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Flow Cytometry

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Updated: May 13, 2026

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Published on: May 5, 2017

Advantages of full spectrum flow cytometry.

Claire K Sanders¹, Judith R Mourant

¹Bioscience Division, Los Alamos National Laboratory, P.O. Box 1663, MS M888, Los Alamos, New Mexico 87544, USA. csanders@lanl.gov

Journal of Biomedical Optics

|March 13, 2013

Summary

A novel charge-coupled device (CCD) flow cytometer offers high spectral resolution for analyzing cellular and bead fluorescence. This system accurately quantifies spectral contributions, enabling observation of dynamic processes like fluorophore binding kinetics.

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Area of Science:

Analytical Chemistry
Biophysics
Instrumentation

Background:

Flow cytometry is a powerful technique for cellular analysis.
Accurate spectral resolution is crucial for distinguishing fluorophores.
Existing methods may have limitations in spectral detail and kinetic analysis.

Purpose of the Study:

To implement and characterize a charge-coupled device (CCD)-based flow cytometer for full spectral measurements.
To evaluate the system's performance in spectral resolution and data analysis.
To demonstrate the capability of analyzing complex kinetic processes involving fluorophores.

Main Methods:

Implementation and characterization of a CCD-based flow cytometer.
Spectral resolution measurement using flow check beads.
Analysis of cell and bead data by fitting to component spectra.
Application of Principle Component Analysis (PCA) and Alternating Least Squares (ALS) for data analysis.
Kinetic studies using ethidium bromide and propidium iodide stained cells.

Main Results:

Achieved spectral resolution better than 1.5 nm with a coefficient of variation of 5% or better for fluorescence.
Demonstrated effective separation of beads with similar spectra using spectral and scatter analysis.
Successfully quantified contributions of spectrally similar fluorophores (ethidium bromide and propidium iodide).
Observed and analyzed the kinetic process of fluorophore dissociation and rebinding.
ALS analysis yielded component spectra highly similar to measured spectra.

Conclusions:

The developed CCD-based flow cytometer provides high spectral resolution and accuracy.
The system is capable of detailed spectral analysis and kinetic studies of cellular processes.
Advanced data analysis techniques like PCA and ALS enhance the utility of spectral flow cytometry.