Two-Photon non-linear microscopy, also called multi-photon microscopy, is a fluorescence imaging technique to image live biological samples, particularly in the neuroscience field. In nonlinear microscopy, contrary to linear confocal microscopy, fluorescence is excited only at the focal point of a femtosecond pulsed IR laser where the intensity is adequate to enable a dye molecule to simultaneously absorb two IR photons as if one single high energy photon had arrived.
The scanning of this focal point in three dimensions results in the acquisition of a 3D image. The added advantages of two-photon microscopy include reduced background interference that leads to higher resolution and reduced photo-induced damage to the sample.
Lasers for Two-Photon Microscopy
An ultrafast femtosecond laser that emits in the infrared region is the commonly used laser for two-photon microscopy. This laser’s repetition rate is typically in the order of 80MHz. However, it has recently been demonstrated that higher rates in the 1GHz range can achieve further reduction of photobleaching and damage utilizing lower peak powers, with the image quality same as with an 80 MHz laser.
This is directly linked to the power laws governing the various processes of two-photon microscopy. The increase in photodamage is an exponent of 2.5 and the bleaching increases cubic, whereas the fluorescence intensity increases quadratically with the illumination intensity.
Laser Quantum Products
Laser Quantum provides a variety of lasers for use in two-photon microscopy. The company’s taccor is an ultrafast laser that operates at a high repetition rate of 1 GHz, which provides a substantial increase in observation time prior to a cell being studied is lethally damaged. This, in turn, minimizes the photodamage by a factor of 2.5 and decreases photobleaching, thereby enabling repeated measurements with high quality. According to recent studies, there is a 5-10 fold increase in measurement times.
The following are the lasers offered by the company for this application:
- taccor c
- taccor s
- gigajet 20
- gigajet 20c
- gigajet 30s
This information has been sourced, reviewed and adapted from materials provided by Laser Quantum.
For more information on this source, please visit Laser Quantum.