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Although many efforts have been made to engineer cell sheets with anisotropic patterns, current techniques still exhibit several shortcomings including uncontrolled harvest time and deformation of pattern by contraction. In this study, we report a simple method to harvest a cell sheet with a striated structure of extracellular matrix (ECM) and myoblasts using a thermosensitive hydrogel micropatterned with different sizes (25 and 80 μm). The hydrogel supported the formation of a confluent monolayer of myoblasts with aligned morphology. When the temperature was reduced from 37 to 4 oC, the size of the hydrogel increased by approximately 1.2-fold within 10 min. In response to this change, a cell sheet was harvested and could be transferred to the desirable substrate through conformal contact between the hydrogel and target. We further examined the effect of pattern size on alignment of ECM/cell assembly by fast Fourier transform (FFT) analysis of fluorescently stained stress fibers and ECM proteins within the harvested cell sheet. The cell sheet maintained alignment of confluent myoblasts after being harvested to the substrate. In addition, the topologic patterns also promoted formation of aligned myotube on the harvested cell sheet, which was important for muscle tissue regeneration. However, the pattern size appeared to have no influence on alignment of cells on the cell sheet. Finally, a bi-layered structure was fabricated in which each layer was stacked with aligned direction on the cell sheet being perpendicularly assembled. Collectively, our platform could be used for rapid harvest of cell sheets with aligned architecture of ECM/cell, which can be applied to fabrication of welldefined 3D tissue for tissue engineering.

Adipose tissue is a dynamic organ that provides endocrine, inflammatory and angiogenic factors, which can assist breast carcinoma cells with invasion and metastasis. Previous studies have shown that adipocytes adjacent to carcinoma, known as cancer-associated adipocytes, undergo extensive changes that correspond to an "activated phenotype," such as reduced size relative to adipocytes in non-neoplastic breast tissue. Optical imaging provides a tool that can be used to characterize adipocyte morphology and other features of the tumor microenvironment. In this study, we used confocal fluorescence microscopy to acquire images of freshly excised breast tissue stained topically with proflavine. We developed a computerized algorithm to identify and quantitatively measure phenotypic properties of adipocytes located adjacent to and far from normal collagen, ductal carcinoma in situ and invasive ductal carcinoma. Adipocytes were measured in confocal fluorescence images of fresh breast tissue collected from 22 patients. Results show that adipocytes adjacent to neoplastic tissue margins have significantly smaller area compared to adipocytes far from the margins of neoplastic lesions and compared to adipocytes adjacent to non-neoplastic collagenous stroma. These findings suggest that confocal microscopic images can be utilized to evaluate phenotypic properties of adipocytes in breast stroma which may be useful in defining alterations in microenvironment that may aid in the development and progression of neoplastic lesions.

Background & Aims Esophageal squamous cell neoplasia has a high mortality rate as a result of late detection. In high-risk regions such as China, screening is performed by Lugol's chromoendoscopy (LCE). LCE has low specificity, resulting in unnecessary tissue biopsy with a subsequent increase in procedure cost and risk. The purpose of this study was to evaluate the accuracy of a novel, low-cost, high-resolution microendoscope (HRME) as an adjunct to LCE. Methods In this prospective trial, 147 consecutive high-risk patients were enrolled from 2 US and 2 Chinese tertiary centers. Three expert and 4 novice endoscopists performed white-light endoscopy followed by LCE and HRME. All optical images were compared with the gold standard of histopathology. Results By using a per-biopsy analysis, the sensitivity of LCE vs LCE + HRME was 96% vs 91% ( P = .0832), specificity was 48% vs 88% ( P P P = .3551), and overall accuracy was 57% vs 90% ( P P = .16), specificity was 29% vs 79% ( P P Conclusions In this trial, HRME improved the accuracy of LCE for esophageal squamous cell neoplasia screening and surveillance. HRME may be a cost-effective optical biopsy adjunct to LCE, potentially reducing unnecessary biopsies and facilitating real-time decision making in globally underserved regions. ClinicalTrials.gov, NCT 01384708.

BACKGROUND Autosomal dominant polycystic kidney disease (ADPKD) volumetry is an important marker for evaluating the progression of disease. Three-dimensional (3D) volumetry is generally more timesaving than 2D volumetry, but its reliability and accuracy are uncertain. METHODS Small and large phantoms simulating polycystic kidneys and 20 patients with ADPKD underwent magnetic resonance imaging (MRI) volumetry. We evaluated the total kidney volume (TKV) and total cyst volume (TCV) using a novel 3D volumetry program (XelisTM) and compared 3D volumetry data with the conventional 2D method (the reference volume values). After upload and threshold setting, the other organs surrounding the kidney were removed by picking and sculpting. The novel method involves drawing of the kidney or cyst and automatic measurement of kidney volume and cyst volume in 3D images. RESULTS The 3D volume estimation of the small and large phantoms differed from the actual values by 6.9% and -8.2%, respectively, for TKV and by 2.1% and 1.4% for TCV. In ADPKD patients, the intra-reader reliability of 3D volumetry was 30 ± 180 mL (1.3 ± 10.3%) and 25 ± 113 mL (1.2 ± 9.4%), respectively, for TKV and TCV. Correlation between 3D volumetry and 2D volumetry of TKV and TCV resulted in a high correlation coefficient and a regression slope approaching 1.00 (r = 0.97 - 0.98). The mean of the volume percentage differences for 3D vs. 2D for TKV : TCV were -6.0 ± 8.9% : 2.0 ± 11.8% in large ADPKD and -16.1 ± 10.4% : 13.2 ± 21.9% in small ADPKD. CONCLUSION Our study showed that 3D volumetry has reliability and accuracy compared with 2D volumetry in ADPKD. 3D volumetry is more accurate for TCV and large ADPKD.

