21 June 2004 Scanning algorithms in high-sensitivity two-photon excitation microscopy for single-molecule investigations
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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.
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Giuseppe Chirico, Giuseppe Chirico, Gabriele Malengo, Gabriele Malengo, Roberto Milani, Roberto Milani, Fabio Cannone, Fabio Cannone, Silke Krol, Silke Krol, Alberto Diaspro, 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); doi: 10.1117/12.537915; https://doi.org/10.1117/12.537915
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