Scanning algorithms in high-sensitivity two-photon excitation microscopy for single-molecule investigations

Giuseppe Chirico; Gabriele Malengo; Roberto Milani; Fabio Cannone; Silke Krol; Alberto Diaspro

doi:10.1117/12.537915

21 June 2004 Scanning algorithms in high-sensitivity two-photon excitation microscopy for single-molecule investigations

Giuseppe Chirico, Gabriele Malengo, Roberto Milani, Fabio Cannone, Silke Krol, Alberto Diaspro

Author Affiliations +

Proceedings Volume 5323, Multiphoton Microscopy in the Biomedical Sciences IV; (2004) https://doi.org/10.1117/12.537915
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

Abstract

We describe and apply two raster scanning algorithms that are suited for fast imaging and for spectroscopy in a two photon microscope. Imaging can be performed at a rate of 1-100 Hz per line with a closed loop piezo-actuator. In order to reach the single molecule sensitivity and to study the dynamics of the fluorescence emission, the detection is performed via avalanche photodiodes. In a slow scanning algorithm we have implemented photon counting histogram and lifetime analysis on the image. In this way we are able to discriminate between local concentration and molecular brightness and to measure lifetimes on extended samples.

Citation Download Citation

Giuseppe Chirico, Gabriele Malengo, Roberto Milani, Fabio Cannone, Silke Krol, and Alberto Diaspro "Scanning algorithms in high-sensitivity two-photon excitation microscopy for single-molecule investigations", Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); https://doi.org/10.1117/12.537915

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KEYWORDS

Luminescence

Molecules

Imaging spectroscopy

Avalanche photodetectors

Analog electronics

Spectroscopy

Confocal microscopy

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