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In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy
Published on: December 20, 2021
Enhancing the resolution of the forward second harmonic imaging using the two-photon laser scanning microscope
1Institute of Physical Biology, University of South Bohemia, Nove Hrady 37333, Czech Republic. maalidph@yahoo.co.uk
Micron (Oxford, England : 1993)
|June 9, 2009
Summary
Researchers enhanced forward second harmonic imaging of chloroplasts in moss leaves by optimizing objective lenses. This technique improves visualization of plant cellular structures using advanced optical methods.
Area of Science:
- Plant Biology
- Microscopy
- Optics
Background:
- Chloroplasts, the sites of photosynthesis in plants, have internal structures like grana.
- Second harmonic generation (SHG) microscopy is a label-free imaging technique capable of visualizing biological structures.
- Understanding plant cellular morphology is crucial for plant science research.
Purpose of the Study:
- To demonstrate the enhancement of forward second harmonic (FSH) imaging of chloroplasts in moss leaves.
- To investigate the impact of objective numerical aperture (NA) and immersion medium on FSH signals.
- To simultaneously record forward and backward SHG signals for comprehensive analysis.
Main Methods:
- Utilized a femtosecond pulsed laser with linear polarization for SHG signal generation.
- Employed high numerical aperture (NA) objectives with varying immersion media (e.g., water).
- Focused on imaging the grana and intergranal regions within chloroplasts of the shade moss *Plagiomnium affine*.
Main Results:
- Demonstrated successful enhancement of FSH images by increasing objective NA and changing immersion medium.
- Showcased improved visualization of grana and intergranal structures within chloroplasts.
- Successfully recorded both forward and backward SHG signals concurrently.
Conclusions:
- Forward second harmonic signals were significantly enhanced by capturing higher-order diffraction rays.
- High numerical aperture water immersion objectives proved effective in improving FSH signal intensity.
- The study highlights the potential of optimized SHG microscopy for plant cellular imaging.
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