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SAIBR: a simple, platform-independent method for spectral autofluorescence correction.

Nelio T L Rodrigues¹, Tom Bland^1,2, Joana Borrego-Pinto¹

¹Francis Crick Institute, London NW1 1AT, UK.

Development (Cambridge, England)

|June 17, 2022

Summary

Autofluorescence (AF) hinders accurate protein quantification in biological imaging. A new method, Spectral Autofluorescence Image Correction By Regression (SAIBR), corrects AF in fluorescence microscopy, enabling reliable detection of weakly expressed proteins.

Keywords:

C. elegans S. pombe Autofluorescence correction Fiji plug-in Starfish

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Area of Science:

* Molecular and Cellular Biology
* Developmental Biology
* Microscopy and Imaging

Background:

* Quantitative biological research requires precise measurement of gene expression and protein levels.
* CRISPR/Cas9 gene tagging facilitates endogenous protein monitoring using fluorescence microscopy.
* Autofluorescence (AF) presents a significant challenge, particularly for weakly expressed proteins and common fluorescent proteins (e.g., GFP, mNeonGreen).

Purpose of the Study:

* To develop and validate a robust method for correcting autofluorescence in biological imaging.
* To enable accurate quantitation of fluorescence signals from endogenously tagged proteins.
* To provide an accessible tool for improving protein detection in diverse cell and developmental systems.

Main Methods:

* Development of Spectral Autofluorescence Image Correction By Regression (SAIBR) protocol and FIJI plugin.
* Utilization of standard filter sets and illumination conditions for AF correction.
* Validation in various biological models including C. elegans embryos, starfish oocytes, and fission yeast.

Main Results:

* SAIBR effectively corrects autofluorescence across different biological samples.
* The method achieves accurate quantitation of fluorophore signals, even with low expression levels.
* Reliable detection and quantification of weakly expressed proteins are enabled by SAIBR.

Conclusions:

* SAIBR is a simple, platform-independent protocol for autofluorescence correction.
* This method enhances the accuracy of fluorescence-based protein quantitation in diverse biological systems.
* SAIBR offers a low-barrier solution for researchers to implement AF correction in their imaging workflows.