Wavefront optimized nonlinear microscopy of ex vivo human retinas

Area of Science:

• Biomedical Optics
• Microscopy
• Ophthalmology

Background:

• Aberrations in ultrafast laser beams can degrade nonlinear microscopy image quality.
• Accurate imaging of human retinal tissues is crucial for understanding ocular health and disease.
• Current methods may require sample fixation or staining, potentially altering biological structures.

Purpose of the Study:

• To develop and evaluate a multiphoton microscope system with integrated Hartmann-Shack wavefront sensing for aberration correction.
• To investigate the impact of laser wavefront aberrations on two-photon autofluorescence imaging of human retinal tissues.
• To demonstrate improved imaging and reduced phototoxicity in retinal tissue analysis.

Main Methods:

• Development of a multiphoton microscope equipped with a Hartmann-Shack (HS) wavefront sensor.
• Realignment of the laser system cavity and active wavefront control to minimize aberrations.
• Two-photon autofluorescence imaging of human retinal tissues.
• Detection and analysis of nonlinear signals from retinal anatomical features.

Main Results:

• Minimizing laser beam aberrations significantly improved nonlinear microscopy image quality of human retinal tissues.
• Nonlinear signals from multiple human retinal anatomical features were detected without fixation or staining.
• The aberration correction approach reduced required excitation power levels.
• Minimized phototoxicity was observed in the imaged retinal samples.

Conclusions:

• The developed multiphoton microscope with HS wavefront control offers enhanced imaging of human retinal tissues.
• This technique allows for label-free, in-situ visualization of retinal structures with improved image fidelity.
• The reduction in excitation power and phototoxicity is beneficial for studying retinal physiology and pathology.