened with a 20ϫ objective. The screening can be repeated over time by imaging extended surfaces or a user-defined group of cells (data not shown). This enables the measurement of temporal responses over a high number of cells.
Figs. 3 and 4 (see also Supplemental Figs. 2-7) exemplify how cellular subpopulations can be analyzed by imaging single cells. The differences between the two co-transfection conditions used would be impossible to resolve when only the averages over these large numbers of cells were considered. The analysis of cell populations is important for the understanding of the regulation and molecular mechanisms of biological events as biological models are usually heterogeneous. The capability of screening and segmenting diverse cellular populations combined with the possibility to detect protein-protein interactions can offer a significant advantage for the fields of cellular proteomics and interactomics. Quantitative multiparametric microscopy and automated unsupervised microscopy are comparatively young techniques that attract a growing number of industrial and academic research groups. This work represents an advance in the combination of these technologies and demonstrates that current technologies can be used for the construction of an unsupervised FLIM system for high throughput and high content screening. Several commercial automated systems could be adapted for lifetime sensing, immediately offering a powerful tool for the screening community. The experiments presented in this work represent well defined benchmarks for the characterization of the quality of the data that are generated and for the application of software solutions for the detailed statistical analyses that can be performed. FRET assays enjoy an increasing popularity in the life sciences and represent the major application of our platform. The feasibility of sensitive FRET assays on our platform is demonstrated by its high quality and sensitivity. A lifetime difference of 300 ps can be clearly separated. Furthermore taking into account the CV, 95% of the cells could be successfully classified. This difference corresponds to a FRET efficiency of ϳ12% with fully separated distributions. In the same experiment, three populations differing by only ϳ6% were still reliably separated. The ubiquitination assay (Fig. 5) shows that differences of ϳ4% can be distinguished (wild type versus A30P mutant) with high statistical significance (p Ͻ 0.01, Student's t test) by the use of the same fluorescence assays used in conventional microscopy.
This remarkable resolution in the biochemical event of protein ubiquitination is only achieved by the automation of the lifetime microscope, combining high throughput with high content information; large cell numbers in the sample were subjected to the uniquely quantitative determination of FRET by lifetime microscopy. The cell-based statistics identify differences in the ubiquitination of disease-related mutant forms of the ␣-synuclein protein. These forms are ubiquitinated less than the wild type ␣-synuclein. All three ␣-synuclein proteins seem to share two basal states of ubiquitination, but the wild type protein possesses an additional high ubiquitination state that drives the average significantly upward. The cellular response to the presence of ␣-synuclein ubiquitination substrates is thus intrinsically heterogeneous, a fact that would be lost if this modification is measured by biochemical means with imaging approaches that do not resolve the individual responses (e.g. plate readers) or by manual acquisition of only a few cells. Because ubiquitination of unwanted proteins, leading up to their proteasomal digestion, is an integral part of the detoxification machinery of the cell, the observed altered behavior of the familial disease-causing ␣-synuclein mutants is important for the understanding of the pathophysiology of Parkinson disease. These novel findings are in agreement with the slower proteolytic degradation of A53T ␣-synuclein observed in pulse-chase biochemical experiments (25). Furthermore the clinical hallmark of this disease is the presence of intracellular inclusion bodies, called Lewy bodies, which contain ubiquitinated ␣-synuclein (26), and ubiquitin-proteasomal dysfunction is generally considered to be an important feature of Parkinson disease (7). However, it is not known in what way these observations are causally connected. The high sensitivity afforded by our screen is crucial because, given the progression of Parkinson disease over decades, even minor impediments of ␣-synuclein degradation could favor the formation of aggregates. The mutations have been suggested to inhibit proteasomal activity (27), but differences in their ubiquitination levels were never reported by conventional biochemical methods. For the first time, it was shown that the ubiquitination level of ␣-synuclein can be quantitatively imaged in cells and that the mutants are significantly less ubiquitinated. As the mutants exhibit an increased tendency toward self-aggregation, giving rise to neurotoxicity, their decreased ubiquitination might indicate that their altered proteolytic processing contributes to aggregation. On the other hand, ubiquitination might represent a mechanism that protects ␣-synuclein from aggregation. These issues warrant further research.