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A custom-made two-photon microscope and deconvolution system.

A Majewska1, G Yiu, R Yuste

  • 1Department of Biological Sciences, Columbia University, New York, NY 10027, USA. akm21@columbia.edu

Pflugers Archiv : European Journal of Physiology
|February 24, 2001
PubMed
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We developed a custom two-photon microscope for deep imaging in scattering samples. This versatile system supports quantitative measurements with various fluorophores and advanced applications like photobleaching recovery.

Area of Science:

  • Biophysics
  • Microscopy
  • Optical Engineering

Background:

  • Advanced microscopy techniques are crucial for visualizing biological structures in scattering samples.
  • Existing systems may have limitations in depth penetration, fluorophore compatibility, and quantitative analysis.

Purpose of the Study:

  • To detail the design and capabilities of a custom-built two-photon microscope.
  • To demonstrate its utility for quantitative measurements in deep, scattering biological samples.

Main Methods:

  • Utilized a modified confocal scanhead (Olympus Fluoview) and a mode-locked Ti:sapphire laser (Coherent Mira 900).
  • Integrated internal detectors, external whole-field detection, and an electro-optical modulator for beam control.
  • Employed the Estimation Maximization algorithm with XCOSM software for image deconvolution.

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Main Results:

  • The custom two-photon microscope enables deep imaging in scattering samples.
  • Demonstrated quantitative measurements using a wide range of fluorophores (e.g., GFP, fura, fluo-3, rhodamine).
  • Successfully applied the system for fluorescent photobleaching recovery and uncaging experiments.

Conclusions:

  • The described two-photon microscope is a versatile tool for advanced biological imaging.
  • Its capabilities extend to quantitative analysis, photobleaching studies, and uncaging in challenging sample types.
  • The system's design and accompanying deconvolution software (XCOSM) facilitate high-quality imaging in deep scattering tissues.