Fig. 3: BrightEyes-TTM for FLISM.
From: The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy
Imaging and FLISM analysis of 100 nm fluorescent beads with a custom-built single-photon laser-scanning microscope equipped with a 5 × 5 SPAD array detector prototype. a Side-by-side comparison of confocal (left, pinhole 0.2 AU), adaptive pixel-reassignment ISM (center), and open confocal (right, pinhole 1.4 AU). AU = Airy unit. Each imaging modality shows both the intensity-based image (top-left corner) and the lifetime image (bottom-right corner). A bidimensional look-up-table represents in the lifetime images both the intensity values (i.e., photon counts) and the excited-state lifetime values (i.e., τfl). The intensity-based images integrate the relative 3D data (x, y, Δt) along the start–stop time dimension Δt. b Histogram distributions showing the number of pixels versus lifetime values—in violet lifetime values which fall out of the selected lifetime interval. The selected interval was chosen by visually inspecting the FLISM image. The same intervals were used for the confocal and open confocal data. The lifetime images report in violet the pixels whose lifetime is in this interval. c Zoomed regions in the white-dashed boxes, with the intensity panels re-normalized to the maximum and minimum values. d Pixel intensity phasor plots, 5% and 10% thresholds respectively in gray and color. Pixel dwell time 100 μs. Scale bars 2 μm.
