Signal-to-noise characterization of time-gated intensifiers used for wide-field time-domain FLIM

Time-gated imaging using gated optical intensifiers provides a means to realize high speed fluorescence lifetime imaging (FLIM) for the study of fast events and for high throughput imaging. We present a signal-to-noise characterization of CCD-coupled micro-channel plate gated intensifiers used with this technique and determine the optimal acquisition parameters (intensifier gain voltage, CCD integration time and frame averaging) for measuring mono-exponential fluorescence lifetimes in the shortest image acquisition time for a given signal flux. We explore the use of unequal CCD integration times for different gate delays and show that this can improve the lifetime accuracy for a given total acquisition time.

[1]  M A A Neil,et al.  Optically sectioned fluorescence lifetime imaging using a Nipkow disk microscope and a tunable ultrafast continuum excitation source. , 2005, Optics letters.

[2]  E. V. van Munster,et al.  Fluorescence lifetime imaging microscopy (FLIM). , 2005, Advances in biochemical engineering/biotechnology.

[3]  Fred S Wouters,et al.  Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening *S , 2007, Molecular & Cellular Proteomics.

[4]  Hans C. Gerritsen,et al.  Fluorescence Lifetime Imaging, a New Tool in Confocal Microscopy , 1995 .

[5]  Klaus Benndorf,et al.  FRET between cardiac Na+ channel subunits measured with a confocal microscope and a streak camera , 2004, Nature Biotechnology.

[6]  A. deMello,et al.  Time-resolved fluorescence imaging of solvent interactions in microfluidic devices. , 2005, Optics express.

[7]  J. D. Hares,et al.  2-D Fluorescence Lifetime Imaging using a Time-Gated Image Intensifier , 1997, Advances in Optical Imaging and Photon Migration.

[8]  F. Wouters,et al.  All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing. , 2005, Optics express.

[9]  Klaus Suhling,et al.  Refractive index sensing using Fluorescence Lifetime Imaging (FLIM) , 2006 .

[10]  R. Malina,et al.  Investigation of Large Format Microchannel Plate Z Configurations , 1985, IEEE Transactions on Nuclear Science.

[11]  C. Harris,et al.  Single-Photon Decay Spectroscopy. II. The Pile-up Problem , 1979 .

[12]  H. Gerritsen,et al.  Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution , 2002, Journal of microscopy.

[13]  Enrico Gratton,et al.  A novel fluorescence lifetime imaging system that optimizes photon efficiency , 2008, Microscopy research and technique.

[14]  T Wilson,et al.  Low‐cost, frequency‐domain, fluorescence lifetime confocal microscopy , 2004, Journal of microscopy.

[15]  John White,et al.  Simultaneous two-photon spectral and lifetime fluorescence microscopy. , 2004, Applied optics.

[16]  P. Bastiaens,et al.  Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. , 1999, Trends in cell biology.

[17]  M. Neil,et al.  Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier , 2004 .

[18]  Dylan M Owen,et al.  Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source. , 2007, Optics letters.

[19]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale , 1993 .

[20]  Govindjee,et al.  Fluorescence Lifetime Imaging (FLI) in Real-Time - a New Technique in Photosynthesis Research , 2000, Photosynthetica.

[21]  Brian Herman,et al.  Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera , 2003 .

[22]  R. Clegg.  Fluorescence resonance energy transfer. , 1995, Current opinion in biotechnology.

[23]  Hans C. Gerritsen,et al.  High frame rate fluorescence lifetime imaging , 2003 .

[24]  M. Gersbach,et al.  A 128 $\times$ 128 Single-Photon Image Sensor With Column-Level 10-Bit Time-to-Digital Converter Array , 2008, IEEE Journal of Solid-State Circuits.

[25]  P. French,et al.  Rapid hyperspectral fluorescence lifetime imaging , 2007, Microscopy research and technique.

[26]  A. W. Parker,et al.  Development of a laser-based fluorescence microscope with subnanosecond time resolution , 1996, Journal of Fluorescence.

[27]  J D Hares,et al.  Fluorescence lifetime imaging with picosecond resolution for biomedical applications. , 1998, Optics letters.

[28]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[29]  P. French,et al.  Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging. , 2007, Optics express.

[30]  C G Morgan,et al.  Direct modulation of the effective sensitivity of a CCD detector: a new approach to time‐resolved fluorescence imaging , 2002, Journal of microscopy.

[31]  E. Eberhardt,et al.  Gain model for microchannel plates. , 1979, Applied optics.

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

[33]  R. Ballew,et al.  An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays , 1989 .

[34]  P. French,et al.  High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events. , 2007, Optics express.

[35]  H Szmacinski,et al.  Fluorescence lifetime imaging. , 1992, Analytical biochemistry.

[36]  A. deMello,et al.  Quantitative 3D mapping of fluidic temperatures within microchannel networks using fluorescence lifetime imaging. , 2006, Analytical Chemistry.

[37]  P. R. Bevington,et al.  Data Reduction and Error Analysis for the Physical Sciences , 1969 .

[38]  Klaus Suhling,et al.  Wide-field photon counting imaging for fluorescence microscopy , 2007, SPIE Optics East.

[39]  M A A Neil,et al.  High-speed wide-field time-gated endoscopic fluorescence-lifetime imaging. , 2004, Optics letters.

[40]  V. Subramaniam,et al.  Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM). , 2002, Biophysical journal.

[41]  M A A Neil,et al.  Toward the clinical application of time-domain fluorescence lifetime imaging. , 2005, Journal of biomedical optics.

[42]  Ewan J McGhee,et al.  High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis , 2008, Journal of biophotonics.

[43]  Georges Wagnières,et al.  Instrumentation for real-time fluorescence lifetime imaging in endoscopy , 1999 .

[44]  J. Siegel,et al.  Wide-Field Time-Resolved Fluorescence Anisotropy Imaging (TR-FAIM) , 2003 .

[45]  P. So,et al.  Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy. , 2000, Biophysical journal.

[46]  Supriya Chakrabarti,et al.  A position sensitive detector for EUV remote sensing , 1989 .