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

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

IRCCS Giovanni Paolo II

Research output: Chapter in Book/Report/Conference proceeding › Conference contribution

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.

Original language	English
Title of host publication	Proceedings of SPIE - The International Society for Optical Engineering
Editors	A. Periasmy, P.T.C. So
Pages	319-326
Number of pages	8
Volume	5323
DOIs	https://doi.org/10.1117/12.537915
Publication status	Published - 2004
Event	Progress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV - San Jose, CA, United States Duration: Jan 25 2004 → Jan 27 2004

Other

Other	Progress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV
Country	United States
City	San Jose, CA
Period	1/25/04 → 1/27/04

Keywords

Fluorescence microscopy
Intensity distributions
Photon counting histogram
Single molecules
Two-photon excitation

ASJC Scopus subject areas

Electrical and Electronic Engineering
Condensed Matter Physics

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10.1117/12.537915

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Scanning algorithms in high sensitivity two-photon excitation microscopy for single molecule investigations. / Chirico, Giuseppe; Malengo, Gabriele; Milani, Roberto; Cannone, Fabio; Krol, Silke; Diaspro, Alberto.

Proceedings of SPIE - The International Society for Optical Engineering. ed. / A. Periasmy; P.T.C. So. Vol. 5323 2004. p. 319-326.

Research output: Chapter in Book/Report/Conference proceeding › Conference contribution

Chirico, G, Malengo, G, Milani, R, Cannone, F, Krol, S & Diaspro, A 2004, Scanning algorithms in high sensitivity two-photon excitation microscopy for single molecule investigations. in A Periasmy & PTC So (eds), Proceedings of SPIE - The International Society for Optical Engineering. vol. 5323, pp. 319-326, Progress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV, San Jose, CA, United States, 1/25/04. https://doi.org/10.1117/12.537915

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title = "Scanning algorithms in high sensitivity two-photon excitation microscopy for single molecule investigations",

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.",

keywords = "Fluorescence microscopy, Intensity distributions, Photon counting histogram, Single molecules, Two-photon excitation",

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AU - Chirico, Giuseppe

AU - Malengo, Gabriele

AU - Milani, Roberto

AU - Cannone, Fabio

AU - Krol, Silke

AU - Diaspro, Alberto

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N2 - 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.

AB - 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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KW - Intensity distributions

KW - Photon counting histogram

KW - Single molecules

KW - Two-photon excitation

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