Laser Scanning Cytometry: Principles and Applications—An Update

  • Piotr Pozarowski
  • Elena Holden
  • Zbigniew Darzynkiewicz
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 931)

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.

Key words

Cytometry Fluorescence Cell cycle Apoptosis Nucleus Nucleolus Micronucleus, cytoplasm Enzyme kinetics 

Abbreviations

Ab

Antibody

ATM

Ataxia Telangiectasia mutated protein kinase

BrdU

Bromodeoxyuridine

CCD

Charge-coupling device

CGH

Comparative genomic hybridization

DF

Differential fluorescence

FCM

Flow cytometry

FIA

Fluorescence image analysis

FISH

Fluorescence in situ hybridization

FITC

Fluorescein isothiocyanate

FLICA

Fluorochrome-labeled inhibitors of caspases

FLM

Fraction of labeled mitoses

FNA

Fine needle aspirate

GFP

Green Fluorescent Protein

LSC

Laser scanning cytometer

mAb

Monoclonal antibody

MP

Maximal pixel

NF-κB

Nuclear factor kappa B

PCNA

Proliferating Cell Nuclear Antigen

PCR

Polymerase chain reaction

PI

Propidium iodide

RT-PCR

Reverse transcription-polymerase chain reaction

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Notes

Acknowledgement

Supported by NCI Grant CA 28704 and by Robert A. Welke Foundation for Cancer Research.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Piotr Pozarowski
    • 1
    • 2
  • Elena Holden
    • 3
  • Zbigniew Darzynkiewicz
    • 1
  1. 1.The Brander Cancer Research Institute, New York Medical CollegeValhallaUSA
  2. 2.Department of Clinical ImmunologySchool of MedicineLublinPoland
  3. 3.CompuCyte CorporationWestwoodUSA

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