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

Abstract

Summary 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.

Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease

“…Additionally, understanding the biological relevance of these biomarkers and their associated regulators is equally important in furthering the therapeutics and diagnostics of CAVD, a disease with no drug-based therapies, or early diagnostic tests (4-7). Advancements in probe design for accessibility and challenges associated with imaging moving objects with multiple confounding autofluorescent and absorbing sources must be overcome for the successful adoption of MPM techniques and metrics for clinical imaging of aortic valves (28,97,(101)(102)(103)(104)(105).…”

“…MPM-based intravital imaging of the ventricular wall has been performed (15,16,99,100); however, in vivo imaging of valves faces challenges due to accessibility, tissue movement, and blood flow (28,97,(101)(102)(103)(104). Limitations of tissue accessibility are being addressed by enhancing flexibility and miniaturization of microendoscopy tools, which will help facilitate preclinical and clinical translation of MPM (28,(101)(102)(103)105).…”

“…Limitations of tissue accessibility are being addressed by enhancing flexibility and miniaturization of microendoscopy tools, which will help facilitate preclinical and clinical translation of MPM (28,(101)(102)(103)105). Work is also focused on developing strategies to overcome motion-based artifacts introduced by heartbeat or physiological geometry changes (104). Additionally, researchers have developed algorithms to account for the NIR signal attenuation by blood (97).…”

3D Whole-Brain Imaging Approaches to Study Brain Tumors

“…In addition, injury from optical imaging setups (e.g., cranial windows) is accompanied by inflammatory responses that may impact studies investigating vascular or immune system involvement in brain tumor pathology [ 54 ]. Thinned cranial windows may injure the pia if re-thinning is required.…”

Machine Learning Aided Photonic Diagnostic System for Minimally Invasive Optically Guided Surgery in the Hepatoduodenal Area

“…Despite the gradual transition from the in vivo single-point measurements towards the in vivo imaging techniques, the use of fibre optical probes for the surgical guidance has great potential to compete with the costly techniques and to significantly improve the outcome of the routine procedures for cancer eradication. Most imaging approaches still need to address major issues concerning the motion artefacts, real time imaging processing and the trade-off between the parameters of resolution, frame rate and aperture diameter [6]. In particular, this set of limitations affects the minimally invasive surgical interventions as one deals with the tools of low outer diameters being applied in confined space of the abdominal cavity [7].…”