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Multiphoton microscopy
uses two or three-photon excitation for fluorophores in a specimen

TECHNOLOGY:

Multiphoton excitation of a fluorophore in a specimen occurs when two or more photos of excitation light are absorbed at the same time. Because the photon energy is inversely proportional to wavelength, longer wavelength light (approximately two times as long) may be used for multiphoton excitation compared with one-photon excitation. A fluorophore, for example, that normally absorbs ultraviolet (UV) light of 350nm can be excited by two photons of near-infrared (IR) light of approximately 700 nm. Because excitation occurs only where photons coincide, the excitation light is not attenuated by fluorphore absorption above and below the point of focus (enabling greater depth penetration). Phototoxicity and photobleaching are also greatly reduced. Multiphoton imaging is generally achieved not only by focusing photons spatially, but also by focusing them in time using a pulsed laser. IR imaging also requires objectives with high IR transmission rates.

mult-photon

Fixed neuronal cells of mouse brain expressing eGFP. Excitation with pulsed IR laser (920 nm) allowed higher S/N ratio imaging of deep brain tissue areas than with a confocal microscope. Photographed with the cooperation of: Dr. Satoru Kondo, Department of Cellular Neurobiology, Graduate School of Medicine, the University of Tokyo.

APPLICATIONS:

Multiphoton microscopy provides three-dimensional optical sectioning similar to that achieved with confocal microscopy, but because it is less damaging than confocal imaging, it is ideal for imaging living specimens especially when deep imaging is required. Key applications include the observation of neural networks in brain slices, microvasculature in tissues or whole organisms, and developmental studies in living embryos.

MICROSCOPE CONFIGURATION:

Nikon’s multiphoton microscope, the A1R MP+, is a combined multiphoton and confocal microscope system that offers high speed (up to 420fps with the high speed resonant scanner), high resolution and high sensitivity multiphoton excitation and confocal fluorescence imaging. Signal-to-noise is increased by a new and highly sensitive, 4-channel Non-Descanned Detector (NDD), which has a wider sensitive area than conventional detectors. The NDD also has a broad spectral range allowing real-time unmixing of closely spaced fluorescence emissions for accurate assignment of fluorescence and high contrast images. Nano crystal coat technology in Nikon’s latest objectives has been developed especially for near IR applications. The ideal partners for the A1R MP+ system, these objectives offer higher light transmission, high contrast image acquisition, faster image capture at lower excitation levels, and reduced photobleaching and damage to live cells.


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