Two-Photon Autofluorescence Imaging of Fixed Tissues: Feasibility and Potential Values for Biomedical Applications.

Area of Science:

• Biomedical Optics
• Cellular Metabolism Imaging
• Histopathology Techniques

Background:

• Optical redox imaging (ORI) uses intrinsic fluorescence of NADH and FAD to assess cellular metabolic states.
• Current ORI methods face limitations in spatial resolution, light penetration depth, and tissue preservation requirements (viable or snap-frozen).
• Existing technologies like the Chance redox scanner and conventional fluorescence microscopy have restricted applications due to these limitations.

Purpose of the Study:

• To investigate the feasibility of performing ORI on unstained, fixed tissues.
• To evaluate the performance of a modern Serial Two-Photon (STP) Tomography scanner for rapid, high-resolution ORI.
• To explore the potential of STP Tomography for extracting biological information from fixed tissue redox states.

Main Methods:

• Mouse muscle, liver, and tumor xenograft tissues were harvested and fixed in 4% paraformaldehyde.
• Fixed tissue blocks were scanned using STP Tomography at room temperature.
• Autofluorescence signals in the NADH (750 nm excitation, blue emission) and FAD (860 nm excitation, green emission) channels were acquired.

Main Results:

• STP Tomography successfully acquired autofluorescence signals from fixed tissues.
• Remarkable intra-tissue heterogeneity was observed in NADH, FAD, and calculated redox ratio (FAD/(NADH+FAD)) images.
• The acquired data demonstrated the potential for detailed metabolic analysis in fixed tissue specimens.

Conclusions:

• ORI is achievable on unstained, fixed tissues using STP Tomography.
• STP Tomography offers a rapid, high-resolution method for assessing tissue metabolic states.
• The observed metabolic heterogeneity warrants further investigation for diagnostic and prognostic applications in pathology.