Laser Scanning Cytometry: Principles and Applications—An Update

Abstract

Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.

Microtubule and cortical forces determine platelet size during vascular platelet production

“…Laser scanning cytometry was performed on the iCys Research Imaging Cytometer (CompuCyte, Westwood, Massachusetts, USA); a microscope-based cytofluorometer that employs wide-field laser-spot scanning illumination, and was used to detect β1-tubulin localization in our samples at 488 nm (ref. 26). Briefly, coverslides were scanned at a ×20 magnification (low-resolution mosaic scan) to generate DIC and fluorescence image maps of the sample slides.…”

Prognostic significance of S-phase fractions in peritumoral invading zone analyzed by laser scanning cytometry in patients with high-grade glioma: A preliminary study

“…LSC was developed to utilize the sensitivity of fluorescence-based assays and the specificity of on-slide measurements (16)(17)(18). LSC involves a microscope-based cytofluorometer that is able to assess flow and image cytometry.…”

“…Alternatively, the entire cell population on the slide may be measured automatically using small samples, including biopsy specimens. Although flow cytometric analysis requires a large number of cells, LSC may be performed with a relatively small number (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

HIPK1 drives p53 activation to limit colorectal cancer cell growth

“…1A-C). Image acquisition and quantitative analyses were performed by laser scanning cytometry using the Icys System, 15,16 as formerly done for HIPK2.…”