TY - GEN
T1 - Analyzing blinking effects in super resolution localization microscopy with single-photon SPAD imagers
AU - Antolovic, Ivan Michel
AU - Burri, Samuel
AU - Bruschini, Claudio
AU - Hoebe, Ron
AU - Charbon, Edoardo
N1 - Publisher Copyright: © 2016 SPIE. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2016
Y1 - 2016
N2 - For many scientific applications, electron multiplying charge coupled devices (EMCCDs) have been the sensor of choice because of their high quantum efficiency and built-in electron amplification. Lately, many researchers introduced scientific complementary metal-oxide semiconductor (sCMOS) imagers in their instrumentation, so as to take advantage of faster readout and the absence of excess noise. Alternatively, single-photon avalanche diode (SPAD) imagers can provide even faster frame rates and zero readout noise. SwissSPAD is a 1-bit 512×128 SPAD imager, one of the largest of its kind, featuring a frame duration of 6.4 μs. Additionally, a gating mechanism enables photosensitive windows as short as 5 ns with a skew better than 150 ps across the entire array. The SwissSPAD photon detection efficiency (PDE) uniformity is very high, thanks on one side to a photon-to-digital conversion and on the other to a reduced fraction of "hot pixels" or "screamers", which would pollute the image with noise. A low native fill factor was recovered to a large extent using a microlens array, leading to a maximum PDE increase of 12×. This enabled us to detect single fluorophores, as required by ground state depletion followed by individual molecule return imaging microscopy (GSDIM). We show the first super resolution results obtained with a SPAD imager, with an estimated localization uncertainty of 30 nm and resolution of 100 nm. The high time resolution of 6.4 μs can be utilized to explore the dye's photophysics or for dye optimization. We also present the methodology for the blinking analysis on experimental data.
AB - For many scientific applications, electron multiplying charge coupled devices (EMCCDs) have been the sensor of choice because of their high quantum efficiency and built-in electron amplification. Lately, many researchers introduced scientific complementary metal-oxide semiconductor (sCMOS) imagers in their instrumentation, so as to take advantage of faster readout and the absence of excess noise. Alternatively, single-photon avalanche diode (SPAD) imagers can provide even faster frame rates and zero readout noise. SwissSPAD is a 1-bit 512×128 SPAD imager, one of the largest of its kind, featuring a frame duration of 6.4 μs. Additionally, a gating mechanism enables photosensitive windows as short as 5 ns with a skew better than 150 ps across the entire array. The SwissSPAD photon detection efficiency (PDE) uniformity is very high, thanks on one side to a photon-to-digital conversion and on the other to a reduced fraction of "hot pixels" or "screamers", which would pollute the image with noise. A low native fill factor was recovered to a large extent using a microlens array, leading to a maximum PDE increase of 12×. This enabled us to detect single fluorophores, as required by ground state depletion followed by individual molecule return imaging microscopy (GSDIM). We show the first super resolution results obtained with a SPAD imager, with an estimated localization uncertainty of 30 nm and resolution of 100 nm. The high time resolution of 6.4 μs can be utilized to explore the dye's photophysics or for dye optimization. We also present the methodology for the blinking analysis on experimental data.
KW - fluorescence microscopy
KW - image quality assessment
KW - imaging ultrafast phenomena
KW - photodetectors
KW - photoncounting
KW - super resolution
UR - http://www.scopus.com/inward/record.url?scp=84983371801&partnerID=8YFLogxK
U2 - https://doi.org/10.1117/12.2211430
DO - https://doi.org/10.1117/12.2211430
M3 - Conference contribution
T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE
BT - Single Molecule Spectroscopy and Superresolution Imaging IX
A2 - Enderlein, Jorg
A2 - Gregor, Ingo
A2 - Gryczynski, Zygmunt Karol
A2 - Erdmann, Rainer
A2 - Koberling, Felix
PB - SPIE
T2 - Single Molecule Spectroscopy and Superresolution Imaging IX
Y2 - 13 February 2016 through 14 February 2016
ER -