Zhifen Chen

Layer-resolved colorectal tissues using nonlinear microscopy

In this work, multiphoton microscopy (MPM), based on the nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to evaluate the feasibility of using MPM to distinguish layers of the bowel wall. It was found that MPM has the ability to identify the four-layer microstructures of colorectal tissues including mucosa, submucosa, muscularis propria, and serosa as there are many intrinsic signal sources in each layer. Our results also showed the capability of using the quantitative analyses of MPM images for quantifying some feature parameters including the nuclear area, nuclear-to-cytoplasmic ratio, and optical redox ratio. This work demonstrates that MPM has the potential in noninvasively monitoring the development and progression of colorectal diseases and then guiding effective treatment.

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.

Label-free monitoring of colorectal adenoma–carcinoma sequence based on multiphoton microscopy

The monitoring and evaluation of colorectal adenoma–carcinoma sequence during endoscopy are important for endoscopic resection of precursor lesions to disrupt the adenoma–carcinoma sequence and halt progression to invasive neoplastic disease. In this study, multiphoton microscopy (MPM) was used to identify different stages during the development of colorectal adenocarcinoma including adenoma with low-grade and high-grade dysplasia, and adenocarcinoma invading the submucosa. It was found that by combining two-photon excited fluorescence (TPEF) imaging and second harmonic generation (SHG) imaging, MPM can reveal the morphological changes of the epithelial cells and glands, identify the invasive position and depth of atypical glands and quantitatively describe the change of the cellular nucleus and the nuclear-to-cytoplasmic ratio during the stepwise progression of colorectal adenocarcinoma. These are important pathological findings for pathologists when diagnosing colorectal lesions. With the advancement of a compact and flexible multiphoton endoscope for in vivo imaging and clinical applications, MPM has the potential to provide immediate histological diagnosis for the monitoring and evaluation of the colorectal adenoma–carcinoma sequence during endoscopy.

Identifying and quantifying the stromal fibrosis in muscularis propria of colorectal carcinoma by multiphoton microscopy

The examination of stromal fibrosis within colorectal cancer is overlooked, not only because the routine pathological examinations seem to focus more on tumour staging and precise surgical margins, but also because of the lack of efficient diagnostic methods. Multiphoton microscopy (MPM) can be used to study the muscularis stroma of normal and colorectal carcinoma tissue at the molecular level. In this work, we attempt to show the feasibility of MPM for discerning the microstructure of the normal human rectal muscle layer and fibrosis colorectal carcinoma tissue practicably. Three types of muscularis propria stromal fibrosis beneath the colorectal cancer infiltration were first observed through the MPM imaging system by providing intercellular microstructural details in fresh, unstained tissue samples. Our approach also presents the capability of quantifying the extent of stromal fibrosis from both amount and orientation of collagen, which may further characterize the severity of fibrosis. By comparing with the pathology analysis, these results show that the MPM has potential advantages in becoming a histological tool for detecting the stromal fibrosis and collecting prognosis evidence, which may guide subsequent therapy procedures for patients into good prognosis.

Assessment of Tumor Invasion Depth in Colorectal Carcinoma Using Multiphoton Microscopy

Assessment of tumor invasion depth prior to making therapeutic decisions in colorectal carcinoma is crucial for both the patient and the physician. In this paper, multiphoton microscopy (MPM) was used to simultaneously label freely image loose areolar connective tissue in the submucosa and intramuscular septa in the muscularis propria to perform assessment of colorectal carcinoma invasion depth. The results indicated that MPM can accurately exhibit whether colorectal carcinoma invades into the submucosa or the muscularis propria. Collagen content alteration and the presence of dirty necrosis can be extracted to serve as quantitatively intrinsic biomarkers for reflecting collagen degradation, the occurrence of desmoplastic reaction, and breakdown of cancer cells, which are tightly related to the prognosis of colorectal carcinoma. With the development and clinical applications of the multiphoton endoscope, in vivo histological-like diagnosis of tumor invasion depth may become its main application in the field of colorectal carcinoma and lead to faster and improved therapeutic decision making in the clinics.

A new method of assessing the surgical margin in rectal carcinoma—using nonlinear optical microscopy

Nowadays, surgical resection is still the most effective treatment strategy for rectal carcinoma and one of the most important factors affecting whether the operation is successful or not is the surgical margin determination, especially in the distal rectal carcinoma which should take the sphincter-preserving issue into consideration. However, until recently no reliable evaluation method has been developed for this purpose. There are some shortcomings in intraoperative negative surgical margin assessment such as either lack of enough detailed information of biological tissues or the fact that it is time-consuming. Multiphoton microscopy (MPM)—nonlinear optical microscopy, which is based on the nonlinear optical process two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), has the ability to label freely and noninvasively visualize tissue micro-architecture at the sub-cellular level. The advantage of providing high contrast and high resolution biomedical image in real time makes MPM have a wide range of applications in life sciences. In this study, we introduced MPM to identify the boundary between normal and abnormal rectal tissues. MPM images clearly exhibit biological tissue microstructure and its morphological changes in the regions of our interest, which enable it to determine the surgical margin in rectal carcinoma. It can be foreseen that once MPM imaging system is used in clinical examination, it will greatly improve the accuracy of surgical resection.

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.

DISTINGUISHING BETWEEN HYPERPLASTIC AND ADENOMATOUS POLYPS AND NORMAL COLONIC MUCOSA BY USING MULTIPHOTON LASER SCANNING MICROSCOPY

Precisely distinguishing between hyperplastic and adenomatous polyps and normal human colonic mucosa at the cellular level is of great medical significance. In this work, multiphoton laser scanning microscopy (MPLSM) was used to obtain the high-contrast images and the morphological characteristics from normal colonic mucosa, hyperplastic polyps and tubular adenoma. By integrating the length and area measurement tools and computing tool, we quantified the difference of crypt morphology and the alteration of nuclei in normal and diseased human colonic mucosa. Our results demonstrated that the morphology of crypts had an obvious tendency to cystic dilatation or elongated in hyperplastic polyps and tubular adenoma. The content and number of mucin droplets of the scattered goblet cells had a piecemeal reduction in hyperplastic polyps and a large decrease in tubular adenoma. The nuclei of epithelial cells might be elongated and pseudostratified, but overt dysplasia was absent in hyperplastic polyps. Nevertheless, the nuclei showed enlarged, crowded, stratified and a rod-like structure, with loss of polarity in tubular adenoma. These results suggest that MPLSM has the capacity to distinguish between hyperplastic and adenomatous polyps and normal human colonic mucosa at the cellular level.

Laparoscopic double-stapled colorectal anastomosis without “dog-ears”

The lateral intersections of double-stapled anastomoses are structural weak spots, and they are considered to be potential ischemic areas leading to anastomotic leakage and possible sites of local recurrence [1, 2]. In 1996, Hazama et al. [3] reported a modified technique for rectal reconstruction during open surgery, and they used a circular stapler to eliminate the staple line on the rectal stump and cut off the dog-ears. But because of the narrowness of the pelvic cavity, it is very difficult to perform this technique in laparoscopic rectal surgery and there is no related report on its application in laparoscopic procedures. In this retrospective study, we evaluate the feasibility of a modified laparoscopic double-staple anastomosis technique, which is similar to Hazama’s procedure during open surgery, to eliminate the ‘dog-ears’ in laparoscopic anterior resection of the rectum or sigmoidectomy by laparoscopic suturing on the staple line. To follow is a description of our technique also illustrated by a video and brief description of our results. Materials and methods