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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: Jun 22, 2025

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Kayvan Samimi¹, Ojaswi Pasachhe¹, Emmanuel Contreras Guzman¹

¹Morgridge Institute for Research, Madison, Wisconsin, USA.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology

|June 29, 2024

Summary

We developed a novel flow cytometer using time-correlated single-photon counting (TCSPC) to measure autofluorescence lifetime in single cells. This high-throughput, label-free system analyzes cellular metabolism and function in real-time.

Keywords:

NAD(P)H flow cytometry fluorescence lifetime label‐free sensing metabolism single‐cell analysis time tagger

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Last Updated: Jun 22, 2025

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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Published on: February 9, 2012

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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Published on: January 18, 2017

Area of Science:

Cellular metabolism and function analysis
Advanced microscopy and flow cytometry techniques
Biophotonics and single-cell analysis

Background:

Autofluorescence lifetime imaging microscopy (FLIM) detects cellular metabolic changes via NAD(P)H co-enzymes.
Traditional FLIM using laser-scanning microscopes is costly, slow, and requires extensive post-processing.
There is a need for higher-throughput, real-time methods for analyzing single-cell metabolic states.

Purpose of the Study:

To develop a fluorescence lifetime-sensitive flow cytometer with high temporal resolution.
To enable real-time, label-free analysis of single-cell metabolism and function.
To overcome the limitations of throughput and cost associated with traditional FLIM.

Main Methods:

Utilized a 375 nm picosecond-pulsed diode laser and time-correlated single-photon counting (TCSPC) detection.
Integrated an FPGA-based time tagger for real-time phasor-based classification of flowing cells.
Employed a microfluidic system with simultaneous brightfield imaging and two-color analysis, achieving high throughput and low light dose.

Main Results:

Demonstrated real-time, phasor-based gating of flowing cells with TCSPC temporal resolution.
Confirmed cellular viability post-measurement and sensitivity to metabolic perturbations in Jurkat T cells.
Successfully differentiated quiescent versus activated states in primary human T cells and mouse neural stem cells, consistent with FLIM studies.

Conclusions:

The developed TCSPC-based autofluorescence lifetime flow cytometer offers a high-throughput, label-free alternative to laser-scanning FLIM.
This system provides valuable real-time insights into single-cell function and metabolism.
The technology has broad applications in cell biology, immunology, and neuroscience research.