The collagen in glioma is changed due to remodeling of the extracellular matrix during the malignant process and plays an important role in the progression of human gliomas. In this paper, multiphoton microscopy (MPM) based on twophoton excited fluorescence (TPEF) and second harmonic generation (SHG) was introduced to image the changes of collagen in normal human brain and gliomas. What’s more, together with the image analysis, the collagen content was quantitatively measured. It was found that in gliomas the collagen content significantly increased compared to normal brain tissue. These results suggest that MPM has the capability to provide collagen signature as a potential diagnostic marker for detection of gliomas.
Accurate histopathological diagnosis is essential for facilitating the optimal surgical management of intracranial germinoma. Current intraoperative histological methods are time- and labor-intensive and often produce artifacts. Multiphoton microscopy (MPM) is a label-free imaging technique that can produce intraoperative histological images of fresh, unprocessed surgical specimens. We employ an MPM based on second-harmonic generation and two-photon excited fluorescence microscopy to image fresh, unfixed, and unstained human germinoma specimens. We show that label-free MPM is not only capable of identifying various cells in human germinoma tissue but also capable of revealing the characteristics of germinoma such as granuloma, stromal fibrosis, calcification, as well as the abnormal and uneven structures of blood vessels. In conjunction with custom-developed image-processing algorithms, MPM can further quantify and characterize the extent of stromal fibrosis and calcification. Our results provide insight into how MPM can deliver rapid diagnostic histological data that could inform the surgical management of intracranial germinoma.
Glioblastoma (GBM) is a highly malignant and rapidly invasive astrocytoma, which has explosive biologic properties with rapid clinical progression leading to death and has a poor clinical outcome. The average survival time of most patients is only 12 to 15 months. GBM is distinguished pathologically from lower grade tumors by ‘pseudopalisading’ necrosis and microvascular hyperplasia. The most exaggerated form of microvascular hyperplasia is called glomeruloid body. MPM is a potential tool for imaging biological tissues at the molecular level. In this paper, MPM based on twophoton excited fluorescence (TPEF) and second harmonic generation (SHG) was applied for identifying the GBM without labeling or fluorescent markers. The results showed that MPM can display the specific histological characteristics of GBM including ‘pseudopalisading’ necrosis and glomeruloid vascular proliferation. The results obtained are consistent with the diagnosis of pathological findings. MPM will become a promising imaging tool for preoperative diagnosis of glioblastoma in the future.
Standard histopathology is well accepted as the gold standard for the diagnosis a wide range of diseases. Despite continuing advances in tissue staining automation, typical histological processing such as formalin-fixed paraffin-embedded are also labour- and time-intensive for treatment decisions in intraoperative histopathologic diagnosis. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), can be a versatile tool that enables label-free mapping of endogenous fluorophores within a fresh specimen, which provides pathology-like images with cellular and subcellular details. Here, we describe the use of label-free MPM for visualizing rat and human ex vivo brain tissue without tissue fixation, processing, and staining. Moreover, MPM is able to identify 6 types of cells in rat cerebrum and cerebellum, including cortical neurons, glia cells, Purkinje cells, pyramidal neurons and granule neurons in hippocampus, as well as epithelial cells in lateral ventricle. In addition, we further demonstrate that MPM can provide definitive pathological features in cerebral ischemia and focal cortical dysplasia (FCD) for assisting pathologic diagnosis. Our work establishes the methodology and augments the diagnostic accuracy of traditional frozen section histopathology. With the development of the miniature two-photon microscope, MPM will show more potential as a practical clinical tool for providing intraoperative reference image guidance of resection in neurosurgery.