Hyperchromatic cytometry is a novel analytical approach intended to significantly increase the depth of information gained on the basis of individual cells by the intelligent combination of different analytical and preparation steps (Figs. 1 and 2). In this paper we have presented different components of the method that can be combined appropriately to achieve this highly multiplexed cell analysis. The ideal instrument for hyperchromatic cytometry is the fluorescent microscope with an imaging system that enables for SBC analysis and repositioning capabilities. Identical cells can be reanalyzed for getting more (virtual) colors, and on the other hand an in-depth view of the detailed morphology of every individual cell can be obtained.
FCM can distinguish more fluorochromes in a single analysis than any SBC instrument. As published by Perfetto et al. in 2004 (12), it allows nowadays to differentiate up to 17 colors and two light scatter signals per measurement. This result is achieved by the use of three lasers (an additional UV laser is applied) for excitation. SBC seemed still far away from these possibilities: the highest number of fluorochromes that can be differentiated with LSC was recently increased to eight (14) in an SBC instrument and also to 8 with a confocal microscope combined with spectral deconvolution (35, 65). Up to now this was the highest number of fluorochromes that could be used for immunophenotyping in a quantitative fluorescence microscopic assay.
By hyperchromatic cytomery, however, virtually anything in one cell becomes measurable.
Iterative restaining is an elegant way to measure subsequently a high number of antigens in the identical specimen. By using just a few fluorochromes at each step and intermittent bleaching steps to get rid of the undesired fluorescence from the previous staining step fluorescence spill-over can be limited to a minimum. This approach was led to the extreme by Schubert, using a single fluorochrome (FITC) (47) or the combination of two chromosomes (FITC and PE) at each step (48) for measuring up to 20 antigens in the identical cell. The author discussed that measuring subsequently up to 100 antigens for Toponomics is feasible. One has here to take into account that after each staining step the detected antigen expression level may appear lower due to altered antigenecity induced by photodestruction, reduced stainability, or other reasons. Concurrently, the authors stressed that controls have to be carefully performed using the same antibody at various steps of iterative restaining in order to ascertain that the antigen is stained similarly independent if it is used in the first or in the last run. We demonstrated for human peripheral blood leukocytes (19) that for most antigens tested fluorescence intensity is clearly reduced in subsequent staining steps. Still, in the case of highly expressed antigens, brightness was sufficient for phenotyping.
Iterative restaining can easily be combined with polychromatic cytometry, i.e. at each step six or more fluorochome labeled detector molecules are applied simultaneously. In order to keep the advantage of low spill-over, dyes that are excited by different lasers should be combined (e.g. blue excitation (FITC, PE, PE-Cy7), green excitation (Texas Red), red excitation (APC, APC-Cy7)). By this improvement, the time needed to analyze the same number of antigens could be reduced by an order of magnitude.
Best suited for iterative restaining are specimens that are sticking strongly to the microscope slide and will not be washed away during restaining (i.e. sections, adherent cells, tissue cultures). Non-adherent cells such as peripheral blood leukocytes as we have used in our experiments are less well suited. These cells have to be immobilized either by centrifugation or by embedding into highly viscous mounting media. Both processes may affect the availability of their antigens for staining.
Differential photobleaching, photoactivation, photodestruction, and photoconversion
The beauty of all methods where fluorescences are modified by light is that no mechanical manipulation is included. This method is not only technically simpler to perform but also non-adherent cells can be easily analyzed hyperchromatically by combinations of polychromatic staining and methods manipulating fluorescences with light.
Differential photobleaching has been first applied in microscopic imaging to combine analysis of two dyes with similar emission spectra (FITC with ALEXA488) (50). We were the first to demonstrate that this approach is also feasible for SBC (51). Differential photobleaching is very conveniently combined with polychromatic cytometry as ALEXA or Cy analogues, with higher photosensitivity existing to virtually all standard organic fluorochromes. Photobleaching can also be easily combined with photoactivation and photoconversion, as in the latter, fluorescence does either not change or increase.
Photoacivation of QDs has several important aspects for hyperchromatic cytometry as detailed in the methodology. Background fluorescence is virtually absent because high Stoke's shift and QDs are virtually unbleachable. The tendency to blink in an irregular fashion at least of some types of QDs (13) may hamper quantitation of fluorescence intensity and requires careful testing. Another aspect of QDs is probably more critical for hyperchromatic analysis. We have observed that excitation of QDs for a long period of time not only increases their brightness but also induces a shift of the emission to shorter wavelengths. The exact reason for this behavior is unclear and may be due to destruction of the outer shell of the particle. Again, careful testing is required when setting up an experimental protocol.
Photodestruction is useful for distinguishing signals of FRET-dyes from those of other conventional fluorochromes. Although the latter will get dimmer, for FRET dyes the fluorescence intensity ratio of donor to acceptor will increase. Thereby a signal of a PE labeled antibody can be distinguished from the PE signal of a PE-Cy5, PE-Cy5.5, or PE-Cy7 labeled antibody. This signal can be subtracted from the original (unbleached) PE signal to yield the PE-only fluorescence intensity.
Photoconversion like photoactivation have both the enchanting properties that signals are virtually invisible before conversion or activation takes place. Because after photocenversion conventional organic dyes fluoresce the problems discussed for QDs will not apply. A photoconversion step can be applied after the original fluorescence from the organic dyes was bleached (and documented) so that the photoconverted signal appears in a background free environment.
