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Two-photon excitation microscopy of mouse intestine.Red: actin.Green: cell nuclei.Blue: mucus of goblet cells.Obtained at 780 nm using a Ti-sapphire laser.. Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness.
In atomic physics, non-degenerate two-photon absorption (ND-TPA or ND-2PA) [1] or two-color two-photon excitation [2] is a type of two-photon absorption (TPA) where two photons with different energies are (almost) simultaneously absorbed by a molecule, promoting a molecular electronic transition from a lower energy state to a higher energy ...
Schematic of energy levels involved in two photons absorption. In atomic physics, two-photon absorption (TPA or 2PA), also called two-photon excitation or non-linear absorption, is the simultaneous absorption of two photons of identical or different frequencies in order to excite an atom or a molecule from one state (usually the ground state), via a virtual energy level, to a higher energy ...
Fluorescence and confocal microscopes operating principle. Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. [1]
A two-photon microscope is also a laser-scanning microscope, but instead of UV, blue or green laser light, a pulsed infrared laser is used for excitation. Only in the tiny focus of the laser is the intensity high enough to generate fluorescence by two-photon excitation , which means that no out-of-focus fluorescence is generated, and no pinhole ...
Confocal microscopy: Fluorescence, scattering 0.2 3-20 0.3-3 10-100 Two-photon microscopy: Fluorescence 0.5-1.0 1-10 0.3-3 10-100 Optical coherence tomography: 300 T Optical scattering 1-2 0.5-10 1-10 20-4.000 Scanning laser acoustic microscopy: 300 M Ultrasonic scattering 1-2 20 20 10 Acoustic microscopy: 50 M Ultrasonic scattering 20 20-100 ...
Researchers have long sought to break the diffraction limit of conventional optical microscopy in order to achieve super-resolution microscopes. One of the first major advances toward this goal was the development of scanning optical microscopy (SOM) by Young and Roberts in 1951. [1]
This is the same phenomenon used in two-photon microscopy (TPM), but there are two key features that distinguish pump–probe microscopy from TPM. First, since the molecule is not necessarily fluorescent, a photodetector measures the probe intensity. Therefore, the signal decreases as two-photon absorption occurs, the reverse of TPM. [3]
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