Background EUS-guided FNA is one of the few techniques that can obtain cells and tissue from the liver and pancreas. However, the technique remains vulnerable to poor specimen quality and sampling error. Objective To evaluate the ability of a high-resolution microendoscope (HRME) to visualize the cellular and architectural features of normal and malignant liver and pancreatic tissue ex vivo, to assess the ability of endosonographers to identify normal and neoplastic tissue by using HRME images, and to demonstrate preliminary technical feasibility of in vivo HRME imaging via EUS fine-needle puncture (FNP). Design Ex vivo pilot feasibility study in human tissue; in vivo swine model. Setting Two academic medical centers. Patients Co-registered HRME images and biopsies were obtained from surgically resected hepatic and pancreatic tissues from 44 patients. Intervention Images were divided into training (12 images) and test (80 images) sets containing a range of normal and pathologic conditions for each organ. After viewing the training sets, 9 endosonographers attempted to distinguish malignant tissue from normal or benign lesions in the test sets, each of which contained 40 unique images with individual diagnoses from pathology. Main Outcome Measurements Image acquisition feasibility, ex vivo and in vivo. Ability of endosonographers to recognize features of normal/benign or malignant tissue from the liver and pancreas. Results Overall, the 9 endosonographers achieved median accuracy figures of 85% in the liver and 90% in the pancreas. The endosonographers with prior experience in reading HRME images achieved accuracy rates between 90% and 95%. Technical feasibility of HRME imaging through a 19-gauge EUS-FNP needle was demonstrated in an in vivo swine model. Limitations Ex vivo study. Conclusion High-resolution microendoscopy allows real-time imaging of cellular-level morphology and tissue architecture in the liver and pancreas. The technique appears to have a short learning curve, after which endosonographers achieved high accuracy rates in distinguishing malignant tissue from normal and benign pathology in both organs. Translating this imaging platform to the in vivo setting appears technically feasible.

A finite element model of the human lumbar spine was developed for the parametric study of the stiffness of a dynamic stabilization device. Spinal segments (L2-L5) were used to investigate the effect of dynamic stabilization and the influence on the mobility of adjacent intervertebral segments. Three spines were analysed and compared: (1) a lumbar spine with intact discs, used as a reference; (2) a fused spine with a fixation device after the the facetectomy and the total laminectomy; and (3) a spine stabilized with a dynamic stabilization device after the facetectomy and the total laminectomy. The Range of Motion (ROM) and the intervertebral disc pressure of L3-L4 and the ROM and disc pressure of the adjacent segments were examined to determine the influence of the implant on the adjacent segments. In the case of the dynamically stabilized spine, the total ROM was greater than that of the fused spine but similar to that of the intact spine. Furthermore, the disc pressure on the adjacent segments in the fused spine was greater than that of the intact spine, but the disc pressure of the dynamically stabilized spine was similar to the intact spine. In particular, the dynamic stabilization device having a stiffness of 10-15 N/mm made the destabilized spine more similar to the intact spine. Results indicated that the use of dynamic stabilization devices restored functionality closer to that of the intact spine as compared to the fused spine. The stiffness value utilized in the device was determined to be an important design parameter in manufacturing the dynamic stabilization device.

Introduction Pathologists currently diagnose breast lesions through histologic assessment, which requires fixation and tissue preparation. The diagnostic criteria used to classify breast lesions are qualitative and subjective, and inter-observer discordance has been shown to be a significant challenge in the diagnosis of selected breast lesions, particularly for borderline proliferative lesions. Thus, there is an opportunity to develop tools to rapidly visualize and quantitatively interpret breast tissue morphology for a variety of clinical applications. Methods Toward this end, we acquired images of freshly excised breast tissue specimens from a total of 34 patients using confocal fluorescence microscopy and proflavine as a topical stain. We developed computerized algorithms to segment and quantify nuclear and ductal parameters that characterize breast architectural features. A total of 33 parameters were evaluated and used as input to develop a decision tree model to classify benign and malignant breast tissue. Benign features were classified in tissue specimens acquired from 30 patients and malignant features were classified in specimens from 22 patients. Results The decision tree model that achieved the highest accuracy for distinguishing between benign and malignant breast features used the following parameters: standard deviation of inter-nuclear distance and number of duct lumens. The model achieved 81 % sensitivity and 93 % specificity, corresponding to an area under the curve of 0.93 and an overall accuracy of 90 %. The model classified IDC and DCIS with 92 % and 96 % accuracy, respectively. The cross-validated model achieved 75 % sensitivity and 93 % specificity and an overall accuracy of 88 %. Conclusions These results suggest that proflavine staining and confocal fluorescence microscopy combined with image analysis strategies to segment morphological features could potentially be used to quantitatively diagnose freshly obtained breast tissue at the point of care without the need for tissue preparation. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0617-9) contains supplementary material, which is available to authorized users.

