Applied Physics B

, Volume 76, Issue 5, pp 483–496

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

Authors

  • P. Urayama
    • Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
  • W. Zhong
    • Dept. of Physics and Astronomy, Dartmouth College, Hanover, NH 03755, USA
  • J.A. Beamish
    • Dept. of Physics and Astronomy, Dartmouth College, Hanover, NH 03755, USA
  • F.K. Minn
    • Dept. of Physics and Astronomy, Dartmouth College, Hanover, NH 03755, USA
  • R.D. Sloboda
    • Dept. of Biological Science, Dartmouth College, Hanover, NH 03755, USA
  • K.H. Dragnev
    • Dept. of Medicine, Dartmouth Medical School, Hanover, NH 03755, USA
  • E. Dmitrovsky
    • Dept. of Medicine, Dartmouth Medical School, Hanover, NH 03755, USA
  • M.-A. Mycek
    • Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
Invited paper

DOI: 10.1007/s00340-003-1152-4

Cite this article as:
Urayama, P., Zhong, W., Beamish, J. et al. Appl Phys B (2003) 76: 483. doi:10.1007/s00340-003-1152-4

Abstract.

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns).

PACS: 87.64.-t; 87.57.-s

Copyright information

© Springer-Verlag 2003