Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy.

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.

[1]  P. Powers.  Third-Harmonic Generation , 2013 .

[2]  Michael D. Young,et al.  Differential Multiphoton Laser Scanning Microscopy , 2012, IEEE Journal of Selected Topics in Quantum Electronics.

[3]  Meng Cui.  A high speed wavefront determination method based on spatial frequency modulations for focusing light through random scattering media. , 2011, Optics express.

[4]  David Kleinfeld,et al.  Chronic optical access through a polished and reinforced thinned skull. , 2010, Nature methods.

[5]  D. Kleinfeld,et al.  Topological basis for the robust distribution of blood to rodent neocortex , 2010, Proceedings of the National Academy of Sciences.

[6]  Ramón Carriles,et al.  Optimizing the Fluorescent Yield in Two-photon Laser Scanning Microscopy with Dispersion Compensation References and Links , 2022 .

[7]  S. Achilefu,et al.  Fluorescence lifetime measurements and biological imaging. , 2010, Chemical reviews.

[8]  Eric Betzig,et al.  Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues , 2010, Nature Methods.

[9]  Ramón Carriles,et al.  Invited review article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy. , 2009, The Review of scientific instruments.

[10]  David Kleinfeld,et al.  MPScope 2.0: A Computer System for Two-Photon Laser Scanning Microscopy with Concurrent Plasma-Mediated Ablation and Electrophysiology , 2009 .

[11]  David Kleinfeld,et al.  In Vivo Two-Photon Laser Scanning Microscopy with Concurrent Plasma-Mediated Ablation Principles and Hardware Realization , 2009 .

[12]  K. Svoboda,et al.  Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window , 2009, Nature Protocols.

[13]  Richard K P Benninger,et al.  Single-photon-counting detector for increased sensitivity in two-photon laser scanning microscopy. , 2008, Optics letters.

[14]  D. Kleinfeld,et al.  Advancing multifocal nonlinear microscopy: development and application of a novel multibeam Yb:KGd(WO4)2 oscillator. , 2008, Optics express.

[15]  Karel Svoboda,et al.  The Spread of Ras Activity Triggered by Activation of a Single Dendritic Spine , 2008, Science.

[16]  F. Helmchen,et al.  Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo. , 2008, Optics express.

[17]  David Kleinfeld,et al.  Chapter 10. In vivo measurements of blood flow and glial cell function with two-photon laser-scanning microscopy. , 2008, Methods in enzymology.

[18]  David Kleinfeld,et al.  In Vivo Measurements of Blood Flow and Glial Cell Function with Two-Photon Laser-Scanning Microscopy , 2008 .

[19]  S. Ly,et al.  Time-gated single photon counting enables separation of CARS microscopy data from multiphoton-excited tissue autofluorescence. , 2007, Optics express.

[20]  Tyson N. Kim,et al.  Spherical aberration correction in nonlinear microscopy and optical ablation using a transparent deformable membrane , 2007 .

[21]  T. Sejnowski,et al.  A Compact Multiphoton 3D Imaging System for Recording Fast Neuronal Activity , 2007, PloS one.

[22]  J. Squier,et al.  Simultaneous imaging of multiple focal planes using a two-photon scanning microscope. , 2007, Optics letters.

[23]  David Kleinfeld,et al.  Penetrating arterioles are a bottleneck in the perfusion of neocortex , 2007, Proceedings of the National Academy of Sciences.

[24]  Klaus Suhling,et al.  Time-resolved fluorescence microscopy , 2007, SPIE Optics East.

[25]  W. Denk,et al.  Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing , 2006, Proceedings of the National Academy of Sciences.

[26]  David Kleinfeld,et al.  MPScope: A versatile software suite for multiphoton microscopy , 2006, Journal of Neuroscience Methods.

[27]  Karel Svoboda,et al.  Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging , 2006, Nature Neuroscience.

[28]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[29]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[30]  P. So,et al.  Single-Photon Counting Multicolor Multiphoton Fluorescence Microscope , 2005, Journal of Fluorescence.

[31]  A. Fabre,et al.  Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy , 2005, Nature Methods.

[32]  Matt Wachowiak,et al.  In Vivo Imaging of Neuronal Activity by Targeted Expression of a Genetically Encoded Probe in the Mouse , 2004, Neuron.

[33]  T. Dalkara,et al.  Intravenously administered propidium iodide labels necrotic cells in the intact mouse brain after injury , 2003, Cell Death and Differentiation.

[34]  T. Jovin,et al.  Spectrally Resolved Fluorescence Lifetime Imaging Microscopy , 2002 .

[35]  L M Loew,et al.  Second-harmonic imaging microscopy of living cells. , 2001, Journal of biomedical optics.

[36]  H. Gerritsen,et al.  Multiple Time-Gate Module for Fluorescence Lifetime Imaging , 2001 .

[37]  E. Neher,et al.  Highly nonlinear photodamage in two-photon fluorescence microscopy. , 2001, Biophysical journal.

[38]  J Mertz,et al.  Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy. , 2000, Optics letters.

[39]  E Neher,et al.  Fast scanning and efficient photodetection in a simple two-photon microscope , 1999, Journal of Neuroscience Methods.

[40]  J. Steinkamp,et al.  Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry. , 1999, Journal of immunological methods.

[41]  Rainer Pepperkok,et al.  Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy , 1999, Current Biology.

[42]  K R Wilson,et al.  Third harmonic generation microscopy. , 1998, Optics express.

[43]  K. Svoboda,et al.  Photon Upmanship: Why Multiphoton Imaging Is More than a Gimmick , 1997, Neuron.

[44]  D. Kleinfeld,et al.  In vivo dendritic calcium dynamics in neocortical pyramidal neurons , 1997, Nature.

[45]  P. So,et al.  Two‐photon excited lifetime imaging of autofluorescence in cells during UV A and NIR photostress , 1996, Journal of microscopy.

[46]  B. Ulfhake,et al.  Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope. , 1995, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[47]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[48]  David M. Coleman,et al.  Time-Resolved Fluorescence Microscopy Using Multichannel Photon Counting , 1990 .

[49]  P. B. Coates,et al.  The correction for photon `pile-up' in the measurement of radiative lifetimes , 1968 .

[50]  M. Kendall.  Theoretical Statistics , 1956, Nature.

[51]  E. L. Lehmann,et al.  Theory of point estimation , 1950 .

[52]  W. Shockley,et al.  A Theory of Noise for Electron Multipliers , 1938, Proceedings of the Institute of Radio Engineers.

[53]  G. A. Morton,et al.  The Secondary Emission Multiplier-A New Electronic Device , 1936, Proceedings of the Institute of Radio Engineers.