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This study demonstrates a new photon-counting imager using single-photon avalanche diodes (SPADs) for advanced fluorescence correlation spectroscopy. The technology enables high-speed, large-scale molecular motion analysis, including in vivo measurements.

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Area of Science:

  • Photonics and Spectroscopy
  • Biophysics and Molecular Imaging
  • Advanced Sensor Technology

Background:

  • Single-photon avalanche diodes (SPADs) are advancing photon-counting imaging with zero readout noise and high temporal accuracy.
  • SPAD imagers have improved spatial resolution, timing accuracy, and sensitivity, reducing spurious signals.
  • These sensors are increasingly used for time-resolved applications like real-time autocorrelation measurements.

Purpose of the Study:

  • To utilize a state-of-the-art 512 × 128 SPAD array for selective plane illumination-fluorescence correlation spectroscopy (SPI-FCS).
  • To enable simultaneous, high-speed fluorescence correlation spectroscopy measurements across a 2D sample slice.
  • To measure molecular motion of small fluorescent particles and assess the system's performance for in vivo applications.

Main Methods:

  • Employed a 512 × 128 SPAD array with microlenses for enhanced photosensitivity and 10^-5–10^-6 s time resolution.
  • Integrated the SPAD imager into a selective plane illumination-fluorescence correlation spectroscopy setup for simultaneous 2D measurements.
  • Utilized global fitting to compensate for detection efficiency inhomogeneities and mitigate afterpulsing effects, with spatial binning for improved results.

Main Results:

  • Successfully measured molecular motion of small fluorescent particles, such as Alexa-488 diffusion.
  • Achieved calibration-free measurements for various samples by compensating for detection inhomogeneities.
  • Recorded the first high-speed SPI-FCS in vivo measurements, demonstrating the diffusion of eGFP oligomers in HeLa cells.

Conclusions:

  • The developed SPAD-based SPI-FCS system enables high-speed, large-scale, and in vivo molecular motion analysis.
  • While particle concentrations may be overestimated compared to confocal setups, calibration allows for absolute measurements.
  • Future SPAD sensor generations promise further improvements in sensitivity and noise reduction for widefield imaging applications.