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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: Sep 19, 2025

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

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Published on: July 17, 2019

Characterization of a Single-Molecule Sensitive Digital Flow Cytometer for Amplification-Free Digital Assays.

Yuanhua Cheng^1,2, Alya Nguyen¹, Wyatt Nelson¹

¹Department of Chemistry, University of Washington, Seattle, Washington 98195, United States.

|June 17, 2025

Summary

A novel digital flow cytometer (dFC) enables direct single-molecule counting for amplification-free digital assays. This technology offers high sensitivity and accuracy for quantifying molecules without complex sample preparation.

Keywords:

absolute quantitation antibody QC/QA digital assay digital flow cytometer (dFC)single-molecule counting

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Last Updated: Sep 19, 2025

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

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Published on: July 17, 2019

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Area of Science:

Biotechnology
Analytical Chemistry
Single-molecule detection

Background:

Digital assays offer superior quantitation but often require amplification, hindering multiplexing and increasing complexity.
Current methods like digital PCR and digital ELISA face limitations due to amplification steps and intricate sample handling.

Purpose of the Study:

To introduce and characterize a novel digital flow cytometer (dFC) for sensitive, amplification-free single-molecule detection.
To demonstrate the dFC's capability for absolute concentration measurements of antibody-dye conjugates.

Main Methods:

Development of a digital flow cytometer (dFC) with optimized microfluidics and optics for enhanced single-molecule sensitivity.
Characterization of the dFC's analytical performance using antibody-dye conjugates with various dyes.
Validation of absolute concentration measurements across a wide dynamic range.

Main Results:

The dFC achieved over 98% single-molecule detection efficiency for 18 different antibody-dye conjugates.
Demonstrated a low false-positive rate, stable baseline signal, and accurate concentration measurements.
Exhibited a dynamic range spanning 4 orders of magnitude for absolute quantitation.

Conclusions:

The dFC provides a sensitive and accurate platform for direct single-molecule counting, enabling amplification-free digital assays.
This technology can be applied to authenticate reagents and develop multiplexed assays for nucleic acids and proteins.
The dFC offers a promising alternative to traditional digital assays, overcoming limitations of amplification and sample preparation.