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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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Related Experiment Video

Updated: Mar 11, 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

Representation Method for Spectrally Overlapping Signals in Flow Cytometry Based on Fluorescence Pulse Time-Delay

Wenchang Zhang^1,2,3, Xiaoping Lou^4,5, Xiaochen Meng^6,7

¹School of Instrumentation Science & Opto-Electronics Engineering, Hefei University of Technology, Hefei 230009, China. baomuayi007@126.com.

Sensors (Basel, Switzerland)

|November 26, 2016

Summary

This study introduces a novel method for accurate fluorescence lifetime analysis in flow cytometry, effectively correcting for spectral overlap using time-delay estimation and digital signal processing. The approach enhances multicolor fluorescence analysis accuracy.

Keywords:

digital signal processing flow cytometry fluorescence lifetime spectrally overlapping signals time-delay estimation

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Area of Science:

Biophotonics and Fluorescence Spectroscopy
Analytical Chemistry and Cytometry
Digital Signal Processing in Biological Applications

Background:

Flow cytometry's utility is expanding due to multicolor fluorescence analysis.
Nonlinearity in detection and spectral overlap between fluorophores hinder accurate intensity measurements and signal representation.
Accurate fluorescence lifetime determination is crucial for reliable multicolor analysis.

Purpose of the Study:

To develop a quantitative method for analyzing and correcting spectral overlap in two-color flow cytometry.
To propose a fluorescence pulse signal representation method based on time-delay estimation.
To eliminate the influence of spectral overlap on fluorescence lifetime measurements.

Main Methods:

Time-delay estimation using modified chirp Z-transform (MCZT) and fine interpolation of the correlation peak (FICP) algorithms.
Hardware influence removal via calibration to obtain original fluorescence lifetimes.
Artificial creation of modulated signals with phase shifts and introduction of reference signals for spectral overlap elimination using digital signal processing.

Main Results:

Successfully rectified fluorescence lifetimes affected by spectral overlap from 8.28 and 4.86 ns to 8.51 and 4.63 ns.
Obtained lifetimes closely match single-color fluorochrome measurements (8.48 and 4.67 ns), validating the method's accuracy.
Demonstrated effective elimination of spectral overlap influence without requiring high-speed analog devices.

Conclusions:

The proposed comprehensive approach accurately determines fluorescence lifetimes by effectively addressing spectral overlap.
Digital signal processing methods simplify the system, making it more flexible and accessible than existing techniques.
This method significantly enhances the reliability and accuracy of multicolor flow cytometry analysis.