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| Use of confocal microscopy to image structures becomes limiting when the fluorescent structures are located deep within a tissue. The deeper the location of the structures, the greater the amount of laser light that is absorbed before the light hits its target. This can cause damage to a sample when imaging live tissues. The greater depth also increases the amount of light that is scattered. With increasing depth, a threshold is reached where the intensity of excitation is no longer sufficient to produce fluorescence that provides a distinct image of the target. |
| Two-photon, also referred to as multi-photon, microscopy is similar to conventional laser scanning confocal microscopy, except that there is no need for pinholes. Optical sectioning of the sample is achieved through the use of a Ti:sapphire laser which operates in the near-infrared. The laser produces a high photon density (tens of kilowatts of peak power in a series of low-energy pulses that are approximately 10nJ per pulse) that is tuned to a wavelength twice that of the intended absorption wavelength of the sample. Consequently, two or more photons are required at a single point to produce an optical signal (i.e. excitation) that can be detected. The probability of such a two-photon event occurring is limited to the focal plane where there is an extremely high photon density. As a result, in two-photon imaging, excitation occurs only at the plane of focus. Fo more information, see the useful info page. |
| Two-photon confocal microscopy has several useful features. First, out of focus bleaching (the loss of optical signal production due to sample damage from the laser) is reduced. Two-photon microscopy also increases sample penetration because of the reduced absorption of near-infrared radiation. This allows thick, live tissues to be imaged with little damage to the sample environment. Two-photon microscopy also increases sensitivity as elimination of the pinhole allows the entire signal to reach the detector . Probably one of the most useful applications of multi-photon laser scanning microscopy is in neurobiology, which requires analysis of networks of sub-cellular features in three dimensions. |
| 'NLO' is an aconym for "non-linear optics". This refers to the fact that the optics of the microscope are compatible with lasers that emit pulses of emission, rather than constant emission. |
| For more information about this system and its capabilities, please contact Dr. Michelle Digman the Director of the Optical Biology Core Facility. |