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Intraoperative confocal microscopy can enable high-resolution cross-sectional imaging of intact tissues as a non-invasive real-time alternative to gold-standard histology. However, all current means of intraoperative confocal microscopy are hindered by a limited field of view (FOV), presenting a challenge for evaluating gliomas, which are highly heterogeneous.

This study explored the use of image mosaicking with handheld dual-axis confocal (DAC) microscopy of fresh human glioma specimens.

In this preliminary technical feasibility study, fresh human glioma specimens from 6 patients were labeled with a fast-acting topical stain (acridine orange) and imaged using a newly developed DAC microscope prototype.

In comparison to individual image frames with small fields of view, mosaicked images from a DAC microscope correlate better with gold-standard H&E-stained histology images, including the ability to visualize gradual transitions from areas of dense cellularity to sparse cellularity within the tumor.

LS-DAC microscopy provides high-resolution, high-contrast images of glioma tissues that agree with corresponding H&E histology. Compared with individual image frames, mosaicked images provide more accurate representations of the overall cytoarchitecture of heterogeneous glioma tissues. Further investigation is needed to evaluate the ability of high-resolution mosaicked microscopy to improve the extent of glioma resection and patient outcomes.