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Counting Proteins in Single Cells with Addressable Droplet Microarrays
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Absolute Quantification of Protein Copy Number in Single Cells With Immunofluorescence Microscopy Calibrated Using

Stelios Chatzimichail1, Pashiini Supramaniam2, Ali Salehi-Reyhani1

  • 1Department of Surgery & Cancer, Imperial College London, London W12 0HS, U.K.

Analytical Chemistry
|April 20, 2021
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Summary
This summary is machine-generated.

This study introduces a method to calibrate fluorescence microscopy using single-molecule microarrays, enabling absolute protein quantitation in single cells. This breakthrough addresses limitations in current single-cell proteomics and microscopy techniques.

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Area of Science:

  • Biotechnology
  • Proteomics
  • Microscopy

Background:

  • Single-cell analysis, especially transcriptomics, has advanced significantly.
  • Proteomics lacks amplification, requiring ultrasensitive single-cell platforms.
  • Current single-cell proteomics platforms lag behind microscopy in throughput and multiplexing.

Purpose of the Study:

  • To assess a microfluidic single-molecule microarray for calibrating fluorescence microscopy data.
  • To enable absolute quantitation of protein abundance in single cells.
  • To bridge the gap between fluorescence intensity and absolute protein copy numbers.

Main Methods:

  • Utilized a microfluidic implementation of single-molecule microarrays.
  • Exploited the equivalence of steady-state protein abundance distributions and microarray data.
  • Developed methods using Green Fluorescent Protein (GFP) as a model.

Main Results:

  • Demonstrated the ability of single-molecule microarrays to calibrate fluorescence microscopy.
  • Showcased a pathway for absolute quantitation in fluorescence and immunofluorescence microscopy.
  • Established a method extendable to various proteins and biomolecules.

Conclusions:

  • Single-molecule microarray data can calibrate fluorescence microscopy for absolute protein quantitation.
  • This approach overcomes limitations of fluorescence intensity as a proxy for protein abundance.
  • The developed methodology offers broad applicability in single-cell biological analyses.