Maximal pixel of fluorescence intensity (MP) is a useful reporter of local hypo- or hyperchromicity, reflecting the degree of concentration (density) of the fluorescent probe either in intracellular compartments or on cell surfaces (e.g., receptor clustering). One of the early applications of LSC along this line was to identify cells with condensed chromatin. Specifically, because DNA in condensed chromatin, such as of mitotic or apoptotic cells, shows increased staining intensity (per unit area of chromatin image, because the same amount of DNA is compacted at higher spatial density), the MP of these cells stained with a DNA fluorochrome is higher than that of cells with the same DNA content but with diffuse chromatin (17, 18). Although mitotic cells can be recognized by cytometry using a variety of markers (reviewed in ref. (19)), an advantage of the MP marker is the use of a single fluorochrome to distinguish G₁ vs. S vs. G₂ vs. M phase cells. This enables one to apply another color fluorochrome(s) to detect other cell constituents. Such an approach has been used to combine pulse labeling of DNA replicating cells with bromodeoxyuridine (BrdU) (detected with anti-BrdU Ab) with identification of mitotic cells to study cell cycle kinetics by the “fraction of labeled mitoses” (FLM) method (20). The FLM assay, designed initially for tritiated thymidine autoradiography, yields a wealth of information on cell cycle kinetics, but in its original version (21) is cumbersome and time consuming. Its adaptation to LSC simplifies the procedure and shortens the time of analysis (20). Similar to mitotic-, BrdU-labeled meiotic-chromosomes were identified by LSC in studies of mutagenesis (22).

Apoptotic cells having condensed chromatin also can be identified by MP of DNA-associated fluorescence (14, 23, 24). It should be cautioned, however, that because mitotic and apoptotic cells both are characterized by high value of MPFI, the distinction of apoptotic—from mitotic—cells is not always possible. This limitation is of particular importance when apoptosis is induced by agents that arrest cells in mitosis, and therefore the sample contains mitotic cells that die by apoptosis (“mitotic catastrophe”). MP has also been found useful to detect localized caspase activity in early apoptotic cells by the analysis of local intracellular accumulation of the caspase-cleavage product of the fluorogenic substrate (25). Likewise, translocation of Bax to, and its accumulation in mitochondria, the event that facilitates the release of cytochrome c and activation of caspases was detected by LSC as an increase in MP of Bax immunofluorescence (26). The MP of DNA-associated fluorescence combined with fluorescence area, the parameter that reflects nuclear size and correlates inversely with chromatin condensation, was used to distinguish lymphocytes from monocytes and from granulocytes, the cell types that differ by the degree of chromatin condensation (27).

The utility of MP of the DNA-bound fluorochrome as measured by LSC was recently underscored in an analysis of cell senescence (28). The attribute of senescent cells is their morphology: enlargement and characteristic “flattening” of the nucleus (Fig. 3). When analyzed by LSC, this was reflected by an increase in nuclear area and a decline in intensity of MP, most pronounced as the decrease of the ratio of MP/nuclear area, providing a very sensitive biomarker of the degree of cell senescence (28). Still another utility of MP analysis by LSC was recently demonstrated by detection of the very early step of DNA damage response (DDR), namely recruitment of the MRN complex of proteins (Mre11, Rad50, Nbs1) to the DNA damage site (13). This event, which is essential for activation of Ataxia Telangiectasia mutated protein kinase (ATM) and initiation of the subsequent steps of DDR, was revealed as the increase in intensity of MP of Mre11 immunofluorescence measured over nuclei of cells subjected to oxidative stress (13, 29).

Fluorochrome-stained DNA provides a good marker defining nuclear boundary. If another color fluorochrome is used to mark other cell constituents, LSC is then able to resolve and separately measure nuclear and cytoplasmic content of such constituents (Fig. 2; peripheral torus). This capability can be used to detect translocation of particular proteins from cytoplasm to nucleus, or vice versa, e.g., to monitor the traffic of signal transduction or activation molecules. A classical example of such a protein is nuclear factor kappa B (NF-κB). This ubiquitous factor is involved in regulation of diverse immune and inflammatory responses and also plays a role in control of cell growth and apoptosis (30). Activation of NF-κB was detected by an increase in its immunofluorescence measured over the nucleus, concomitant with a decrease in fluorescence over the cytoplasm, which was reflected by a large increase in the nuclear to cytoplasmic fluorescence ratio (see ref. (31); Fig. 4). One of the virtues of this assay is that NF-κB activation can be correlated with cell morphology, immunophenotype, or cell cycle position (31). The assay of NF-κB translocation by LSC has been found more sensitive than any of the four alternative methods (32). This application of LSC can be extended to monitor other transcription factors that upon activation undergo translocation to the nucleus, such as tumor suppressor p53 and specific signal transduction or cell cycle regulatory molecules. For example, the upregulation and translocation of p53 from cytoplasm to nucleus in response to DNA damage by topoisomerase I inhibitor camptothecin was detected and measured by LSC (33). The nuclear location of proliferating cell nuclear antigen (PCNA), detected immunocytochemically by LSC, provided a good discrimination of the proliferating potential in histopatological analysis of renal cell carcinoma (34).

