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Epifluorescence microscopy with a pulsed nitrogen tunable dye laser source.

F van Geel, B W Smith, B Nicolaissen

    Journal of Microscopy
    |February 1, 1984
    PubMed
    Summary
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    This study introduces a novel epifluorescence system utilizing a pulsed laser for precise quantitative fluorescence measurements. This advanced system enhances accuracy, especially for low-intensity intrinsic fluorescence, by minimizing errors from excitation light reflection.

    Area of Science:

    • Optical microscopy
    • Spectroscopy
    • Biophysics

    Background:

    • Conventional epifluorescence microscopy often faces challenges with low signal-to-noise ratios.
    • Reflection of excitation light in traditional systems can introduce significant errors in quantitative measurements.
    • Intrinsic fluorescence measurements are particularly susceptible to these limitations.

    Purpose of the Study:

    • To describe a novel epifluorescence system for quantitative fluorescence measurements.
    • To highlight the advantages of using a pulsed laser source over continuum spectral sources.
    • To demonstrate improved accuracy in detecting low-intensity fluorescence signals.

    Main Methods:

    • Development of an epifluorescence system employing a pulsed laser as the excitation source.

    Related Experiment Videos

  • Utilizing a fast detection system for fluorescence measurement.
  • Achieving selective excitation wavelengths from 337 nm and 357 nm to 710 nm using different laser dyes, with a spectral bandwidth of 0.1-3 nm.
  • Illuminating a precise 1-micron diameter portion of the sample with high-intensity, short light pulses.

    • Main Results:

    • The pulsed laser system enables high-intensity, short-pulse illumination of small sample areas (1 micron).
    • Selective excitation across a broad spectral range (337 nm to 710 nm) with narrow bandwidth (0.1-3 nm) is achieved.
    • The system demonstrates significant advantages over continuum sources, particularly for low-intensity intrinsic fluorescence measurements.

    Conclusions:

    • The described epifluorescence system offers superior performance for quantitative fluorescence measurements.
    • Its ability to minimize excitation light reflection errors enhances accuracy, especially for intrinsic fluorescence.
    • This pulsed laser-based approach represents a significant advancement for sensitive fluorescence detection.