Xingfu Wang

Multiphoton microscopy for tumor regression grading after neoadjuvant treatment for colorectal carcinoma

AIM To evaluate the feasibility of using multiphoton microscopy (MPM) to assess a tumor regression grading (TRG) system. METHODS Fresh specimens from seven patients with colorectal carcinoma undergoing neoadjuvant radiochemotherapy at the Fujian Medical University Union Hospital were obtained immediately after proctectomy. Specimens were serially sectioned (10 µm thickness) and used for MPM or stained with hematoxylin and eosin for comparison. Sections were imaged by MPM using 810 nm excitation, and images were collected in two wavelength channels corresponding to second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals. The ratio of these signal intensities was used to distinguish fibrosis from normal mucosal and serosal tissues. RESULTS TRG of specimens assessed by MPM were in complete agreement with histologic grading performed by a consulting pathologist. SHG and TPEF images clearly revealed collagen fibers and fragmented elastic fibers in the muscularis propria specimens following neoadjuvant radiochemotherapy. Additionally, blood vessel hyperplasia was observed as thickening and fibrosis of the intima and media, which was accompanied by minimal inflammatory cell infiltration. Furthermore, the SHG/TPEF ratio in stromal fibrosis (4.15 ± 0.58) was significantly higher than those in the normal submucosal (2.31 ± 0.52) and serosal (1.47 ± 0.10) tissues (P < 0.001 for both). Analysis of emission spectra from cancerous tumor cells revealed two peaks corresponding to nicotinamide adenine dinucleotide hydrogen and flavin adenine dinucleotide signals; the ratio of these values was 1.19 ± 0.02, which is close to a normal metabolic state. CONCLUSION MPM can be used to perform real-time diagnosis of tumor response after neoadjuvant treatment, and can be applied to evaluate TRG.

Rapid, label‐free identification of cerebellar structures using multiphoton microscopy

The cerebellum is the prominent laminar structure of the mammalian brain that has been implicated in various psychiatric and neurological diseases. Although clinical brain imaging techniques have provided precise anatomic images of cerebellar structures, a definitive diagnosis still requires adequate resolution to identify individual layers in cerebellar cortex, the extent of tumor, even requires the histological tissue examination during surgical procedures. In this study, multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), was perform on the rat cerebellar structures and pathology with the combination of image analysis methods. Results show that MPM can reveal the cerebellar vermis, hemispheres, medulla, and ventricle, as well as axon bundles, Purkinje cells, capillaries, and the pia mater of the cerebellum. Together with custom-developed image processing algorithms, MPM could further differentiate between the gray and white matter, as well as evaluate the Purkinje cell layer, identify the cerebellar tumor boundary, and distinguish between the tumor core and peritumor regions. Our results establish a direct visualization and rapid assessment approach for the cerebellar structures, as well as suggest the feasibility of in vivo multiphoton microendoscopes and fiberscopes as clinical tools for neuropathological diagnoses.

Visualization of Tumor Response to Neoadjuvant Therapy for Rectal Carcinoma by Nonlinear Optical Imaging

The continuing development of nonlinear optical imaging techniques has opened many new windows in biological exploration. In this study, a nonlinear optical microscopy-multiphoton microscopy (MPM) was expanded to detect tumor response in rectal carcinoma after neoadjuvant therapy; especially normal tissue, pre- and post-therapeutic cancerous tissues were investigated in order to present more detailed information and make comparison. It was found that the MPM has ability not only to directly visualize histopathologic changes in rectal carcinoma, including stromal fibrosis, colloid response, residual tumors, blood vessel hyperplasia, and inflammatory reaction, which had been proven to have important influence on estimation of the prognosis and the effect of neoadjuvant treatment, but also to provide quantitative optical biomarkers including the intensity ratio of SHG over TPEF and collagen orientation index. These results show that the MPM will become a useful tool for clinicians to determine whether neoadjuvant therapy is effective or treatment strategy is approximate, and this study may provide the groundwork for further exploration into the application of MPM in a clinical setting.

Detection of morphologic alterations in rectal carcinoma following preoperative radiochemotherapy based on multiphoton microscopy imaging

BackgroundPreoperative radiochemotherapy improves outcomes in patients with locally advanced rectal carcinoma, and has been used increasingly in patient management. However, there is a strong clinical need to assess tumor response to neoadjuvant treatment, and a non-invasive technique that allows the precise identification of morphologic changes in tumors would be of considerable clinical interest.MethodsIn this study, we used multiphoton microscopy (MPM) to detect morphologic alterations in rectal adenocarcinomas in patients treated with preoperative radiochemotherapy.ResultsMPM was able to identify histopathologic alterations in rectal cancer following preoperative radiochemotherapy, and allowed the qualitative assessment of treatment efficacy and feasibility in relation to dose or strategy.ConclusionThese findings may provide the groundwork for evaluating tumor response to neoadjuvant treatment, thus allowing the tailoring of effective treatment doses and strategies.

