Multi‐Photon Microscopy

Abstract

In this series of papers on light microscopy imaging, we have covered the fundamentals of microscopy, super-resolution microscopy, and lightsheet microscopy. This last review covers multi-photon microscopy with a brief reference to intravital imaging and Brainbow labeling. Multi-photon microscopy is often referred to as two-photon microscopy. Indeed, using two-photon microscopy is by far the most common way of imaging thick tissues; however, it is theoretically possible to use a higher number of photons, and three-photon microscopy is possible. Therefore, this review is titled "multi-photon microscopy." Another term for describing multi-photon microscopy is "non-linear" microscopy because fluorescence intensity at the focal spot depends upon the average squared intensity rather than the squared average intensity; hence, non-linear optics (NLO) is an alternative name for multi-photon microscopy. It is this non-linear relationship (or third exponential power in the case of three-photon excitation) that determines the axial optical sectioning capability of multi-photon imaging. In this paper, the necessity for two-photon or multi-photon imaging is explained, and the method of optical sectioning by multi-photon microscopy is described. Advice is also given on what fluorescent markers to use and other practical aspects of imaging thick tissues. The technique of Brainbow imaging is discussed. The review concludes with a description of intravital imaging of the mouse.

Streamlined intravital imaging approach for long-term monitoring of epithelial cell dynamics on an inverted confocal microscope

“…While we demonstrate that this confocal-based method can achieve stable longterm imaging deep into intact tissue, it should be noted that multiphoton microscopes cause less photodamage and penetrate to greater depths compared with confocal instruments 17 . Therefore, the utility of this tool will rely on the strength of the fluorescent signal in the transgenic animal model of choice as well as the tissue depth required to achieve experimental goals.…”

Imaging actin organisation and dynamics in 3D

“…Multiphoton instruments have historically been the gold standard for deep imaging owing to the reduction in background signal and longer wavelengths used, which reduces scattering However, higher laser power is often required to achieve sufficient signal in twophoton imaging, and the signal degrades with increasing depth (Sanderson, 2023). Adaptive optics can be introduced to correct for sample-based aberrations, such as those typically seen when imaging at depth.…”

A Residual Dense Attention Generative Adversarial Network for Microscopic Image Super-Resolution

“…Regardless of the magnification and numerical aperture of the objective lens used, the imaging throughput of current microscopes is typically only in the order of ten megapixels [ 1 ]. This leads to a compromise between high resolution and Field of View (FOV) when imaging.…”