Multiphoton intravital microscopy in small animals: motion artefact challenges and technical solutions

Since its invention 29 years ago, two-photon laser-scanning microscopy has evolved from a promising imaging technique, to an established widely available imaging modality used throughout the biomedical research community. The establishment of two-photon microscopy as the preferred method for imaging fluorescently labelled cells and structures in living animals can be attributed to the biophysical mechanism by which the generation of fluorescence is accomplished. The use of powerful lasers capable of delivering infrared light pulses within femtosecond intervals, facilitates the nonlinear excitation of fluorescent molecules only at the focal plane and determines by objective lens position. This offers numerous benefits for studies of biological samples at high spatial and temporal resolutions with limited photo-damage and superior tissue penetration. Indeed, these attributes have established two-photon microscopy as the ideal method for live-animal imaging in several areas of biology and have led to a whole new field of study dedicated to imaging biological phenomena in intact tissues and living organisms. However, despite its appealing features, two-photon intravital microscopy is inherently limited by tissue motion from heartbeat, respiratory cycles, peristalsis, muscle/vascular tone and physiological functions that change tissue geometry. Because these movements impede temporal and spatial resolution, they must be properly addressed to harness the full potential of two-photon intravital microscopy and enable accurate data analysis and interpretation. In addition, the sources and features of these motion artefacts are varied, sometimes unpredictable and unique to specific organs and multiple complex strategies have previously been devised to address them. This review will discuss these motion artefacts requirement and technical solutions for their correction and after intravital two-photon microscopy.