Rapid tissue histology using multichannel confocal fluorescence microscopy with focus tracking.

Area of Science:

• Biomedical Optics
• Pathology Imaging
• Microscopy Techniques

Background:

• Traditional hematoxylin and eosin (H&E) staining for Mohs surgery is time-consuming.
• Frozen sectioning artifacts can compromise diagnostic accuracy.
• A faster, more precise tissue evaluation method is needed for intraoperative pathology.

Purpose of the Study:

• To develop and evaluate a rapid multichannel confocal microscopy system for cancerous tissue evaluation.
• To create H&E-like histology images from freshly excised tissue using optical sectioning.
• To assess the system's potential as a diagnostic tool for intraoperative use.

Main Methods:

• Utilized multichannel confocal microscopy with Hoechst 33342 and Eosin-Y staining for rapid tissue evaluation.
• Employed optical sectioning, line scanning, and stitching to image large tissue areas.
• Developed a focus tracking method to maintain image focus on thick tissues without mechanical tilting.

Main Results:

• Successfully imaged freshly excised mouse organs (stomach, tumor, heart) within minutes.
• Converted confocal grayscale images to pseudocolor to mimic H&E histology.
• Achieved high-resolution imaging of both large structures (ventricles) and small details (nuclei, muscle fibers).

Conclusions:

• Confocal fluorescence microscopy with optical sectioning, line scanning, and focus tracking provides rapid pathology results.
• The developed technique enables intraoperative histology.
• This method has the potential to significantly reduce patient stress during surgeries requiring repeated histology examinations.