Journal: Journal of Medicinal Chemistry
Article Title: Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules
doi: 10.1021/acs.jmedchem.4c00339
Figure Lengend Snippet: Estimation of Alexa-C3 labeling efficiency using photobleaching step-analysis. In order to obtain fluorescence data on a single molecule level, CHO K1 cells were incubated at 4 °C with 200 nM Alexa488-C3 or 200 nM Alexa568-C3, respectively, fixed using 4% PFA/0.2% glutaraldehyde solution and subsequently captured in TIRF-M (total internal reflection fluorescence microscopy) time lapse recordings. (A) Left panel shows recordings of non-transfected cells, overlaid with cell outlines from corresponding bright field images (not shown). The panel in the middle depicts typical single molecule complexes 48 h after transfection with 50 ng wild type BACE1. The right panel shows the same image after rolling background subtraction using ImageJ to improve the signal-to-background ratio prior to semiautomatic analysis. The insets highlight single fluorescence spots at higher magnification. Scale bar represents 10 μm. (B) Single fluorescence spots were located and fluorescence intensities over time were extracted (see methods). Subsequently, bleaching steps were identified and counted (red: idealized time series). (C) Distributions of step counts proved similar for both Alexa488-C3 ( n = 15 cells, on average 360 tracks per cell) and Alexa568-C3 ( n = 12, on average 330 tracks per cell). (D) In order to estimate Alexa-C3 labeling efficiencies, the observed distributions were fitted with a sum of n th order binomial distributions for possible BACE1 stoichiometries, calculating the respective residual error: sum of 2nd, 4th, and 6th order for dimers (black line); sum of 3rd and 6th order for trimers (red line); and sum of 2nd, 3rd, 4th, 5th, and 6th order for a combination of dimer and trimers (blue line). Spots with more than six bleaching steps were excluded from analysis.
Article Snippet: Single molecule TIRF microscopy was performed using an inverse-stage TIRF setup (Nikon Eclipse Ti-E) using a 488 nm laser (Coherent, 150 mW) set to 20% power output and a 561 nm laser (Coherent, 50 mW).
Techniques: Labeling, Fluorescence, Incubation, Microscopy, Transfection
