Extended Data Fig. 2: Post-image analysis for counting the number of single-molecule fluorescence spots.
a, Setting the cutoff to eliminate background signals for post image analysis. We used 8 to 10 control images captured with a 100 ms time resolution from empty reaction chambers to establish a background limit. The background limit (cutoff=1,015 A.U.) was determined from the signal distribution recorded in each pixel of the control images, effectively eliminating 99% of background signals. b, Identification of diffraction-limited fluorescence spots. Three sequential images captured with a 300 ms time frame were averaged to create a single image upon subtracting the background. From the averaged image, the number of diffraction-limited fluorescence spots were counted (single-molecule counting). c, Identified single-molecule counts and integration of individual pixel signals of the imaging area (total fluorescence integration) from the analyzed TIRF images. The TIRF images were obtained by surface IP of BCLxL-eGFP expressed in HEK293T cell extracts (Scale bar: 10 μm) (a-c). d-g, Calibration of single-molecule count from the total fluorescence integration. (d) Single-molecule count dependent on the total fluorescence integration of surface-immobilized BCL2-mCherry obtained from the analyzed images (Scale bar: 10 μm). (e) Photobleaching kinetics of surface-immobilized BCL2-mCherry signals depending on the bleaching time. (f) The linear correlation between single-molecule count and total fluorescence integration of surface-immobilized BCL2-mCherry after photobleaching. (g) Calibration of single-molecule count from total fluorescence integration by extrapolating. The linear correlation was obtained in (f). The TIRF images were obtained by surface IP of BCL2-mCherry expressed in HEK293T cell extracts (d-g).
