Fig. 6: Fluorescence fluctuation spectroscopy on living cells.
From: The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy
a ISM (bottom-left corner) and FLISM (top-right corner) images of a HEK293T cell expressing eGPF. b, c Start–stop time histograms (central pixel in black) and intensity time traces for all 25 channels of a 100 s FLFS measurement. The blue circle in a depicts the position in the cell where the measurement is performed. d Autocorrelation curves (scattered points) and fits (lines) for the central pixel, sum 3 × 3, and sum 5 × 5 curves obtained from (c). e Spot-variation analysis: the dashed black line represents the average (D = 34 ± 12 μm2/s, N = 5) of the dashed light-gray lines. Each dashed light-gray line represents a different position within the same cell. f Spot-variation analysis as a function of the measurement time-coarse. Data from (c). The intensity time traces are divided into chunks of 5 s, each chunk is analyzed by means of spot-variation FCS and generates a dashed light-gray line. The dashed black line represents the average (D = 32 ± 5 μm2/s, N = 14) of the dashed light-gray lines. Error bars in (e, f) represent standard deviations. g Ratio between the diffusion coefficients measured for the central pixel and sum 5 × 5 (Dcentral/D5×5), overlapped with the fluorescence lifetime as a function of the measurement time-coarse. Data from (f). Scale bar 5 μm. Pixel dwell time 100 μs. The data were acquired from five different cells, in each cell multiple positions (from 3 to 5) were sampled. Setup: custom-built single-photon laser-scanning microscope equipped with a 5 × 5 SPAD array detector prototype.
