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

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• 作者：P. Urayama ; W. Zhong ; J.A. Beamish ; F.K. Minn ; R.D. Sloboda ; K.H. Dragnev ; E. Dmitrovsky and M.-A. Mycek
• 关键词：PACS ; 87.64. ; t ; 87.57. ; s
• 刊名：Applied Physics B: Lasers and Optics
• 出版年：2003
• 出版时间：May 2003
• 年：2003
• 卷：76
• 期：5
• 页码：483-496
• 全文大小：2,341 KB
• 刊物类别：Physics and Astronomy
• 刊物主题：Physics
  Electromagnetism, Optics and Lasers
  Physical Chemistry
  Laser Technology and Physics and Photonics
  Quantum Optics, Quantum Electronics and Nonlinear Optics
  Optical Spectroscopy and Ultrafast Optics
  Physics and Applied Physics in Engineering
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0649

文摘

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).