Fluorescence lifetime imaging FLIM is very demanding from a technical and computational perspective, and the output is usually a compromise between acquisition/processing time and data accuracy and precision. We present a new approach to acquisition, analysis, and reconstruction of microscopic FLIM images by employing a digital micromirror device DMD as a spatial illuminator. In the first step, the whole field fluorescence image is collected by a color charge-coupled device CCD camera. Further qualitative spectral analysis and sample segmentation are performed to spatially distinguish between spectrally different regions on the sample. Next, the fluorescence of the sample is excited segment by segment, and fluorescence lifetimes are acquired with a photon counting technique. FLIM image reconstruction is performed by either raster scanning the sample or by directly accessing specific regions of interest. The unique features of the DMD illuminator allow the rapid on-line measurement of global good initial parameters GIP, which are supplied to the first iteration of the fitting algorithm. As a consequence, a decrease of the computation time required to obtain a satisfactory quality-of-fit is achieved without compromising the accuracy and precision of the lifetime measurements

BEDNARKIEWICZ Artur;  WHELAN Maurice; 

2009-01-30

SPIE-INT SOCIETY OPTICAL ENGINEERING

JRC47645

1083-3668,   

https://publications.jrc.ec.europa.eu/repository/handle/JRC47645,