Accessing nano- to femtosecond molecular relaxation phenomena for fluorescence imaging through double-pulse saturation excitation

G. J. Brakenhoff; Arjan H. Buist; Michiel Mueller; E. J. Gijsbers; Thomas S. Sosnowski; Theodore B. Norris; Jeffrey A. Squier

doi:10.1117/12.271251

10 April 1997 Accessing nano- to femtosecond molecular relaxation phenomena for fluorescence imaging through double-pulse saturation excitation

G. J. Brakenhoff, Arjan H. Buist, Michiel Mueller, E. J. Gijsbers, Thomas S. Sosnowski, Theodore B. Norris, Jeffrey A. Squier

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Proceedings Volume 2984, Three-Dimensional Microscopy: Image Acquisition and Processing IV; (1997) https://doi.org/10.1117/12.271251
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States

Abstract

It is shown that nanosecond to picosecond fluorescence relaxation phenomena can be accessed for imaging after double pulse saturation excitation. This new technique has been introduced before as fluorescence lifetime imaging (DPFLIm) (Mueller et al, 1995). An OPA laser system generating ultra short, widely tunable, high power optical pulses provides the means for the selective excitation of specific fluorophores at sufficient excitation levels to obtain the necessary (partial) saturation of the optical transition. A key element in the developed method is that the correct determination of fluorescence relaxation times does allow for non-uniform saturation conditions over the observation area. This is true for the validation demonstration experiments reported here as well as for imaging applications at a later stage. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique. As only integrated signal detection is required no fast electronics are needed, making the technique suitable for fluorescence lifetime imaging in confocal microscopy, especially when used in combination with bilateral scanning and cooled CCD detection.

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G. J. Brakenhoff, Arjan H. Buist, Michiel Mueller, E. J. Gijsbers, Thomas S. Sosnowski, Theodore B. Norris, and Jeffrey A. Squier "Accessing nano- to femtosecond molecular relaxation phenomena for fluorescence imaging through double-pulse saturation excitation", Proc. SPIE 2984, Three-Dimensional Microscopy: Image Acquisition and Processing IV, (10 April 1997); https://doi.org/10.1117/12.271251

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KEYWORDS

Luminescence

Molecules

Confocal microscopy

Fluorescence lifetime imaging

Charge-coupled devices

Femtosecond phenomena

Rhodamine B

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