Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

Michael Wahl; Hans-Juergen Rahn; Uwe Ortmann; Rainer Erdmann; Martin Boehmer; Joerg Enderlein

doi:10.1117/12.463829

28 March 2002 Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

Michael Wahl, Hans-Juergen Rahn, Uwe Ortmann, Rainer Erdmann, Martin Boehmer, Joerg Enderlein

Proceedings Volume 4634, Methods for Ultrasensitive Detection II; (2002) https://doi.org/10.1117/12.463829
Event: High-Power Lasers and Applications, 2002, San Jose, California, United States

Abstract

Fluorescence lifetime measurement of organic fluorophores is a powerful tool for distinguishing molecules of interest from background or other species. This is of interest in sensitive analysis and Single Molecule Detection (SMD). A demand in many applications is to provide 2-D imaging together with lifetime information. The method of choice is then Time-Correlated Single Photon Counting (TCSPC). We have devloped a compact system on a single PC board that can perform TCSPC at high throughput, while synchronously driving a piezo scanner holding the immobilized sample. The system allows count rates up to 3 MHz and a resolution down to 30 ps. An overall Instrument Response Function down to 300ps is achieved with inexpensive detectors and diode lasers. The board is designed for the PCI bus, permitting high throughput without loss of counts. It is reconfigurable to operate in different modes. The Time-Tagged Time-Resolved (TTTR) mode permits the recording of all photon events with a real-time tag allowing data analysis with unlimited flexibility. We use the Time-Tag clock for an external piezo scanner that moves the sample. As the clock source is common for scanning and tagging, the individual photons can be matched to pixels. Demonstrating the capablities of the system we studied single molecule solutions. Lifetime imaging can be performed at high resolution with as few as 100 photons per pixel.

Citation Download Citation

Michael Wahl, Hans-Juergen Rahn, Uwe Ortmann, Rainer Erdmann, Martin Boehmer, and Joerg Enderlein "Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity", Proc. SPIE 4634, Methods for Ultrasensitive Detection II, (28 March 2002); https://doi.org/10.1117/12.463829

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