It should be mentioned that in the case of asymmetrically shaped cells (e.g., fibroblasts, neurons) or cells with acentric position of the nucleus (e.g., muscle cells), the integration of fluorescence from the entire cytoplasm is problematic. Partial solution to the problem may involve trypsinization followed by cell deposition on slides by cytocentrifugation. The cells that grow asymmetrically on slides become then more spherical, with the nucleus centrally located.

The micronucleus assay is widely used to assess the chromosomal or mitotic spindle damage induced by ionizing radiation or mutagenic agents in vivo or in vitro. Because visual scoring of micronuclei is cumbersome, semi-automatic procedures that rely either on FCM or image analysis were developed. LSC was adapted for the analysis of micronucleation induced by genotoxic agents in vivo in mouse erythrocytes (35), as well as in vitro, in cultured cells (12, 36, 37). The ability of LSC to relocate micronuclei for visual examination was useful in confirming their identification. Multiparameter characterization of micronuclei that took into an account their DNA content and protein/DNA ratio (Fig. 5) made it possible to establish the gating parameters that excluded objects that were not micronuclei (36). The percentage of micronuclei assayed by LSC correlated well with that estimated visually by microscopy in published studies (36, 37). LSC, thus, can be used to obtain an unbiased estimate of the frequency of micronuclei more rapidly than by conventional examination of the preparations by microscopy. Furthermore, unlike FCM, LSC allows one to characterize individual cells with respect to frequency and DNA content of micronuclei residing in these cells, and furthermore can be applied to the cytokinesis-blocked (e.g., by cytocholasin B) micronuclei assay (36, 37). Assessment of DNA damage is another measure of genotocicity or can be used as a marker of cell death (apoptosis). LSC has found an application for this purpose as well, specifically, for measuring the extent of DNA degradation and electrophoretic mobility from individual cells in the “comet” (38) or “halo” (39) assays.

During the past 5 years, LSC has been extensively used in studies of DDR (12, 40–45). In these studies, the DNA damage signaling was detected immunocytochemically using phospho-specific Abs reactive with activated members of the signaling pathways: histone H2AX phosphorylated on Ser139 (γH2AX), ATM phosphorylated on Ser1981, p53 phosphorylated on Ser15, and Chk2 phosphorylated on Thr68. The advantage of LSC in these studies stems from the possibility of cell imaging, which allows one to identify cells having punctate (foci) distribution of γH2AX, considered to be a marker of double-strand DNA breaks (46). The multiparameter analysis of DNA damage signaling of cells treated with a variety of genotoxic agents correlated with identification of the cell cycle phase and active DNA replication detected by the “click chemistry” provided a wealth of information on mechanisms of DDR involving activation of cell cycle checkpoints, induction of apoptosis, and recruitment of the repair machinery (12, 40–45).

LSC has been adapted to perform routine immunophenotyping. Multi-chamber microscope slides were developed which can be used to automatically screen cells against up to 36 antibodies on a single slide by LSC (57–62). The chambers are filled with cell suspension by capillary action. In the absence of serum or other proteins in the suspension, the cells strongly attach to the floor of the chambers by electrostatic interactions (57–60). Various antibody combinations are then introduced into the chambers, the cells are incubated in their presence for 30–60 min, and following the rinse, their fluorescence is measured. The rate of analysis is relatively fast, as it takes ∼20 min to screen the cells distributed in 12 wells labeled with a panel of 36 antibodies (three antibodies at a time), measuring 3,000–5,000 cells/well (57–60).

Although the rate of measurement by LSC is slower than FCM, and the lack of side (90° angle) light scatter analysis precludes discrimination of lymphocytes from monocytes and granulocytes, certain advantages of LSC may outweigh these deficiencies. Thus, LSC is preferred for hypocellular samples which cannot tolerate repeated centrifugations that lead to cell loss. It should be stressed that loss of cells during centrifugations, as required for FCM analysis, is not random but preferential to different cell types (27). LSC analysis is also economical, since small sample size reduces the cost of reagents (Abs) by over 80% compared to FCM (57–60). Furthermore, the LSC provides the possibility to relocate immunophenotyped cells for additional analysis or archival preservation.