Third harmonic generation microscopy of a mouse retina

Area of Science:

• Ophthalmology
• Biomedical Optics
• Microscopy

Background:

• Retinal imaging is crucial for understanding ocular health and disease.
• Current methods often require exogenous contrast agents, which can introduce artifacts or toxicity.
• Developing label-free imaging techniques is essential for accurate in vivo studies.

Purpose of the Study:

• To demonstrate the capability of third harmonic generation (THG) microscopy for lipid-specific imaging of the retina.
• To utilize THG's inherent tissue contrast generated from optical inhomogeneities for retinal visualization.
• To perform simultaneous THG and two-photon autofluorescence (TPAF) imaging of retinal structures.

Main Methods:

• A custom fiber laser and multiphoton microscope were engineered and optimized.
• Simultaneous TPAF and THG imaging were performed on fixed-frozen mouse eye sections.
• No exogenous fluorescent dyes were used; nuclear location was verified with a nuclear stain.

Main Results:

• All retinal layers were visualized with subcellular resolution using simultaneous THG and TPAF imaging.
• In BALB/c mice, THG signals originated from lipidic organelles within cellular and nuclear membranes.
• In C57BL/6 mice, THG signals in retinal pigment epithelium (RPE) cells were attributed to pigmented granules.

Conclusions:

• Third harmonic generation (THG) microscopy enables label-free, lipid-specific imaging of the mouse retina.
• The technique leverages inherent tissue contrast, eliminating the need for fluorescent dyes or recombinant proteins.
• THG microscopy offers a promising approach for high-resolution visualization of retinal structures.