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Proceedings Paper

Microscopic fluorescence lifetime and hyperspectral imaging with digital micromirror illuminator
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Paper Abstract

New approach to acquisition, analysis and reconstruction of Microscopic Fluorescence Lifetime Images (FLIM) and Hyper Spectral Images (HSI) is presented. Spatial selectivity is obtained with a Digital micro-Mirror Device Illuminator (DMDI) combined with a fluorescence microscope. More specifically spatially selective illumination is achieved by tilting the relevant group of micro-mirrors to reflect the excitation light from a UV picosecond laser diode towards chosen regions on the sample. In the first step, the whole field fluorescence image is collected by a color CCD camera for further qualitative spectral analysis and sample segmentation. In the next step fluorescence of the sample is excited segment by segment and acquired with a single detector (e.g. photomultiplier in photon counting mode for FLIM, CCD spectrophotometer for HSI) from the whole field of view. The acquired fluorescence is analyzed in following step for further FLIM or HSI image reconstruction. This can be facilitated by either raster scanning over the sample or by directly accessing specific regions of interest. The unique features of the DMD illuminator allow to Globally Analyze (GA) the sample and supply on-line good initial values for fitting algorithms associated with the subsequent raster-scanning, which in turn decreases the computation time needed to obtain a satisfactory quality-of-fit. FLIM/HIS images acquired on phantoms and on biological samples demonstrate the possibilities for temporal and spectral "unmixing".

Paper Details

Date Published: 12 July 2007
PDF: 12 pages
Proc. SPIE 6630, Confocal, Multiphoton, and Nonlinear Microscopic Imaging III, 66300A (12 July 2007); doi: 10.1117/12.728422
Show Author Affiliations
Artur Bednarkiewicz, Institute of Health and Customer Protection, European Joint Research Ctr. (Italy)
Institute for Low Temperature and Structure Research (Poland)
Maurice P. Whelan, Institute of Health and Customer Protection, European Joint Research Ctr. (Italy)

Published in SPIE Proceedings Vol. 6630:
Confocal, Multiphoton, and Nonlinear Microscopic Imaging III
Tony Wilson; Ammasi Periasamy, Editor(s)

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