Femtosecond fiber lasers provide imaging depth, focus, and contrast that is significantly improved in two-photon microscopy. The thing is that two-photon microscopy allows performing deep-tissue imaging right in thick/live samples. Herewith, two-photon excitation needs for ultrafast-pulsed laser systems that are able to generate high peak laser beam power with low pulse energy to get rid of the degradation of living cells.
To be more precise, fiber laser systems generate ultrashort femtosecond laser beam pulses at around 800 nm, however, higher wavelengths at 920 nm and in the 1030–1120 nm wavelength range are now more preferable to decrease scattering and optical damage and to excite fluorescent proteins. The thing is that the majority of two-photon volumetric microscopy applications need for watt-level average power with <150 fs pulse duration and repetition rates in the 80 MHz range.
Therefore, such fiber lasers allow generating <100 fs ultrashort pulses with up to 4 W average power at 920 or 1064 nm peak wavelengths with an 80 MHz laser beam pulse repetition rate because of their dispersion-compensation tailoring. Additionally, laser beam pulse duration may enlarge essentially after propagation through complex optical systems due to group-velocity dispersion. It is possible to monitor dispersion in a range from 0 to -90,000 fs2 to minimize fiber laser pulse duration at the sample and to maximize contrast on the analyzed sample.
It should be noted that the developed laser module is less than 25 cm in length and it transmits femtosecond laser beam pulses through a single-mode polarization-maintaining optical fiber that plays a crucial role in live-animal imaging because of its low weight and physical flexibility and reliability. Advanced two-photon microscopy or optogenetics applications allow exciting different cells or areas applying various laser beam pulse energy levels by the new fiber laser system.
The operating principle of the fiber laser is based on a fast acousto-optic modulator, the laser module that offers “>1 MHz bandwidth with active modulation using an analog electrical signal combined with a TTL signal to enable fast pulse gating.” The laser system has been already tested and demonstrated a two-photon microscopy system employing laser beams utilizing axicon technology to enlarge the depth of field at a given acquisition time when imaging green fluorescent protein-labeled microglial cells in zebrafish.
Such features as a combination of high peak power, GVD precompensation, rapid pulse modulation, and laser beam delivery makes the fiber laser systems are perfect in two-photon microscopy applications while simultaneously providing a smaller form factor with reduced maintenance and overall lower cost of ownership.
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