Abstract
BackgroundWe provide an update of our two-photon laser scanning microscope by compressing or reducing the broadening of the pulse width of ultrafast laser pulses for dispersion precompensation, to enable the pulses to penetrate deeply inside the sample.FindingsThe broadening comes as the pulses pass through the optical elements. We enhanced and modified the quality and the sharpness of images by enhancing the resolution using special polarizer namely Glan Laser polarizer GL10. This polarizer consists of two prisms separated by air space. This air separation between the two prisms uses to delay the red wavelength when the light leaves the first prism to the air then to second prism. We note a considerable enhancing with using the GL polarizer, and we can see the details of the leaf structure in early stages when we trying to get focus through z-stacks of images in comparison to exactly the same measurements without using GL polarizer. Hence, with this modification we able to reduce the time of exposure the sample to the laser radiation thereby we will reduce the probability of photobleaching and phototoxicity. When the pulse width reduced, the average power of the laser pulses maintained at a constant level. Significant enhancement is found between the two kinds of images of the Two-Photon Excitation Fluorescence (TPEF).ConclusionIn summary reduction the laser pulse width allowed to collect more diffraction orders which will used to form the images. The more diffraction orders the higher resolution images.
Highlights
Non-linear optical techniques have been exploited to develop a new generation of optical microscopes with unprecedented capabilities
Genetic variability in responses to toxicants can occur in Lemna, and there are insufficient data to recommend a specific clone for testing
Only 100 mW is really needed from the laser at most wavelengths, because the loss of most laser power in the optical equipments, we will get only 1/3 of the laser average power at the sample
Summary
Non-linear optical techniques have been exploited to develop a new generation of optical microscopes with unprecedented capabilities. These new capabilities include the ability to use near-infrared (IR) light to induce absorption, and fluorescence, from fluorophores that absorb in the ultraviolet wavelength region. Two-photon excitation offers major advantages when working in the thick tissue, such as brain slices or developing embryos, due to the dramatically reduced effects of light scattering. This is partly because the longer red and near-IR wavelengths used for two photon illumination penetrate deeper into biological tissue with less absorption and scattering. We provide an update of our two-photon laser scanning microscope by compressing or reducing the broadening of the pulse width of ultrafast laser pulses for dispersion precompensation, to enable the pulses to penetrate deeply inside the sample
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