By spectral fingerprinting one gets rid of the uncertainty as to what extent a given dye spills over into the emission signal from another dye. This approach was successfully applied by Ecker and Steiner (65) who were able to distinguish up to eight different colors in tissue sections. However, there is also an advantage in using different PMTs for the different colors. In many spectral imaging microscopes the fluorescence spectrum is detected by an array of PMTs that are set to the same sensitivity. If now a bright signal from one cell constituent is measured in parallel with a dim signal from another, the first may be out of range if we want to detect the second or vice versa. With multiple separate PMTs, the sensitivity of each of them can be set to fit the brightness of the signal in the cell.
Calibration and standardization are key issues and special care needs to be taken for them in hyperchromatic cytomery. By principle, all (optical) manipulations that affect the fluorescence emission are contradictory to cytometric analysis because the measured fluorescence intensities will be modified and will not strictly represent antigen expression levels. When samples are iteratively restained, those antigens that are stained in later steps may artificially present reduced fluorescence intensity (19). This is either due to loss of antigen availability, photodamaging of the antigen, or other reasons. Therefore, we recommend using the fluorescence intensity signals before further manipulation to quantify expression levels. The signals detected after manipulation serve as phenotypic markers in order to pin-point to which sub-type the cells belong.
With some additional effort, however, these signals may be standardized to yield cytometric data. To this end, one can apply control cells of known antigen expression that are treated identically to the unknown specimen. For example, samples from healthy individuals can be used for the calibration of antigen expression levels on human leukocytes (68). Alternatively, all antigen expressions are tested in a permutating order within the flow of the hyperchromatic staining and changes are precisely recorded (i.e. without manipulation vs. after manipulation). These data serve for each antigen as a calibration data base that then can be used to recalibrate fluorescence intensities. Essential is that all preparation and manipulation steps are strictly standardized and reproducible. This demand could be fulfilled by automating the whole process of hyperchromatic preparation steps.
One can expect that there will be a natural upper limit of the number of parameters that can be measured in a cell. This is not so much due to the technical limitations of the instruments but to sterical hindrance of the reporter molecules that are placed into the cell. Using non-saturating concentrations of antibodies or other markers can help to avoid this problem (48), but it has to be taken into account that fluorescence intensities may be affected. It would be useful for iterative restaining to remove all previous markers from the cell before the staining step. But this will need the use of proteases or detergents with the unpredictable side-effect of destructing antigens of interest.
Another possible problem that could occur in hyperchromatic cytometry and may need careful analysis is the phenomenon of unwanted FRET in the case that two different molecules are located very close to each other. If the expression level of each of the molecules should be recorded this is clearly not desired. Therefore, careful experiments have to be done to rule out fluorescence quenching (or enhancement) by FRET.
Repositioning of the objects after manipulation with a precision of ±1 μm is sufficient if measurements are performed in a low resolution as is the case with the LSC. Here, the major issue is that cells from two measurements are identified as being identical based on their position in an area of about 5 μm. If, however, a high structural resolution is required as in the case of toponomics or location proteomics, a highly precise pixel based recognition of identical positions from different images of the same object is crucial. Because of imprecision of motorized microscope stages errors in repositioning will naturally accumulate the more often we reanalyze the sample. This will even become worse if we have to remove the specimen from the stage for staining and then replace it again. Obviously, intelligent software solutions are necessary to unequivocally combine the different images acquired by hyperchromatic cytometry.
The advantages of SBC systems for high-content cytomics analysis by hyperchromatic cytometry are evident. The cells of interest remain immobilized on the slide. This on the one hand is pivotal to make hyperchomatic detection possible at all. On the other hand, relocation is important for the visual inspection of cells of interest and their morphological documentation. This forms an input for the automated high-resolution structural analysis of the specimen. Structural analysis requires high spatial resolution but only to a lesser extent unperturbed fluorescence intensity. Therefore, cytometric data should be obtained first, followed by structural analysis. The combination of both cytometric and quantitative structural data tremendously boosts the information gained per cell.
To perform hyperchromatic cytometry as a routine technique in future, new instruments and new computational solutions are required. Instruments have to be developed that enable automatize staining, analysis, and photo-manipulation. Presently, several slide based instruments are under development that can at least partially fulfill this demand [e.g. 34]. New fluorochromes that have very specific features (e.g. photoconversion at very specific wavelengths) need to be developed to more specifically modify fluorescences. New detection molecules smaller in size such as RNA aptamers (69) are essential to reduce sterical hindrance. Finally, solutions for combining, analyzing, and documenting the tremendous amount of data collected by hyperchromatic analysis have to be found [examples approaching this point in Refs.48, 62, 70].
The possibility to analyze genomic and proteomic properties of whole cell populations on the single cell level in their natural environment is important to unravel the complexity of cells and cell systems. It opens the way to better understand complex processes in health and disease and could be an important tool for predictive and preventative medicine. Cytomics and systems biology can show information of the present status and diagnosis, and consequently allow an individualized therapy as a general practice of medicine (2, 4, 7, 31, 32, 71). Furthermore, with hyperchromatic cytometry novel cell types might be recognized not detectable with presently available methods. With the proposed methods in theory, the amount of detectable cell characteristics is only limited by sterical hindrance. Hyperchromatic cytometry is, therefore, conceived as a joint cross-disciplinary effort for cytomics, systems biology, and high-throughput-oriented research for basic, clinical, and industry scientists [1–4, 7, 8, 26, 30–32].