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Proceedings Paper

Confocal fluorescence microscopy using intensity-modulated multiple-wavelength scanning (IMS): evaluation of results from spectral and lifetime imaging
Author(s): Kjell Carlsson; Anders Liljeborg
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Paper Abstract

In this paper we summarize the imaging possibilities and performance offered by the Intensity-modulated Multiple- wavelength Scanning (IMS) technique. The IMS technique is based on intensity-modulated laser illumination of the specimen in combination with lock-in detection of the fluorescent light. We have used this technique with multiple illumination wavelengths, spectral separation of the fluorescent light, and multiple detectors. It is then possible to substantially extend the imaging possibilities of confocal fluorescence microscopy. Among the new possibilities are: 1) improved spectral separation when recording the light intensities of multiple fluorophores, 2) separation of intensity signals from multiple fluorophores based on lifetime differences, 3) simultaneous lifetime recording of multiple fluorophores. We have investigated how different system parameters influence the channel separation and signal-to-noise ratio (SNR). In order to maximize the SNR, factors such as fluorophore lifetimes and modulation frequency of the laser light are important. In our investigations we have obtained good agreement between theoretical and experimental results. In summary, our results show that when using the IMS technique to scan multiple-fluorophore specimens, very high channel separation can be obtained for both lifetime and intensity images. For lifetime images, however, the SNR is worse by a factor of approximately four compared with intensity images.

Paper Details

Date Published: 9 June 1998
PDF: 7 pages
Proc. SPIE 3261, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing V, (9 June 1998); doi: 10.1117/12.310534
Show Author Affiliations
Kjell Carlsson, Royal Institute of Technology (Sweden)
Anders Liljeborg, Royal Institute of Technology and Karolinska Institute (Sweden)


Published in SPIE Proceedings Vol. 3261:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing V
Thomas Taiwei Lu; Jeremy M. Lerner; Carol J. Cogswell; Jose-Angel Conchello; Jeremy M. Lerner; Thomas Taiwei Lu; Tony Wilson, Editor(s)

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