title: Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy.
abstract: We present a high-speed photon counter for use with two-photon microscopy. Counting pulses of photocurrent, as opposed to analog integration, maximizes the signal-to-noise ratio so long as the uncertainty in the count does not exceed the gain-noise of the photodetector. Our system extends this improvement through an estimate of the count that corrects for the censored period after detection of an emission event. The same system can be rapidly reconfigured in software for fluorescence lifetime imaging, which we illustrate by distinguishing between two spectrally similar fluorophores in an in vivo model of microstroke.
authors.affiliation: Department of Physics, University of California at San Diego, La Jolla, CA 92093-0374, USA.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Driscoll
authors.forename: Andy Y
authors.initials: AY
authors.lastname: Shih
authors.forename: Satish
authors.initials: S
authors.lastname: Iyengar
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Field
authors.forename: G Allen
authors.initials: GA
authors.lastname: White
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Squier
authors.forename: Gert
authors.initials: G
authors.lastname: Cauwenberghs
authors.forename: David
authors.initials: D
authors.lastname: Kleinfeld
mesh_terms: D000703:Analog-Digital Conversion; D000818:Animals; D001921:Brain / Q000166:cytology*; D016923:Cell Death; D003952:Diagnostic Imaging / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007395:Interneurons / Q000502:physiology*; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017785:Photons; D012815:Signal Processing, Computer-Assisted
chemical_list: D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins