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Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM
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Super-multiplexed fluorescence microscopy via photostability contrast.

Antony Orth1, Richik N Ghosh2, Emma R Wilson1

  • 1ARC Centre of Excellence for Nanoscale BioPhotonics, School of Science, RMIT University, Melbourne, VIC 3001, Australia.

Biomedical Optics Express
|July 10, 2018
PubMed
Summary
This summary is machine-generated.

Researchers can now triple fluorescence microscopy multiplexing. Differences in photostability, combined with a new algorithm, distinguish labels within a single spectral channel, enabling more detailed cellular imaging.

Keywords:
(110.4234) Multispectral and hyperspectral imaging(170.2520) Fluorescence microscopy

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Area of Science:

  • Cell Biology
  • Microscopy Techniques
  • Biophotonics

Background:

  • Fluorescence microscopy is essential for visualizing cellular structures and biomolecules.
  • Current methods using spectrally distinct labels are limited to 4-5 multiplexed targets due to overlapping emission spectra.
  • Advanced multiplexing requires additional contrast dimensions beyond spectral separation.

Purpose of the Study:

  • To introduce a novel method for increasing multiplexing capabilities in fluorescence microscopy.
  • To demonstrate the use of photostability differences as a new dimension for distinguishing fluorescent labels.
  • To develop an unmixing algorithm capable of resolving labels with similar emission spectra.

Main Methods:

  • Utilized photobleaching characteristics of fluorophores as a distinguishing feature.
  • Developed and applied a novel spectral unmixing algorithm.
  • Tested the technique on organic dyes, autofluorescent biomolecules, and fluorescent proteins.

Main Results:

  • Successfully resolved up to three fluorescent labels within a single spectral channel.
  • Demonstrated unmixing of fluorescent labels with nearly identical emission spectra.
  • Validated the approach across diverse fluorescent labeling agents.

Conclusions:

  • Photostability differences offer a viable method to enhance multiplexing in fluorescence microscopy.
  • The developed unmixing algorithm effectively separates spectrally similar fluorophores.
  • This technique can potentially triple multiplexing capacity for standard fluorescence microscopes without hardware upgrades.