Abstract
Fluorescence microscopy is widely used to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously in a sample by using spectrally distinct fluorescent labels. The wide emission spectra of most fluorophores limits spectral multiplexing to four or five labels in a standard fluorescence microscope. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.
Highlights
Fluorescence microscopy is widely used in biological research for high contrast imaging with unrivalled specificity [1]
We show that one may discriminate between fluorescent species by leveraging a photophysical process inherent in all organic fluorophores – photobleaching
The beads have peak emission wavelengths ranging from 500 - 700 nm, and are imaged simultaneously in yellow (570 - 620 nm) and red (663 - 738 nm) spectral channels using a confocal microscope (Fig. 1(a))
Summary
Fluorescence microscopy is widely used in biological research for high contrast imaging with unrivalled specificity [1]. Fluorescent emission from a given fluorescent species contributes to the signal in multiple emission channels This spectral cross-talk can be significant when using more than 3 fluorescent probes, necessitating unmixing algorithms for successful separation [3,4]. Even with a large number of spectral channels it is extremely challenging to identify more than 4-5 fluorescent species in a sample, regardless of the spectral measurement scheme [5,6]. This is because of the wide spectral width of each fluorophore (~40-50 nm with broad tails), the need for excitation windows (~20 nm minimum per excitation source) and the finite spectral bandwidth of the visible spectrum (300 nm). In many areas of biology there is a growing need to separate more objects or structures or to perform several fluorescence-based sensing experiments simultaneously to increase the information content derived from fluorescence microscopy assays
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