Label-free imaging of brain and brain tumor specimens with combined two-photon excited fluorescence and second harmonic generation microscopy

Abstract

Label-free imaging techniques are gaining acceptance within the medical imaging field, including brain imaging, because they have the potential to be applied to intraoperative in situ identifications of pathological conditions. In this paper, we describe the use of twophoton excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy in combination for the label-free detection of brain and brain tumor specimens; gliomas. Two independently detecting channels were chosen to subsequently collect TPEF/SHG signals from the specimen to increase TPEF/SHG image contrasts. Our results indicate that the combined TPEF/SHG microscopic techniques can provide similar rat brain structural information and produce a similar resolution like conventional H&E staining in neuropathology; including meninges, cerebral cortex, white-matter structure corpus callosum, choroid plexus, hippocampus, striatum, and cerebellar cortex. It can simultaneously detect infiltrating human brain tumor cells, the extracellular matrix collagen fiber of connective stroma within brain vessels and collagen depostion in tumor microenvironments. The nuclear-to-cytoplasmic ratio and collagen content can be extracted as quantitative indicators for differentiating brain gliomas from healthy brain tissues. With the development of twophoton fiberscopes and microendoscope probes and their clinical applications, the combined TPEF and SHG microcopy may become an important multimodal, nonlinear optical imaging approach for real-time intraoperative histological diagnostics of residual brain tumors. These occur in various brain regions during ongoing surgeries through the method of simultaneously identifying tumor cells, and the change of tumor microenvironments, without the need for the removal biopsies and without the need for tissue labelling or fluorescent markers.

Comparison of optically‐derived biomarkers in healthy and brain tumor tissue under one‐ and two‐photon excitation

“…This study, among others , shows that qualitative examination of TPEF images not only mirrors the histological features observed by the gold standard (anaplasia, necrosis, and increased cellular density), but rather provides additional information related to tissue architecture such as the (dis)organization of blood vessels and collagen structures surrounding tumor cells (Figure C‐E). The added advantage of interrogating tissue non‐invasively, and in real‐time, without the need of exogenous, contrast further motivates the development of a non‐linear multimodal endomicroscope for intraoperative pathological assessment in lieu of repeat biopsy sampling.…”

“…As eluded above, two‐photon imaging (TPEF and SHG) can provide detailed cytological, histological, and molecular information competitive with histopathological staining with the added advantage of being label‐free and non‐invasive . Low TPEF signals in neoplastic cells typically correspond to hypercellularity and angiogenesis, while high SHG intensities can indicate the presence of microcalcifications, increased vasculature and dense extracellular matrices characteristic of infiltrative and metastatic phenotypes . Overall, this endomicroscope will sample the brain at the tissue, cellular, and subcellular scale to identify microstructural, metabolic, and physiological properties pertaining to different brain tumor types.…”

Quantitative assessment of microenvironment characteristics and metabolic activity in glioma via multiphoton microscopy

“…Due to the histological structure specificity and quantitative biochemical information it can provide, MPM is particularly suitable for investigation of unstained tissue. However, until now, MPM mainly focuses on the tissue anatomy and macroscopic tissue characteristics so as to create the images just like that in hematoxylin and eosin (H&E)-stained histology [24][25][26][27]. Only a few publications reported on the imaging of tumor microenvironment and characterization of tumor metabolism in gliomas.…”

Rapid, label‐free detection of intracranial germinoma using multiphoton microscopy

“…Owing to these significant advantages over confocal laser microscopy, MPM is widely applied in the investigation of healthy versus diseased brain tissues. [12][13][14][15][16][17] In this exploratory analysis, we first assessed MPM's capability of characterizing pathological features associated with intracranial germinoma and comparing the yielded images to gold standard histopathology. Second, using custom-developed image-processing algorithms, we then assessed MPM's capability of quantifying the extent of stromal fibrosis and calcification in intracranial germinoma, which are the two most significant factors that affect surgeries.…”