A novel method of virtual histopathology using laser-scanning confocal microscopy in-vitro with untreated fresh...

Histopathological examination for superficial gastrointestinal lesions has been mainly based upon the light microscopic examination of thin-slice specimens with hematoxylin and eosin (H&E) staining. However, it takes at least a couple of days to create a slide-glass for microscopic study. In order to obtain immediate microscopic images for untreated specimens, the authors used laser-scanning confocal microscopy (LCM) to study fresh samples of gastrointestinal mucosa.

Fresh untreated mucosal specimens from the esophagus, stomach, and colon, obtained by endoscopic pinch biopsy, polypectomy, or endoscopic mucosal resection (EMR), were fixed in normal saline and examined by LCM collecting the reflective light of a 488-nm wavelength argon laser beam. Findings from the LCM image were compared with those of conventional H&E staining in all specimens. For objective evaluation of the similarity of both pictures, the nucleus-to-cytoplasm ratio (N/C) of normal mucosa and that of cancer of the esophagus were calculated and statistically analyzed. The overall diagnostic accuracy for cancer was evaluated.

The average scanning time to obtain the LCM image of a specimen was 1.6 seconds. The LCM images acquired corresponded well to the conventional H&E light microscopic images in the esophagus, stomach, and colon. Cell wall, nucleus, cytoplasm, and tissue structural elements were simultaneously visualized by LCM scanning. A difference in N/C ratios between normal mucosa and cancer in the esophagus was statistically apparent when Welch's test (P=0.05) was applied. The overall diagnostic accuracy of the LCM study for cancer was 89.7%.

This novel method enables us to obtain an immediate serial virtual microscopic section through a fresh specimen, which has not actually been cut, although the resolution of the image obtained is still limited. These early results encourage us to develop imaging relevant to conventional histopathology alongside the development of LCM technology in the near future. We should aim at the in vivo application of LCM coupled to probes which can be introduced through the working channel of endoscopes.