Spatial and temporal identification of cerebral infarctions based on multiphoton microscopic imaging

Ischemic stroke is a leading cause of death and permanent disability worldwide. Middle cerebral artery occlusion (MCAO) of variable duration times could be anticipated to result in varying degrees of injury that evolve spatially over time. Therefore, investigations following strokes require information concerning the spatiotemporal dimensions of the ischemic core as well as of perilesional areas. In the present study, multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was applied to image such pathophysiological events. The ischemic time-points for evaluation were set at 6, 24, 48, and 72 hours after MCAO. Our results demonstrated that MPM has the ability to not only identify the normal and ischemic brain regions, but also reveal morphological changes of the cortex and striatum at various times following permanent MCAO. These findings corresponded well with the hematoxylin and eosin (H&E) stained tissue images. With the technologic progression of miniaturized imaging devices, MPM can be developed into an effective diagnostic and monitoring tool for ischemic stroke.

Label-free imaging of cortical structures with multiphoton microscopy

Cortical structures in the central nervous system exhibit an ordered laminar organization. Defined cell layers are significant to our understanding of brain structure and function. In this work, multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), which was applied for qualitatively visualizing the structure of cerebral and cerebellar cortex from the fresh, unfixed, and unstained specimen. MPM is able to effectively identify neurons and neurites in cerebral cortex, as well as glial cells, Purkinje cells, and granule cells in cerebellar cortex at subcellular resolution. In addition, the use of automated image processing algorithms can quantify the circularity of neurons and the density distribution of neurites based on the intrinsic nonlinear optical contrast, further providing quantitative characteristics for automatically analyzing the laminar structure of cortical structures. These results suggest that with the development of the feasibility of two-photon fiberscopes and microendoscope probes, the combined MPM and image analysis holds potential to provide supplementary information to augment the diagnostic accuracy of neuropathology and in vivo identification of various neurological illnesses in clinic.

Monitoring neoadjuvant therapy responses in rectal cancer using multimodal nonlinear optical microscopy

Most patients with rectal cancer have a better prognosis after receiving neoadjuvant therapy because of its remarkable curative effect. However, no device delivers real-time histopathologic information on treatment response to help clinicians tailor individual therapeutic strategies. We assessed the potential of multimodal nonlinear optical microscopy to monitor therapeutic responses, including tumoral and stromal responses. We found that two-photon excited fluorescence imaging can, without labeling, identify colloid response, inflammatory cell infiltration, vascular proliferation, and tumor regression. It can also directly detect rare residual tumor cells, which may be helpful for distinguishing tumor shrinkage from tumor fragmentation. In addition, second harmonic generation imaging shows the ability to monitor three types of fibrotic responses: mature, intermediate, and immature. We also determined nonlinear spectra, collagen density, and collagen orientation indexes to quantitatively analyze the histopathologic changes induced by neoadjuvant therapy in rectal cancer. Our work demonstrates that nonlinear optical microscopy has the potential to become a label-free, real-time, in vivo medical imaging technique and provides the groundwork for further exploration into the application of nonlinear optical microscopy in a clinical setting.

Real-time monitoring of tumor response to preoperative radiochemotherapy for rectal carcinoma by nonlinear optical microscopy

The continuing advancement of nonlinear optical imaging techniques has opened many new windows in biological exploration. In this work, the nonlinear optical microscopy, based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to probe tumor response to preoperative radiochemotherapy (RCT) for rectal carcinoma. It was found that MPM has the ability of direct visualization of histopathologic changes in rectal carcinoma following preoperative RCT including stromal fibrosis, colloid response and residual tumors. Our results also showed the capability of MPM using the quantitative analyses of images to quantify these changes. This work may provide the groundwork for further exploration into the application of multiphoton-based endoscopy in a clinical setting.

Assessment of colloid response by nonlinear optical microscopy after preoperative radiochemotherapy for rectal carcinoma.

Abstract. Colloid response is a type of tumor response that occurs after preoperative radiochemotherapy for rectal carcinoma. Given its important influence on survival, the colloid response should be considered when estimating histopathological reactions. Here, multiphoton microscopy (MPM) was applied to evaluate the colloid response ex vivo. This study demonstrated that MPM has the capability to visualize the colloid response in the absence of labels and can, in particular, identify rare residual carcinomatous cells in mucin pools. These results highlight the potential of this nonlinear optical technique as a diagnostic tool for tumor response after neoadjuvant treatment.