Enabling Multiphoton and Second Harmonic Generation Imaging in Paraffin-Embedded and Histologically Stained Sections

Area of Science:

• Biomedical Imaging
• Microscopy Techniques
• Histopathology

Background:

• Nonlinear microscopy, including multiphoton imaging and second harmonic generation (SHG), is a key technique for noninvasive extracellular matrix (ECM) imaging.
• Current methods typically involve in vivo measurements or analysis of fresh tissues and biopsies.
• Paraffin-embedded tissues are widely archived, offering potential for retrospective analysis.

Purpose of the Study:

• To investigate the impact of rehydration on nonlinear microscopy signals from paraffin-embedded tissue sections.
• To evaluate the effect of destaining hematoxylin and eosin (H&E)-stained sections on multiphoton and SHG imaging.
• To determine the feasibility of obtaining enhanced nonlinear imaging data from archived tissue samples.

Main Methods:

• Nonlinear microscopy (multiphoton and SHG) was applied to paraffin-embedded tissue sections.
• Sections were subjected to a rehydration protocol to restore a physiological state.
• H&E-stained sections were imaged before and after a destaining procedure.

Main Results:

• Rehydrating paraffin-embedded sections significantly improved both multiphoton and SHG emission signals.
• SHG signal enhancement was particularly notable after rehydration.
• Destaining H&E-stained sections further improved SHG signals, enabling detailed collagen analysis.
• Nonlinear images were successfully acquired from destained H&E sections.

Conclusions:

• Rehydration and destaining protocols can substantially enhance nonlinear microscopy signals from paraffin-embedded tissues.
• These methods allow for improved retrospective analysis of the ECM in archived and rare tissue samples.
• This approach provides valuable additional information for researchers and pathologists using existing tissue repositories.