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

Research output: Chapter in Book/Report/Conference proceedingConference 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 languageEnglish
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
EditorsA. Periasmy, P.T.C. So
Pages319-326
Number of pages8
Volume5323
DOIs
Publication statusPublished - 2004
EventProgress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV - San Jose, CA, United States
Duration: Jan 25 2004Jan 27 2004

Other

OtherProgress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV
CountryUnited States
CitySan Jose, CA
Period1/25/041/27/04

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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