Optical technologies for non-linear microscopy have transformed neurobiology, cancer biology, and developmental biology by enabling visualization of cellular structures in behaving animals. Two-photon microscopy stands out amongst non-linear microscopy methods for its wide adoption and utility. It is now a go-to technique for three-dimensional imaging sub-cellular organization up to depths of 500 microns. Two-photon microscopy relies on the excitation of a fluorophore by two photons of half the energy than required for one-photon microscopy. To elicit practically useful numbers of photons from a fluorophore located within the focal plane, it is necessary to boost the probability of two photons interacting with the same fluorophore at the same time. This is done using mode-locked pulsed lasers, instead of continuous wave lasers typically used in confocal microscopy. In this Biomedical Optics Express article, Perillo et al. developed a custom mode-locked ytterbium fiber laser that provides enough power to image neurons in whole mouse brains up to 900 microns, with sub-micron resolution up to the depth of 200 microns. The total cost of the laser is $13,000, which is less than half the cost of lasers with similar capability available in the market.
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