Optical microscopes have proven their use as a powerful tool for studying a variety of biological samples. In spite of many successes, there are still numerous obstacles limiting practical applications. Most limiting are the inherent background of physiological samples, photobleaching, and phototoxicity. To allow studies of long lasting processes such as drag delivery, three-dimensional cellular structures, embryogenesis, we have combined a technique called Single Plane Illumination Microscopy (SPIM) with Multi-Pulse Pumping with Time-Gated Detection (MPP-TGD) in order to enhance the signal relative to background. This new method allows for a decrease in light exposure times and improves image quality. This combination allows a new outlook into a variety of important, long-lasting biological processes at a level of detection previously unattainable. Multi-pulse pumping is a burst of excitation pulses instead of a single pulse which enhances the excited state population of a long-lived label. This label is chosen so that its lifetime is at least 5 times longer than that of typical autofluorescence. The pulse separation within the burst is chosen so that it is at least 5 times shorter than the lifetime of the label. In this case only the population of the fluorescent label is increased and the background remains the same. By subtracting the image acquired with the burst from an image with a single pulse, we were able to increase the signal-to-background ratio of about 100 fold.

Date Published: 4 March 2019
PDF
Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII, 1088405 (4 March 2019); doi: 10.1117/12.2514298

Published in SPIE Proceedings Vol. 10884:
Single Molecule Spectroscopy and Superresolution Imaging XII
Zygmunt Karol Gryczynski; Ingo Gregor; Felix Koberling, Editor(s)