Widefield High Frame Rate Single-Photon SPAD Imagers for SPIM-FCS

Photon-counting sensors based on standard complementary metal-oxide-semiconductor single-photon avalanche diodes (SPADs) represent an emerging class of imagers that enable the counting and/or timing of single photons at zero readout noise (better than high-speed electron-multiplying charge-coupling devices) and over large arrays. They have seen substantial progress over the last 15 years, increasing their spatial resolution, timing accuracy, and sensitivity while reducing spurious signals such as afterpulsing and dark counts. They are increasingly being applied for time-resolved applications with the added advantage of enabling real-time options such as autocorrelation. We report in this study on the use of such a state-of-the-art 512 × 128 SPAD array, capable of a time resolution of 10−5–10−6 s for full frames while retaining acceptable photosensitivity thanks to the use of dedicated microlenses, in a selective plane illumination-fluorescence correlation spectroscopy setup. The latter allows us to perform thousands of fluorescence-correlation spectroscopy measurements simultaneously in a two-dimensional slice of the sample. This high-speed SPAD imager enables the measurement of molecular motion of small fluorescent particles such as single chemical dye molecules. Inhomogeneities in the molecular detection efficiency were compensated for by means of a global fit of the auto- and cross-correlation curves, which also made a calibration-free measurement of various samples possible. The afterpulsing effect could also be mitigated, making the measurement of the diffusion of Alexa-488 possible, and the overall result quality was further improved by spatial binning. The particle concentrations in the focus tend to be overestimated by a factor of 1.7 compared to a confocal setup; a calibration is thus required if absolute concentrations need to be measured. The first high-speed selective plane illumination-fluorescence correlation spectroscopy in vivo measurements to our knowledge were also recorded: although two-component fit models could not be employed because of noise, the diffusion of eGFP oligomers in HeLa cells could be measured. Sensitivity and noise will be further improved in the next generation of SPAD-based widefield sensors, which are currently under testing.


Published in:
Biophysical Journal, 114, 10, 2455-2464
Year:
May 22 2018
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 Record created 2018-08-13, last modified 2018-12-03


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