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A custom confocal and two-photon digital laser scanning microscope.

W G Wier1, C W Balke, J A Michael

  • 1Department of Physiology and Medicine, University of Maryland School of Medicine, Baltimore 21201, USA. gwier001@umaryland.edu

American Journal of Physiology. Heart and Circulatory Physiology
|June 14, 2000
PubMed
Summary

We developed a custom laser scanning microscope combining one-photon (confocal) and two-photon excitation. This high-sensitivity system offers improved efficiency and resolution for advanced biological imaging, especially in challenging samples.

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Area of Science:

  • Biophotonics
  • Microscopy
  • Laser Scanning Systems

Background:

  • Confocal and two-photon microscopy are vital for biological research.
  • Existing systems face limitations in sensitivity, photon counting rates, and excitation efficiency.
  • Need for advanced microscopy solutions for low fluorescence and thick scattering specimens.

Purpose of the Study:

  • To design and characterize a versatile, all-digital laser scanning microscope.
  • To integrate both one-photon (confocal) and two-photon excitation capabilities.
  • To enhance sensitivity and efficiency for challenging imaging applications.

Main Methods:

  • Constructed a custom all-digital laser scanning microscope with photon counting detectors.
  • Utilized two avalanche photodiodes (APDs) for enhanced confocal fluorescence detection.

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  • Implemented a single-mirror system for seamless switching between one-photon and two-photon excitation modes (Ti:sapphire laser).

    • Main Results:

    • The confocal component demonstrated ~9x greater efficiency compared to a commercial system using fluo-4.
    • Achieved high resolution: 0.25 µm lateral and 0.52 µm axial for confocal; 0.28 µm lateral and 0.82 µm axial for two-photon.
    • System operates with 109 fs laser pulses and ~5 mW average power in two-photon mode.

    Conclusions:

    • The custom microscope offers superior sensitivity and efficiency for fluorescence detection.
    • The dual-mode system is advantageous for low-fluorescence, thick, or scattering biological specimens.
    • This advanced microscopy platform facilitates high-resolution imaging under demanding conditions.