Low‐cost, frequency‐domain, fluorescence lifetime confocal microscopy

We describe the theory and implementation of a frequency‐domain fluorescence lifetime confocal microscope using switched diode laser illumination. Standard, communications‐type, radio‐frequency electronics are used to provide inexpensive modulation references and to perform phase‐sensitive detection. This allows the rapid acquisition of fluorescence intensity and lifetime images and their display in real time. We show fluorescence lifetime images of bead objects and fluorescence lifetime images of biological specimens from a single confocal scan.

[1]  T. Wilson,et al.  Real time 3D fluorescence microscopy by two beam interference illumination , 1998 .

[2]  Valentina Emiliani,et al.  Low-intensity two-dimensional imaging of fluorescence lifetimes in living cells , 2003 .

[3]  M. Kozubek,et al.  Efficient real-time confocal microscopy with white light sources , 1996, Nature.

[4]  Kjell Carlsson,et al.  Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation , 1997 .

[5]  Gary M. Carter,et al.  Low cost phase-modulation measurements of nanosecond fluorescence lifetimes using a lock-in amplifier , 1999 .

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

[7]  Robert M. Clegg,et al.  Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications , 1997 .

[8]  S W Hell,et al.  Four-dimensional multiphoton microscopy with time-correlated single-photon counting. , 2000, Applied optics.

[9]  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.

[10]  Jürgen Wolfrum,et al.  Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC) , 1999 .

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

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

[13]  T. Jovin,et al.  Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression. , 2001, Cytometry.

[14]  T Wilson,et al.  Whole-field optically sectioned fluorescence lifetime imaging. , 2000, Optics letters.