Contact us
EN
RU

Angewandte Physik
&
Elektronik GmbH

Angewandte Physik
&
Elektronik GmbH

Dispersion compensation for
3P microscopy

Three-photon excitation microscopy (3P) is a fluorescence imaging technique that allows imaging of living tissue up to 1 millimeter thick.
Unlike two-photon microscopy, the working wavelength range is from 1300 to 1700 nm, which helps to reduce scattering and absorption in tissues. To compensate for the dispersion of the three-photon excitation, it is necessary to control the short pulses so that they reach the sample and do not broaden as they pass through the microscope.

Frequency modulation is a non-uniformity of frequency throughout the pulse. Appearance of frequency modulation with unchanged pulse duration leads to spectrum broadening. However, in the case of dispersion, the pulse duration increases simultaneously with frequency modulation. As a result, the spectrum of the signal does not change.


Maintaining short pulses in 3P microscopy is difficult for two reasons. The first is that signal generation in 3P-microscopy is proportional to the inverse square of the pulse width, whereas signal generation in 2P-microscopy is proportional to the inverse square of the pulse width. 2P-microscopy is proportional only to the inverse of the pulse width. Very short pulses ( ~ 40-70 fs) are required to generate a sufficient signal Small changes in pulse width have a large effect on the signal.

Pulse broadening is caused by the group velocity dispersion (GVD) of the spectral components of the pulse. This material parameter, which has a physical unit of measurement
[fs²/mm] and describes the relationship between dispersion and optical path length. The microscope combines optics from different materials, so each element affects the shape of the light pulse.
The second is that the short pulse width generated by the laser must be maintained after passing through the microscope and at sample entry. The pulse duration at sample entry is the result of a combination of the laser's initial pulse duration and broadening as it passes through the microscope's optical materials. Additional broadening occurs when passing through immersion fluids, water, and when entering the biological specimen itself.
Compensating for pulse broadening requires complex optical compensation circuitry. Special passive devices, dispersion compensators, designed to correct the shape of optical signals, are used to introduce an angular dispersion equal in magnitude and opposite in sign. The total dispersion of the system becomes zero.
Required GVD for dispersion compensation in a standard 2P microscope configuration. The upper line marks the required GVD for dispersion compensation of a microscope with high dispersion optics. The lower line marks the required GVD for dispersion compensation of a microscope with low dispersion optics.
Required GVD for dispersion compensation in a standard 3P-microscope configuration. Upper line and lower line are positive and negative dispersion components.
To compensate for dispersion in three-photon microscopy, it is necessary to use schemes different from those of two-photon microscopy
The intensity and contrast of imaging with three-photon fluorescence excitation depends on the pulse power. The shorter the pulse reaching the substance, the more contrasting and informative the resulting picture.

For very short pulses (50 fs and below), the effect of third-order dispersion, which causes significant broadening and reduction of peak power, must be taken into account.

APE provides research solutions for three-photon and two-photon excitation microscopy.
Always available in stock
Wide range of nonlinear crystals for OPO/OPA in the IR range

To familiarize yourself with the full range of products, please contact us or ask a question through the form
Contact us
Leave your contact info and our manager will contact you
Always available in stock
Wide range of nonlinear crystals for OPO/OPA in the IR range
To familiarize yourself with the full range of products, please contact us or ask a question through the form