What is multiphoton fluorescence microscopy?
Multiphoton fluorescence microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with highly specific fluorophores.
What does multiphoton mean?
: using or involving more than one photon multiphoton interactions multiphoton ionization.
What is multiphoton microscopy used for?
Main. Multiphoton microscopy (MPM) is regarded as the method of choice for imaging of living, intact biological tissues on length scales from the molecular level through the whole organism.
What is the function of multiphoton excitation microscope?
Multiphoton excitation microscopy provides high axial (optical sectioning) and lateral resolution, reduced out of focus photobleaching/photodamage, and is optimal for deep tissue imaging (Helmchen & Denk, 2005).
What is the main advantage of 2 photon microscopy over multiphoton confocal microscopy and why?
As described above, two-photon excitation is more effective than confocal microscopy in thick samples for several reasons: scattered excitation light does not excite any fluorescence, scattering fluorescence emission can still be detected, and infrared wavelengths scatter less than visible wavelengths.
What is the resolution of two-photon microscopy?
STED microscopy has been done with two-photon excitation and has achieved a spatial resolution of ~60 nm in optical systems with two lasers [13–15] and a single laser [16,17]. However, the imaging depth of two-photon STED microscopy is still limited to being less than ~100 µm.
Can a confocal microscope be used for multiphoton imaging?
A confocal laser scanning microscope must be modified and equipped with a specialized near-infrared pulsed laser system in order to perform multiphoton imaging. The following characteristics should be used as a guide when comparing multiphoton excitation with conventional laser scanning confocal microscopy.
What are the advantages of two photon excitation microscopy?
Many advantageous effects result from the localization of excitation in the two-photon microscopy technique. Perhaps the most important is that the three-dimensional resolution of a two-photon excitation microscope is identical to that achieved in an ideal confocal microscope.
How does a laser scanning confocal microscope work?
Laser scanning confocal microscopes employ a pair of pinhole apertures to limit the specimen focal plane to a confined volume approximately a micron in size.
How to increase photomultiplier gain in confocal microscopy?
To increase the photomultiplier gain in the confocal portion of the tutorial, translate the PMT Red Gain and PMT Green Gain sliders by moving one or both bars with the mouse cursor. Moving the sliders to the right will increase photomultiplier gain, while moving the sliders to the left decreases the gain.
