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”.

© 2007 SPIE