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

Area of Science:

• Biomedical Imaging
• Optical Microscopy
• Neuroscience

Background:

• Two-photon microscopy is a widely adopted technique for imaging fluorescently labeled cells in living animals.
• Its advantages include high spatial/temporal resolution, limited photodamage, and superior tissue penetration.
• These features make it ideal for studying biological phenomena in vivo.

Purpose of the Study:

• To review the motion artifacts that limit two-photon intravital microscopy.
• To discuss the sources and characteristics of these motion artifacts.
• To present technical solutions for correcting motion artifacts in live-animal imaging.

Main Methods:

• Nonlinear excitation of fluorescent molecules at the focal plane using femtosecond infrared laser pulses.
• High spatial and temporal resolution imaging with limited photodamage and deep tissue penetration.
• Identification and characterization of physiological motion sources in various organs.

Main Results:

• Two-photon microscopy is established for live-animal imaging due to its biophysical mechanism.
• Tissue motion from physiological processes (heartbeat, respiration, etc.) significantly impedes imaging resolution.
• Diverse and unpredictable motion artifacts necessitate complex correction strategies.

Conclusions:

• Motion artifacts are a critical limitation for two-photon intravital microscopy.
• Addressing these artifacts is essential for accurate data interpretation and harnessing the technique's full potential.
• This review provides an overview of motion artifacts and their correction strategies.