Counting fluorescent emitters with a single photon avalanche diode array
- 1Department of Physics, Indiana University
- 2Department of Pediatrics, Indiana University School of Medicine
- 3Department of Physics and Astronomy, Purdue University
Abstract
For decades, photon statistics has been used to quantify fluorophore properties in a diffraction-limited focal volume. Techniques such as photon count histogram (PCH) analysis and coincidence measurements exploit the stochastic nature of photon emission to estimate quantities including molecular concentration, brightness, and fluorophore number. However, these approaches have been largely confined to confocal microscopy due to the lack of fast and single photon sensitive camera sensors which can capture the photon emission statistics of individual fluorophores. Here, we explore avenues towards quantitative analysis of the active fluorophore number by leveraging recent advancements in single photon avalanche diode (SPAD) array technology. Binary exposures of a SPAD array can be synchronized with picosecond laser pulses to measure the PCH in a widefield setting. Then, by modeling the statistical relationship between the active fluorophore number and the PCH in a region of interest following a laser pulse, we can perform Bayesian inference of this number. The model is demonstrated experimentally by counting quantum dots and various numbers of fluorescent dye molecules bound to DNA origamis.
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BibTeX
@article{seitz2026counting,
title = {{Counting fluorescent emitters with a single photon avalanche diode array}},
author = {Clayton Seitz and Carmella Evans-Molina and Jing Liu},
journal = {Scientific Reports},
year = {2026},
month = aug,
doi = {10.1038/s41598-026-66870-x},
url = {https://doi.org/10.1038/s41598-026-66870-x},
note = {CC BY},
}
RIS
TY - JOUR
TI - Counting fluorescent emitters with a single photon avalanche diode array
AU - Clayton Seitz
AU - Carmella Evans-Molina
AU - Jing Liu
PY - 2026
DA - 2026/08/01
JO - Scientific Reports
DO - 10.1038/s41598-026-66870-x
UR - https://doi.org/10.1038/s41598-026-66870-x
AB - For decades, photon statistics has been used to quantify fluorophore properties in a diffraction-limited focal volume. Techniques such as photon count histogram (PCH) analysis and coincidence measurements exploit the stochastic nature of photon emission to estimate quantities including molecular concentration, brightness, and fluorophore number. However, these approaches have been largely confined to confocal microscopy due to the lack of fast and single photon sensitive camera sensors which can capture the photon emission statistics of individual fluorophores. Here, we explore avenues towards quantitative analysis of the active fluorophore number by leveraging recent advancements in single photon avalanche diode (SPAD) array technology. Binary exposures of a SPAD array can be synchronized with picosecond laser pulses to measure the PCH in a widefield setting. Then, by modeling the statistical relationship between the active fluorophore number and the PCH in a region of interest following a laser pulse, we can perform Bayesian inference of this number. The model is demonstrated experimentally by counting quantum dots and various numbers of fluorescent dye molecules bound to DNA origamis.
SN - 2045-2322
ER -