Two-photon microscopy uses near-infrared light to excite fluorophores via the simultaneous absorption of two lower-energy photons. This process only occurs efficiently at the focal point, which means two-photon imaging naturally provides optical sectioning without the need for a pinhole. The longer wavelengths penetrate deeper into tissue, making this technique ideal for imaging thick samples and live biological tissues.

Second harmonic generation (SHG) imaging is a nonlinear optical microscopy technique that enables label-free visualisation of specific biological structures based on how they interact with intense, pulsed laser light. In SHG, two photons interacting with a non-centrosymmetric material combine to generate a new photon with exactly twice the energy (and half the wavelength) of the original light. This signal only arises in highly ordered structures, making SHG inherently contrast-specific.

A key advantage of SHG is that it does not require fluorescent dyes or genetic labels, which makes it especially useful for imaging living tissue in a minimally perturbative way. It is most commonly used to visualise fibrillar collagen in connective tissues, as well as myosin in muscle fibres and microtubule structures under certain conditions.

When combined with other modalities such as single or multi-photon fluorescence microscopy, SHG enables multiparametric and multispectral imaging.

Two-photons imaging is possible on our inverted Leica Stellaris Falcon DIVE system.

 

Banner image courtesy of Aneesa Shaikh