3D imaging of hematoxylin and eosin stained thick tissues with a sub-femtoliter resolution by using Cr:forsterite-laser-based nonlinear microscopy (Conference Presentation)

Abstract

Intraoperative assessment of excision tissues during cancer surgery is clinically important. The assessment is used to be guided by the examination for residual tumor with frozen pathology, while it is time consuming for preparation and is with low accuracy for diagnosis. Recently, reflection confocal microscopy (RCM) and nonlinear microscopy (NLM) were demonstrated to be promising methods for surgical border assessment. Intraoperative RCM imaging may enable detection of residual tumor directly on skin cancers patients during Mohs surgery. The assessment of benign and malignant breast pathologies in fresh surgical specimens was demonstrated by NLM. Without using hematoxylin and eosin (H and E) that are common dyes for histopathological diagnosis, RCM was proposed to image in vivo by using aluminum chloride for nuclear contrast on surgical wounds directly, while NLM was proposed to detect two photon fluorescence nuclear contrast from acrdine orange staining. In this paper, we propose and demonstrate 3D imaging of H and E stained thick tissues with a sub-femtoliter resolution by using Cr:forsterite-laser-based NLM. With a 1260 nm femtosecond Cr:forsterite laser as the excitation source, the hematoxylin will strongly enhance the third-harmonic generation (THG) signals, while eosin will illuminate strong fluorescence under three photon absorption. Compared with previous works, the 1260 nm excitation light provide high penetration and low photodamage to the exercised tissues so that the possibility to perform other follow-up examination will be preserved. The THG and three-photon process provides high nonlinearity so that the super resolution in 3D is now possible. The staining and the contrast of the imaging is also fully compatible with the current clinical standard on frozen pathology thus facilitate the rapid intraoperative assessment of excision tissues. This work is sponsored by National Health Research Institutes and supported by National Taiwan University Hospital.