Schematic of IVM and basic optics of confocal/two-photon microscopy. (A) Comparison of conventional confocal microscopy and confocal IVM. The optics of the two imaging systems are not significantly different. However, whereas a conventional confocal microscope can visualize fixed tissue sections or organs extracted from an animal, confocal IVM allows the obtaining of images from the tissue of a live animal. Therefore, IVM can be equipped with additional devices, such as a heating pad or anaesthetic system, in order to make sure that living objects can breathe comfortably for undisrupted imaging. Schematics of the optics of confocal (B) and two-photon microscopy (C). (B) In confocal microscopy, a single photon has enough energy to excite the sample. The emitted photon has slightly lower energy and frequency (w) than the original photon (frequency w1). In two-photon microscopy, the summed energy of the two photons (frequency ϖ2) is enough to excite the sample to emit fluorescence. The emitted photon has slightly lower energy and frequency (w) than the sum of the two photons (frequency 2ϖ2). Also, with two-photon microscopy, second-harmonic generation (SHG) can be observed when two photons with the same wavelength interact with a non-linear material and then generate a new photon with twice as much energy as the initial photons. The virtual state is not the energy level of the atom, but rather represents the combined energy of photons. Meanwhile, unlike a single photon, a two-photon microscope does not require a pinhole to exclude out-of-focus background signals. Since two-photon excitation generates fluorescence only at the focal plane, there is no background signal. 2PF, two-photon fluorescence; SHG, second-harmonic generation.
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