Moscardini, A. | bioRxiv Science

bioRxiv2023

Quantitative determination of fluorescence labeling implemented in cell cultures

BackgroundLabeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative. ResultsWe present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We show the ability of the protocol to extract reliable quantification for different fluorescent probes, reagents concentrations, reaction timing and to optimize labeling performance. As paradigm, we consider the labeling of the membrane-receptor TrkA through 4-phosphopantetheinyl transferase Sfp in living cells, visualizing the results by TIRF microscopy. This investigation allows us to find conditions for demanding single and multi-color single-molecule studies requiring high degrees of labeling. ConclusionsThe developed method allows the quantitative determination and the optimization of staining efficiency in any labeling strategy based on stable reactions.

biophysics↗

bioRxiv2023

Optimized two-color single-molecule tracking of fast-diffusing membrane receptors

Single particle tracking (SPT) combined with total internal reflection fluorescence (TIRF) microscopy is an outstanding approach to decipher crucial molecular mechanisms on the cell membrane at the nanometric scale. In multicolor configurations it can even be the ideal tool to investigate interactions, but this is hindered by a number of experimental challenges. We systematically and quantitatively analyze the impact of all the necessary sub-elements of any SPT-TIRF setup on signal-to-noise ratio, especially in dynamic studies using minimally-invasive dyes for biomolecule labeling. We show that the dominant limiting factor is the autofluorescence originating from the commonly-used optical glass. We identify and test a different material and show a significant improvement of signal-to-noise ratio in a multichannel TIRF configuration employing the new glass covers. We also address the problem of photobleaching of fluorescent probes by presenting effective approaches suited to a multicolor implementation that requires simultaneous stabilization of multiple dyes. We apply the developed protocol to the analysis of p75 receptors labeled by two fluorophores on the membrane of living cells. Our strategy yields reliable, simultaneous two-color SPT, even for fast-diffusing receptors, enabling this study under conditions not accessible with standard experimental configurations. We argue that the present protocol can pave the way for multicolor super-resolved localization and tracking of single molecules by TIRF microscopy, much expanding the potential of SPT.

biophysics↗