Confocal fluorescence microscopy using intensity-modulated multiple-wavelength scanning (IMS): evaluation of results from spectral and lifetime imaging

Kjell Carlsson; Anders Liljeborg

doi:10.1117/12.310534

9 June 1998 Confocal fluorescence microscopy using intensity-modulated multiple-wavelength scanning (IMS): evaluation of results from spectral and lifetime imaging

Kjell Carlsson, Anders Liljeborg

Author Affiliations +

Proceedings Volume 3261, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing V; (1998) https://doi.org/10.1117/12.310534
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

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.

Citation Download Citation

Kjell Carlsson and Anders Liljeborg "Confocal fluorescence microscopy using intensity-modulated multiple-wavelength scanning (IMS): evaluation of results from spectral and lifetime imaging", Proc. SPIE 3261, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing V, (9 June 1998); https://doi.org/10.1117/12.310534

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