Multiphoton microscopy includes a variety of nonlinear optical techniques such as 2PF, 3PF, SHG, THG, CARS, SRS, and others. The key advantage of nonlinear methods is the interaction between a tiny volume of light and matter, which delivers a lower background noise and better spatial resolution.
Some nonlinear techniques are label-free, and reduce the toxication of tissue under observation. They also mirror the biochemical processes closest to those occurring in the living organisms. The high peak intensity of ultrashort laser pulses enable an increased data acquisition rate, and significantly reduce tissue heating.
Biolit 2 is great for multiphoton microscopy. The laser delivers a sub-80fs pulse duration, with a nearly perfect Gaussian-like pulse shape. The lack of a pulse pedestal and sideband satellites enable highly efficient and precise useful signal generation in the tissue under investigation, and also suppress of background noise. The platform allows a wide range of pulse repetition rates ranging from 15 to 40 MHz.
The lower pulse repetition rate compared to common Ti:Sapphire oscillators provides higher peak power at the same avg. power level. This prevents thermal damage of the tissue under investigation. High peak power also guarantees better SNR (up to 5x than the traditional 80 MHz PRR). Another important effect is light scattering by the tissue, which is inversely proportional to the wavelength used for illumination. When emitting at 1045nm, Biolit 2 allows 3x deeper tissue penetration than Ti:Sapphire lasers, which is significantly deeper when compared to conventional microscopes using visible light sources.
The compact laser head and passive air cooling simplifies the integration with a microscope. With Biolit 2 you’ll get brighter and deeper images, faster.
Credits: Ludo van Haasterecht, Max Blokker, prof. dr. Marloes Groot, Vrije Universiteit Amsterdam, Frank van Mourik, Femto Diagnostics b.v.
The image of skin tissue, obtained with a multiphoton microscope using Biolit 2 femtosecond laser: red fibers – collagen bundles, blue lines – elastin fibers, green blobs – fat cells. Field of view – 500 micrometers.
Epilepsy brain tissue multi-contrast imaging
Application case using Biolit 2
We are thankful to our customers from Vrije Universiteit Amsterdam for sharing nice images of human brain tissue taken using different multi-contrast nonlinear imaging techniques with help of Biolit 2. Normal appearing white matter was obtained from an epilepsy patient and a tissue block imaged over time while perfused with artificial cerebrospinal fluid. Multiphoton depth stacks were acquired with 3 channels: THG (green), SHG (red), and TPEF (blue). Colorful structures appear in this stack: some capillaries in red showing up in the first few micrometers. This is followed by myelinated axons (starting at z = 30 um) in green.
Courtesy: Laura van Huizen, prof. dr. Marloes Groot, Vrije Universiteit Amsterdam, Geert Schenk, Antonio Luchicchi, Amsterdam UMC, Clinical Neuroscience.