Excitation photons

The excitation photons Microscopic Parameter (also referred to as "Photon Count" is the number of photons with a given Excitation Wavelength that are simultaneously absorbed by a fluorophore in a fluorescence event.

A fluorescence molecule is usually excited by absorbing a single photon of a particular energy, corresponding to a particular wavelength. However, excitation is also possible by simultaneous absorption of two (n) photons, each of half (1/n) the energy and therefore twice (n times) the wavelength. Because this requires all the absorbed photons to be in the vicinity of the molecule within a limited time frame, the chance for a multi-photon excitation event to occur is much lower than the chance for a single photon event. The chance of a single photon event is proportional to the number of photons 'hitting' the molecule, and therefore proportional to the intensity. The chance of two photons hitting the molecule in a short interval is proportional to the square of this hit rate, so proportional to the square of the intensity.

Raising the photon probability distribution to the n-th power reduces de Point Spread Function and increases the resolution and the bandwidth, thus the Nyquist rate for ideal sampling changes accordingly.

Importantly, the 3D shape of the band-pass volume is very different: while the widefield microscope area has a wedge at the center causing the large widefield blur cones, the multi-photon bandpass volume has no such defects.

Multi-photon excitation reduces the bleaching effects due to the lower excitation wavelength used. It also has less scattering and background noise than single photon confocals and widefield systems. Therefore Multi-photons are ideal for imaging thicker 3D samples.

See Multi-Photon microscopy to find out how to use Huygens with your multi-photon microscope.