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

• Published in: Applied physics. B, Lasers and optics Vol. 76; no. 5; pp. 483 - 496
• Main Authors: URAYAMA, P, ZHONG, W, BEAMISH, J. A, MINN, F. K, SLOBODA, R. D, DRAGNEV, K. H, DMITROVSKY, E, MYCEK, M.-A
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01.05.2003
• Subjects: Biological; Biological and medical applications; Biological and medical sciences; Decay; Delay; Dynamical systems; Dynamics; Fluorescence; Fundamental and applied biological sciences. Psychology; General aspects; General aspects, investigation technics, apparatus; Imaging; Instrumentation. Materials. Reagents. Research laboratory organization; Lasers; Medical imaging: general; Spectroscopic and microscopic techniques in biophysics and medical physics; Tissues, organs and organisms biophysics; Human; Time resolution; Lifetime; Endogenous; Instrumentation; Medical imagery; Cell; Time response; Spatial resolution; Fluorescence microscopy; ps range; Luminescence decay
• ISSN: 0946-2171; 1432-0649
• DOI: 10.1007/s00340-003-1152-4

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 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-*mm 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).