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

Abstract

Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, due to typically lower expression levels, experiments using endogenously-tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image correction By Regression (SAIBR), a simple, platform-independent protocol and FIJI plugin to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source, and achieves accurate quantitation of fluorophore signal and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible, low barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

Cleavage furrow-directed cortical flows bias mechanochemical pathways for PAR polarization in theC. elegansgerm lineage

“…Raw or SAIBR processed images were used for quantification (Rodrigues et al, 2022). In order to measure cortical concentrations, a 100-pixel-wide (15.5 μm) line following the membrane around the embryo was computationally straightened, and a 20-pixel-wide (3.1 μm) rolling average filter was applied to the straightened image.…”

Design principles for selective polarization of PAR proteins by cortical flows

“…Raw images were processed using SAIBR using N2 or NWG0038 animals for the autofluorescence calibration as required and ortical concentrations were obtained as described previously (Rodrigues et al, 2022). Briefly, a 50-pixel-wide (12.8 µm) line following the membrane around the embryo was extracted and computationally straightened, and a 20-pixel-wide (5.1 µm) rolling average filter was applied to the straightened image to reduce noise.…”

Nonlinear readout of spatial cues underlies robustness of asymmetric cell division

“…Subtraction can be done on a pixel-by-pixel basis (allowing for spatial signal correction) or on an embryo-by-embryo basis (e.g., to quantify whole-embryo fluorescence intensities, as indicated above). Note that SAIBR is also compatible when quantifying GFP fluorescence in embryos that co-express additional, spectrally-distinct fluorophores (such as mCherry), as detailed in (Rodrigues et al, 2022).…”

“…To address these questions, we set out to explicitly measure the sensitivity of embryos to perturbations in PAR protein dosage. By combining established methods for manipulation of protein dosage in C. elegans (Oegema, 2006) with a recently developed image quantitation-based workflow (Rodrigues et al, 2022), we were able to directly relate dosage to phenotype in individual embryos. Our data support a model in which pathway responses are highly canalized against variation in spatial signals at multiple levels, leading to quantitative decoupling between symmetry-breaking, polarity, spindle positioning and fate segregation modules.…”