Two-photon non-linear microscopy, sometimes referred to as multi-photon microscopy is a fluorescence technique that enables the imaging of living biological samples especially in the area of neuroscience. In contrast to linear confocal microscopy, a nonlinear microscope excites fluorescence only at the focal point of a femtosecond pulsed IR laser where the intensity is high enough to allow a dye molecule to absorb two IR photons simultaneously, as if one single high energy photon had arrived. A three dimensional image is acquired by scanning this focal point in three dimensions. Two photon microscopy has the added benefits of reducing background interference leading to higher resolution and reducing photo-induced damaged to the sample.
The laser used for two-photon microscopy is commonly an ultrafast femtosecond laser emitting in the IR region with a repetition rate in the order of 80MHz. Recently it has been shown that higher pulse rates (in the 1GHz range) have advantages for researchers interested in imaging living cells. The lower peak powers and higher repetition rates result in a reduction in photo damage to the sample and lower photo-bleaching effects, while maintaining image quality compared to the more conventional lasers.
The fluorescence intensity scales quadratically with the illumination intensity, whereas the photo damage increases with an exponent of 2.5 and the bleaching increases cubically. The high repetition rate offered by Laser Quantum’s taccor thus permits a significant increase in observation time before a cell under study is lethally damaged. These studies have reported increased imaging time for living samples of between 5 and 10 times.
It has also been shown in a number of studies (see downloadable whitepaper) that the use of ultra-short pulses has multiple benefits to two photon microscopy applications. The short pulses create higher peak powers for a given average power leading to higher resolution and reduced scattering allowing for deeper images. Due to using the entire Ti:Sapphire spectral emission, short pulse lasers are capable of simultaneous excitation of multiple fluorophores. There is also evidence of the short pulses promoting the auto-fluorescence of un-labelled cells. The venteon range of lasers offer the widest spectral output of any on the market and are ideal for two photon fluorescence.
With an extension to the range of wavelengths available from the taccor to 920nm, the taccor is now suitable for use with over 95% of commonly used fluorescent dyes.
taccor - Turn-key femtosecond laser with 1GHz repetition rates and 15fs pulses
gecco - 84MHz laser with integrated pump and low user intervention requirements
venteon - Ultra-short pulse lasers with broad spectral output able to excite multiple fluorophres without tuning
|Whitepaper - The benefits of ultra-short pulses in Two Photon Microscopy||418KB|
|Scientific paper - Two photon microscopy||287KB|
|Abstract - High repetition rate for nanomedicine two photon microscopy||351KB|
|Scientific paper - Multifarious control of two-photon excitation||1092KB|
|Abstract - Measurement of two-photon excitation spectra||299KB|
|Scientific paper - Single pulse coherent anti-Stokes Raman scattering microscopy||369KB|