title: SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking.
abstract: sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm.
authors.affiliation: Applied Quantum Architectures, Department of Quantum Engineering, TU Delft, Netherlands.
authors.forename: Ivan Michel
authors.initials: IM
authors.lastname: Antolovic
authors.affiliation: School of Engineering, EPFL, Switzerland.
authors.forename: Samuel
authors.initials: S
authors.lastname: Burri
authors.affiliation: School of Engineering, EPFL, Switzerland.
authors.forename: Claudio
authors.initials: C
authors.lastname: Bruschini
authors.affiliation: Cellular Imaging - Core Facility, Academic Medical Centre, University of Amsterdam, Netherlands.
authors.forename: Ron A
authors.initials: RA
authors.lastname: Hoebe
authors.affiliation: Applied Quantum Architectures, Department of Quantum Engineering, TU Delft, Netherlands.|School of Engineering, EPFL, Switzerland.
authors.forename: Edoardo
authors.initials: E
authors.lastname: Charbon