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

Abstract

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.

Quantitative determination of fluorescence labeling implemented in cell cultures

“…Trolox (Sigma-Aldrich) and  n-propyl gallate (Sigma-Aldrich) are included in the imaging medium to limit photobleaching effects over time [8].…”

“…Simultaneous double excitation was achieved with the internal laser line at 565 nm plus an external 488 nm laser (iFLEX-iRIS, Qioptiq) connected through a laser combiner (iFLEX-adder, QiOptiq), using kineFLEX polarization-maintaining fibers (QiOptiq) and kineMATIX fiber couplers (QiOptiq) [6,8]. Typically, laser powers at the objective were 3.2 mW and 3.5 mW for 565 nm and 488 nm lasers, respectively.…”

“…Powers at the objective were typically 3.5 mW and 3 mW for 561 nm and 635 nm laser respectively. In measurements on cells labeled with Atto 565 and one 488-dye, the two channels were detected simultaneously using a Quad filter cube and a Dual View (Optical Insights DV-CC, with filters Chroma ET525-50 and Semrock 600/52 nm BrightLine® and dichroic beam splitter 565dcxr) placed in front of the camera after an Optomask adjustable field mask (OPTMSK-L, Andor) [6,8]. The Dual View windows in the different channels were aligned using bright field imaging of the supplied grid, leaving some unilluminated pixels between the two detection windows.…”

“…spatiotemporal resolutions [1,2]. Advances in labeling strategies and microscopy yielded access to the single-molecule level and to the direct observation of biomolecule dynamics and interactions in physiological environments [3][4][5][6][7][8]. These homo-and hetero-interactions strongly correlate with molecular functions, as in the case of signal transduction via membrane receptors of different families, whose activity is mainly regulated by interactions with ligands, coreceptors and other membrane and intracellular molecules [9][10][11][12][13].…”

Quantitative determination of fluorescence labeling implemented in cell cultures

“…Fluorescent labeling of cellular components is of utmost importance for studying biological processes since it enables the investigation of a variety of biological processes in sundry kinds of samples with high sensitivity, specificity, and spatiotemporal resolutions [1,2]. Advances in labeling strategies and microscopy yielded access to the single-molecule level and to the direct observation of biomolecule dynamics and interactions in physiological environments [3][4][5][6][7][8]. These homo-and hetero-interactions strongly correlate with molecular functions, as in the case of signal transduction via membrane receptors of different families, whose activity is mainly regulated by interactions with ligands, coreceptors, and other membrane and intracellular molecules [9][10][11][12][13].…”