SignificanceTo prevent meningioma recurrence, it is necessary to detect and remove all corresponding tumors intraoperatively, including those in the adjacent dura mater.AimCurrently, the removal of meningiomas from the dura mater depends solely on cautious visual identification of lesions by a neurosurgeon. Inspired by the requirements for resection, we propose multiphoton microscopy (MPM) based on two-photon-excited fluorescence and second-harmonic generation as a histopathological diagnostic paradigm to assist neurosurgeons in achieving precise and complete resection.ApproachSeven fresh normal human dura mater samples and 10 meningioma-infiltrated dura mater samples, collected from 10 patients with meningioma, were acquired for this study. First, multi-channel mode and lambda mode detection were utilized in the MPM to characterize the architectural and spectral features of normal and meningioma-infiltrated dura mater, respectively. Three imaging algorithms were then employed to quantify the architectural differences between the normal and meningioma-infiltrated dura mater through calculations of the collagen content, orientation, and alignment. Finally, MPM was combined with another custom-developed imaging algorithm to locate the meningioma within the dura mater and further delineate the tumor boundary.ResultsMPM not only detected meningioma cells in the dura mater but also revealed the morphological and spectral differences between normal and meningioma-infiltrated dura mater, providing quantitative information. Furthermore, combined with a self-developed image-processing algorithm, the precise borders of meningiomas in the dura mater could be accurately delineated.ConclusionsMPM can automatically detect meningiomas in the dura mater label-free. With the development of advanced multiphoton endoscopy, MPM combined with image analysis can provide decision-making support for histopathological diagnosis, as well as offer neurosurgeons more precise intraoperative resection guidance for meningiomas.
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.
Calcifications within brain tumors may be an indicator of a relatively long survival because a long time is required for the formation of calcium deposits, and may present a novel biomarker associated with response and improved outcome of therapy. In this paper, we describe the use of two-photon excitation fluorescent (TPEF) microscopy combined second harmonic generation (SHG) microscopy for high-resolution imaging that can be applied in identification of intratumoral calcifications. Our results demonstrate that the calcification has stronger TPEF signal than the area around it and the emission spectra shows the difference between the two areas clearly. The TPEF image of calcified region corresponds well with the corresponding H&E stained image. In this work, we present that the label-free imaging technique is able to distinguish the calcified mass lesions in intracranial neoplasms reliably.
An optical system for measurement of the intracellular pH (pHi) in a single living cell by using the fluorescent probe, 5(6)-carboxyfluorescein diacetate (CFDA) and fiber optic nanoprobe was demonstrated in this work. The CFDA probe is used to determine pHi in the yeast, Saccharomyces cerevisiae 97 while fiber optic nanoprobe is used to guide excitation light and receive emission light within a single cell. Experimental results showed that our system had higher detection sensitivity than other standard spectrometer, which is important to single-cell analysis, especially for the microanalysis in a single-cell.
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