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Two-photon Laser Scanning Microscope

Instrument information:

1,Provider: OLYMPUS

 2. Model: FV1000MPE

 3.Charge: 150 Yuan/h


Specifications:

Laser Unit:

1. IR pulsed laser with negative chirp for multiphoto excitation:

    a.Mode-locked Ti: sapphire laser [femtosecond laser (equipped  with a group velocity dispersion correction/control      

       device)]

    b.MaiTai HP DeepSee-OL: 690-1040nm

2. Visible light laser AOFT laser combiner

    a. LD laser: 405nm

    b. LD (R) laser: 635nm

    c.  Multi Ar laser: 458nm, 488nm, 514nm

    d. HeNe(G) laser: 543 nm

    e.Modulation: Continuously adjustable via an AOTF (0.1% to 100% in 0.1% increments)


Scanning Unit

1. Confocal detector

     a. Detector: 3 channels for fluorescence detection (photomultipliers);

     b. Wavelength resolution: 2nm; Wavelength switching speed: 100nm/ms;

     c. Field Number:18.

2. Scanning modes

      a. Pixel size: 64×64—4096×4096 pixels;

      b. Scanning speed: (pixel time): 2μs-200μs;

      c. High-speed scanning mode:16 frames/s (256×256);

      d. Scanning Dimensions: Time, Z-axis, wavelength or their random combination;

      e. Scanning in line: straight line or random line.

Z-drive

A motorize focus module inside the microscope is used

Minimum increment: 10 nm


Microscope

Upright microscopes

Objects: 5×, 10×, 20×, 40×;60×, 100×(Oil);10×, 20×, 25×, 40×, 60× (Water)


Software

FV10-ASW Ver. 2.0


Main features and Service

Two-Photon Fluorescence Microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning confocal microscopy with the long wavelengths of two-photon excitation. It is based on the phenomenon that at high photon density, two photons can be simultaneously absorbed (combining their energies) to provoke the electronic transition of a fluorophore to the excited state. Because the energy of a photon is inversely proportional to its wavelength, the two photons have approximately twice the wavelengths of light required for single-photon excitation. Two-photon excitation high photon density is necessary to ensure a sufficient level of fluorophore excitation. To prevent photo-damage, the two-photon microscope utilizes high-power mode-locked pulsed laser, which generates a significant amount of power during pulse peaks, but has an average power that is sufficiently low as not to damage the specimen. A typical pulsed laser configuration employs short duty cycles of around 100 femtoseconds with a repetition rate of 80 to 100 megahertz. Because of high numerical aperture objectives, two-photon excitation occurs only at the focal point of a diffraction-limited microscope, a detecting pinhole is not need in two-photon microscope, thereby increasing detection efficiency. Two-photon microscopy has many advantages over confocal microscopy:

1.Long wavelength infrared light scatters less than light of shorter wavelengths and penetrates through specimen more easily;

2.lack of absorption from fluorophores positioned outside the focal plane allows more excitation light to penetrate through the specimen and reach the plane of focus;

3. long wavelength infrared laser is less phototoxic than lasers of shorter wavelengths;

4. photobleaching and phototoxicity occurs only at the focal point.


For these reasons, two-photon microscopy is particularly suitable for experiments including long term live cell imaging, imaging in deep tissues and photobleaching or other manipulations in small localized regions.