Two-channel fluorescence lifetime microscope with two colour laser excitation, single-molecule sensitivity, and submicrometer resolution

We present results from a two channel confocal microscope set-up allowing one to efficiently record two-colour as well as polarization resolved time-correlated single molecule fluorescence data. In addition to their spectral characteristics, single molecules can be distinguished by their fluorescence lifetime and polarization. This provides independent distinctive information and results in enhanced detection sensitivity. The set-up we present uses two picosecond diode lasers (440nm and 635 nm) for fluorescence excitation and a piezo scanner for sample movement. A learning scan algorithm permits very fast piezo scanner movement and offers a superior positioning accuracy on single molecules. The time-correlated photon counting system uses Time-Tagged Time-Resolved (TTTR) data aquisition, in which each photon is recorded individually. This method allows for the reconstruction not only fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function on a single spot of interest. Cross-correlation between two channels can be used to eliminate detector artifacts. Finally, fluorescence antibunching can also be analyzed. We show results obtained with immobilized and diffusing red and blue excited fluorescently labelled latex microspheres, as well as from single fluorophore molecules.

[1]  Joerg Enderlein,et al.  High-speed electronics for the detection of time-resolved fluorescence in a continuous flow system , 1996, Photonics West.

[2]  D S Elson,et al.  Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser. , 2002, Optics letters.

[3]  Richard A. Keller,et al.  Ultrasensitive detection of single molecules in flowing sample streams by laser-induced fluorescence , 1993, Photonics West - Lasers and Applications in Science and Engineering.

[4]  W. P. Ambrose,et al.  A maximum likelihood estimator to distinguish single molecules by their fluorescence decays , 1997 .

[5]  M Dahan,et al.  Ratiometric single-molecule studies of freely diffusing biomolecules. , 2001, Annual review of physical chemistry.

[6]  Steven A. Soper,et al.  An all solid‐state near‐infrared time‐correlated single photon counting instrument for dynamic lifetime measurements in DNA sequencing applications , 1996 .

[7]  T. Rahkonen,et al.  ECL and CMOS ASICs for time-to-digital conversion , 1989, Proceedings., Second Annual IEEE ASIC Seminar and Exhibit,.

[8]  Jan Siegel,et al.  Wide-field fluorescence lifetime imaging with optical sectioning and spectral resolution applied to biological samples , 2002 .

[9]  Michael Wahl,et al.  Hardware solution for continuous time-resolved burst detection of single molecules in flow , 1998, Photonics West - Biomedical Optics.

[10]  Rainer Erdmann,et al.  Time-resolved confocal scanning device for ultrasensitive fluorescence detection , 2001 .

[11]  R. Erdmann,et al.  Simple Near-Infrared Time-Correlated Single Photon Counting Instrument with a Pulsed Diode Laser and Avalanche Photodiode for Time-Resolved Measurements in Scanning Applications , 1999 .

[12]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[13]  Richard A. Keller,et al.  Analysis of fluorescence lifetime data for single rhodamine molecules in flowing sample streams , 1994 .

[14]  Joerg Enderlein,et al.  Lifetime-based identification of single molecules , 1995, Photonics West.

[15]  P ? ? ? ? ? ? ? % ? ? ? ? , 1991 .

[16]  Joerg Enderlein,et al.  All-solid-state TCPC instrument for dynamic lifetime measurements in sensitive DNA analysis , 1997, Photonics West - Biomedical Optics.

[17]  D. V. Vanden Bout,et al.  Fluorescence lifetime imaging with near-field scanning optical microscopy. , 2001, Analytical chemistry.

[18]  S. Lane,et al.  Controlled non-classical photon emission from single conjugated polymer molecules , 2003 .

[19]  Rainer Erdmann,et al.  Time-resolved fluorescence correlation spectroscopy , 2002 .

[20]  M. Sauer,et al.  Photophysical Dynamics of Single Molecules Studied by Spectrally-Resolved Fluorescence Lifetime Imaging Microscopy (SFLIM) , 2001 .

[21]  M. Sauer,et al.  Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting , 2000 .

[22]  W. P. Ambrose,et al.  Progress towards DNA sequencing at the single molecule level , 1995 .