System for fluorescence lifetime imaging microscopy

Lambertus K. van Geest; Frank R. Boddeke; Pieter W. van Dijk; Arjen F. Kamp; Cornelius J. R. van der Oord; Karel W. J. Stoop

doi:10.1117/12.347590

6 May 1999 System for fluorescence lifetime imaging microscopy

Lambertus K. van Geest, Frank R. Boddeke, Pieter W. van Dijk, Arjen F. Kamp, Cornelius J. R. van der Oord, Karel W. J. Stoop

Author Affiliations +

Proceedings Volume 3605, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VI; (1999) https://doi.org/10.1117/12.347590
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

One of the promising recent developments in fluorescence microscopy is fluorescence lifetime imaging microscopy (FLIM). In this technique the fluorescence lifetime (ns range) of molecules is expressed in the image rather than the intensity of the light emitted by these molecules. This physical property is of interest as it gives information about the local environment of the molecule, such as molecular concentration of O₂, Ca²⁺, pH, and conjugation. We develop an affordable, robust and easy-to-use FLIM workstation which is completely automated and does not need any difficult calibration procedure. The system consists of a standard fluorescence microscope, a modulated excitation light source, a camera, a modulation signal generator and acquisition/processing software. The camera contains an Intensified CCD of which the image intensifier gain is modulated. Depending on the application different light sources can be selected. The current light source contains a 12 mW modulated laser-diode emitting at 635 nm. A homodyne detection scheme with modulation frequencies of 1 to 100 MHz is applied, aiming at a resolution of 0.1 ns or better. High level image acquisition strategies are implemented in software, along with the low level image processing routines for lifetime estimation, calibration and correction. An evaluation of the system and its critical components will be presented in this paper.

Citation Download Citation

Lambertus K. van Geest, Frank R. Boddeke, Pieter W. van Dijk, Arjen F. Kamp, Cornelius J. R. van der Oord, and Karel W. J. Stoop "System for fluorescence lifetime imaging microscopy", Proc. SPIE 3605, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VI, (6 May 1999); https://doi.org/10.1117/12.347590

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