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Reduction of the pulse duration of the ultrafast laser pulses of the Two-Photon Laser Scanning Microscopy (2PLSM).

Reshak AH - BMC Res Notes (2008)

Bottom Line: 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).In summary reduction the laser pulse width allowed to collect more diffraction orders which will used to form the images.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Physical Biology-South Bohemia University, Institute of System Biology and Ecology-Academy of Sciences - Nove Hrady 37333, Czech Republic. maalidph@yahoo.co.uk

ABSTRACT

Background: 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.

Findings: The 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).

Conclusion: In 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.

No MeSH data available.


Related in: MedlinePlus

The structure of the leaf using modified 2PLSM.
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Figure 3: The structure of the leaf using modified 2PLSM.

Mentions: Figure 2, 3, and the additional files 1 and 2, show the leaf structure of duckweed Lemna minuta excited by laser beam without and with using GL polarizer. In Figure 3 and the additional file 2, 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 going though the leaf from the dorsal surface to ventral surface in comparison to the exactly the same measurements without using GL polarizer. In this case we can see the structure of the leaf (which we saw it in the first slices with using GL polarizer) approximately after 15 slices, i.e we have to go deeply inside the sample about 7.5 μm (separation between the slices is 0.5 μm) before using the 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. If we look at Figure 2 and the additional file 1, we note that without using the GL polarizer the images will be blurred and we need more focusing to get clearer images resulting to exposure the sample more time to the laser radiation. This improvement over the normal 2PLSM will enable us to get quick focus through the sample with high resolution and give us more opportunity to go deeply in side the tissue to see clearer details structures with less damage to the living cells. We provided two movies (additional files 1 and 2) which show the leaf structures with and without using GL polarizer. These movies clearly show the resolution enhancement with GL polarizer.


Reduction of the pulse duration of the ultrafast laser pulses of the Two-Photon Laser Scanning Microscopy (2PLSM).

Reshak AH - BMC Res Notes (2008)

The structure of the leaf using modified 2PLSM.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2519073&req=5

Figure 3: The structure of the leaf using modified 2PLSM.
Mentions: Figure 2, 3, and the additional files 1 and 2, show the leaf structure of duckweed Lemna minuta excited by laser beam without and with using GL polarizer. In Figure 3 and the additional file 2, 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 going though the leaf from the dorsal surface to ventral surface in comparison to the exactly the same measurements without using GL polarizer. In this case we can see the structure of the leaf (which we saw it in the first slices with using GL polarizer) approximately after 15 slices, i.e we have to go deeply inside the sample about 7.5 μm (separation between the slices is 0.5 μm) before using the 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. If we look at Figure 2 and the additional file 1, we note that without using the GL polarizer the images will be blurred and we need more focusing to get clearer images resulting to exposure the sample more time to the laser radiation. This improvement over the normal 2PLSM will enable us to get quick focus through the sample with high resolution and give us more opportunity to go deeply in side the tissue to see clearer details structures with less damage to the living cells. We provided two movies (additional files 1 and 2) which show the leaf structures with and without using GL polarizer. These movies clearly show the resolution enhancement with GL polarizer.

Bottom Line: 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).In summary reduction the laser pulse width allowed to collect more diffraction orders which will used to form the images.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Physical Biology-South Bohemia University, Institute of System Biology and Ecology-Academy of Sciences - Nove Hrady 37333, Czech Republic. maalidph@yahoo.co.uk

ABSTRACT

Background: 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.

Findings: The 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).

Conclusion: In 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.

No MeSH data available.


Related in: MedlinePlus