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Multiphoton microscopes, also commonly called Two-Photon Microscopes, or being labeled as Non Linear Optics (NLO), rapidly scan the single beam of a pulsed, ultrafast and tunable laser across the sample. In this respect they are a form of LSM. The IR excitation wavelengths range from about 700 nm to over 1000 nm. A high output power of the laser is required to achieve the necessary photon density in the focus. Only then it becomes highly probable that two or more photons excite a fluorophore in a similar way to single photon with half the wavelength. However, outside the focus the laser intensity drops exponentially.
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Principal (projection) neurons in cortex of a transgenic mouse expressing YFP.
A region of the caudal part of the cortex was imaged from the transverse cut surface to a depth of 260 microns using multiphoton excitation (930 nm).
Specimen: S. Turney, Harvard, USA. |
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Thus the laser light is too weak outside the focus to create emission of fluorescence. With the restriction of the excitation process to the focal spot, the presence of a confocal aperture becomes superfluous as it is no longer necessary to reject out-of-focus light. This defined focal excitation provides some crucial advantages: The emitted light can be collected much more efficiently, and the infrared wavelengths used penetrate much deeper into tissue. Together these aspects mean multiphoton systems are the state of the art for imaging deep tissue layers with subcelluar resolution.
Products:
LSM 7 MP
LSM 710 NLO and LSM 780 NLO | |
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