Multiphoton, or non-linear, microscopy, is ideal for capturing high-resolution three-dimensional (3D) images with reduced photobleaching and phototoxicity compared to traditional confocal microscopy techniques. This is the preferred method for analyzing thick samples of living tissue because of the technique’s advanced optical sectioning without out-of-focus absorption.
Multiphoton microscopy is a subset of fluorescence microscopy that uses similar imaging techniques of conventional widefield fluorescence microscopy to differentiate structures in living cells or tissues with various fluorescent tags known as fluorophores. Laser wavelengths of sufficient energy will excite the fluorophores to emit light that generates an image. Only one photon is needed to excite a fluorophore in conventional techniques.
Out-of-focus fluorescence is often an issue in standard fluorescence microscopy due to fluorescence from other planes interfering with the focal plane. Traditional confocal microscopy combats this issue by blocking any out-of-focus light with a pinhole in front of the detector. However, this technique is not ideal for imaging thick specimens because of scattering and absorption. The image quality starts to deteriorate at thicknesses of around 100µm, but in certain samples image quality can be maintained as far as 700µm.