Absolute Quantification of Protein Copy Number in Single Cells With Immunofluorescence Microscopy Calibrated Using Single-Molecule Microarrays

Abstract

Great strides toward routine single-cell analyses have been made over the last decade, particularly in the field of transcriptomics. For proteomics, amplification is not currently possible and has necessitated the development of ultrasensitive platforms capable of performing such analyses on single cells. These platforms are improving in terms of throughput and multiplexability but still fall short in relation to more established methods such as fluorescence microscopy. However, microscopy methods rely on fluorescence intensity as a proxy for protein abundance and are not currently capable of reporting this in terms of an absolute copy number. Here, a microfluidic implementation of single-molecule microarrays for single-cell analysis is assessed in its ability to calibrate fluorescence microscopy data. We show that the equivalence of measurements of the steady-state distribution of protein abundance to single-molecule microarray data can be exploited to pave the way for absolute quantitation by fluorescence and immunofluorescence microscopy. The methods presented have been developed using GFP but are extendable to other proteins and other biomolecules of interest.