To increase the levels of detectable complexity and interrelationship of single cells in mixed cell populations from body fluids or other biological sources, several cutting edge technical and analytical approaches are scrutinized. Using fluorescence labeling of target molecules, one way is to increase on the instrumentation side, the number of detectors for simultaneously quantifying a multitude of fluorochromes (1). However, this approach seems prone to error at its upper limits. Mass cytometry is an innovative way of getting out of this trap as was shown recently by the Nolan group from Stanford, CA (2). Alternatively, several cutting edge laboratories replaced individual photomultipliers (PMTs) that detect a single emission range by a PMT array enabling the detection of the whole emission spectrum of fluorochromes that were then mathematically distinguished using sophisticated deconvolution algorithms (3). The recent advances made by Nolan and collaborators from San Diego, CA (this issue, page 253) explore the potential of visible and near infrared excitation and detection of whole spectra with a spectral resolution of around 11 nm and 3 nm in comparison to standard instrument configuration with discrete detection bands. Using specially developed mathematical approaches for spectral unmixing, results from the different instrument modalities could be compared. The authors demonstrate that in the visible range results are obtained by spectral measurements that are comparable to the traditional (one PMT for one dye) set-up. Advantages of spectral flow cytometry are simplified optical paths and thereby reduced light loss of the emitted light and possibly better separation of different colors by their emission spectra. The latter could allow measurement of more fluorochromes in combination in that emission range. In the near infrared setup, additional fluorochrome combination could be measured than hitherto possible. Furthermore, the traditional calibration beads may be used for instrument setup and quality control.
In fact, synthetic microparticles in the size of a cell or slightly below are for many years common standards for instrument control as well as the documentation of instrument performance with regard to fluorescence sensitivity and resolution for long-term follow-up experiments (4). If measurements of particles in the dimension of sub-eukaryotic cells' size and also substantially lower fluorescence intensity such as bacteria, platelets, or microvesicles, are performed, calibration of small nanosized synthetic particles is needed. These particles would be in the same size range as the analyzed biological objects, thus in the sub μm-diameter range. Unfortunately, the usual triggering that is based on forward angle light scatter (FSC) is useless in standard flow cytometry instruments when particle size is below that of the excitation wavelength. Therefore, the study of Nolan and Stoner from San Diego, CA (this issue, page 301) is important for all doing flow on nanosized particles who show, in brief, that triggering on FSC is worthless for fluorescent calibration particles as the frequency of doublets is over-estimated and fluorescence intensities are over-estimated.
Using infrared as an excitation source for fluorochromes is one important application. The second is to exploit intrinsic fluorescence of biological specimens for a label-free identification of biological processes. Infrared spectroscopic imaging of biological tissues has been around for a number of years and recent advances make it realistic to expect its maturation as a future tool for clinical diagnostics of altered and malignant tissues. One part of implementation into clinical routine is simplification and standardization. To this end, Nallala and colleagues from Reims, France (this issue, page 294) developed a concept for the identification of colon tumors based on infrared spectroscopy of unstained tissue sections and spectral barcoding of statistically different regions of the emission spectrum enabling tumor fingerprinting. When tested and validated by large randomized clinical studies such approaches may in the future, speed up the work of pathologists and render objective and reproducible results. The authors speculate that by the combination with Raman spectroscopy, the precision of identification and prediction may further be facilitated.
Multicolor measurements, that may be done by image or flow cytometry, are based on optimized protocols and clearly pivotal for precision of biological and basic clinical research as well as present and future diagnostics and predictive medicine (5). For example, Goldeck and coworkers from Tübingen, Germany and Singapore (this issue, page 265) realized that polychromatic phospho-flow may suffer from the drawback that not all surface antigens (or the respective antibodies and dyes for their labeling) survive the stress procedures needed for intracellular staining of the signaling molecules. To overcome this obstacle, they optimized the protocol enabling up to 11-color flow cytometry by using several different surface and intracellular markers simultaneously with a substantially increased degree of freedom of antibody combinations and variations.
Steric hindrance of different ligands that are in close proximity to each other on or in the cell is also a potential impairment of results in polychromatic setups. A useful example is the work by Jalbert and colleagues from Honolulu, HI (this issue, page 280). They observed that staining of different chemokine receptors on the surface of peripheral blood macrophages renders different MFI values if the full (11-color) panel is applied or if FMO controls or single antibody stained samples were measured for CCR2 and CXCR1. By sequential staining with different antibody combination, they could identify that the CCR5 antibody was hindering the binding to the aforementioned receptors. This study has two important conclusions. On the one hand, it is essential when setting up polychromatic panels to test results with single antibody stained samples, oligochromatic panels, and FMOs. This may lead not only to optimized protocols but also to basic knowledge about steric relationships of molecules. On the other hand, sequential staining is another tool to optimize protocols for ligands that are in close proximity to each other.
It is common knowledge, that the more complex polychromatic experiments get, the more complex the emission interactions of the dyes involved are. The issue of compensation is an issue since multicolor experiments started, way back in the 1980s. Now, with 10- to 11-colors being standard, the demand for simplification and automation of data evaluation becomes increasingly pressing for research and industry (6). Therefore, the approach of Nguyen and colleagues from Bethesda, MD (this issue, page 306) to simplify spreading of spectral overlap is highly valuable. This they do by their developed spillover spreading matrix that is determined from the original measurements and is visualized in a grey shade coded matrix. This is a useful new way to visualize instrument performance on a single glance.