Background and Aims Previous studies show that microendoscopic images can be interpreted visually to identify the presence of neoplasia in patients with Barrett's esophagus (BE), but this approach is subjective and requires clinical expertise. This study describes an approach for quantitative image analysis of microendoscopic images to identify neoplastic lesions in patients with BE. Methods Images were acquired from 230 sites from 58 patients by using a fiberoptic high-resolution microendoscope during standard endoscopic procedures. Images were analyzed by a fully automated image processing algorithm, which automatically selected a region of interest and calculated quantitative image features. Image features were used to develop an algorithm to identify the presence of neoplasia; results were compared with a histopathology diagnosis. Results A sequential classification algorithm that used image features related to glandular and cellular morphology resulted in a sensitivity of 84% and a specificity of 85%. Applying the algorithm to an independent validation set resulted in a sensitivity of 88% and a specificity of 85%. Conclusions This pilot study demonstrates that automated analysis of microendoscopic images can provide an objective, quantitative framework to assist clinicians in evaluating esophageal lesions from patients with BE. (Clinical trial registration number: NCT01384227 and NCT02018367.)

We developed an automated frame selection algorithm for high-resolution microendoscopy video sequences. The algorithm rapidly selects a representative frame with minimal motion artifact from a short video sequence, enabling fully automated image analysis at the point-of-care. The algorithm was evaluated by quantitative comparison of diagnostically relevant image features and diagnostic classification results obtained using automated frame selection versus manual frame selection. A data set consisting of video sequences collected in vivo from 100 oral sites and 167 esophageal sites was used in the analysis. The area under the receiver operating characteristic curve was 0.78 (automated selection) versus 0.82 (manual selection) for oral sites, and 0.93 (automated selection) versus 0.92 (manual selection) for esophageal sites. The implementation of fully automated high-resolution microendoscopy at the point-of-care has the potential to reduce the number of biopsies needed for accurate diagnosis of precancer and cancer in low-resource settings where there may be limited infrastructure and personnel for standard histologic analysis.

We performed quantitative measurement of adipocyte area in confocal microscopy images to evaluate adipocyte phenotype adjacent to and 2 mm (a narrow resection margin) away from invasive breast carcinoma and benign, fibrous breast tissue.

This paper describes a warpage study on LOC-TSOP memory devices. The main objectives of this study are to evaluate the impacts of epoxy mold compounds on package warpage with different sized dies. It was found that the balance of the bending between the edge side region and the die attach region controls the package warpage. The package tends to bend convex upwards in the edge side region and concave downwards in the die attach region. The various mold compounds were prepared to study the affect on the components, particularly the resin and the filler. It has been shown that the optimum mold compound can be changed according to the inner structure of the LOC-TSOP. The effect of post-mold cure on package warpage was also examined based on the cure rate.

Quantitative analysis of non-proliferative ducts, ductal hyperplasia, and ductal carcinoma in situ in breast tissue demonstrates that confocal fluorescence images display morphologic features associated with malignant progression that could be used to objectively identify DCIS.

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is useful to obtain specimens from lesions underlying deep parts of the liver and spleen. However, the development of novel ancillary techniques must be explored to reduce the number of needle passes and potential adverse effects during this procedure. We conducted an animal study using a swine to demonstrate technical feasibility of in vivo cytological observation of liver and spleen using the high-resolution microendoscopy (HRME) system under EUS guidance. We successfully performed the study. No significant acute adverse events occurred during the procedure. The HRME system could obtain clear images representing cytology-level morphology of spleen and liver. Hence, it is found out that in vivo cytological observation of liver and spleen using the HRME system under EUS guidance is technically feasible.

This study introduces an approach for quantitative analysis of microendoscopic images to identify esophageal neoplasia. The approach quantifies morphologic parameters which distinguish esophageal neoplasia accurately in an independent validation set

This prospective pilot study evaluates the potential of high-resolution fiber optic microscopy (HRFM) to identify lymph node metastases in breast cancer patients. 43 lymph nodes were collected from 14 consenting breast cancer patients. Proflavine dye was topically applied to lymph nodes ex vivo to allow visualization of nuclei. 242 images were collected at 105 sites with confirmed histopathologic diagnosis. Quantitative statistical features were calculated from images, assessed with one-way ANOVA, and were used to develop a classification algorithm with the goal of objectively discriminating between normal and metastatic tissue. A classification algorithm using mean image intensity and skewness achieved sensitivity of 79% (27/34) and specificity of 77% (55/71). This study demonstrates the technical feasibility and diagnostic potential of HRFM with fluorescent contrast in the ex vivo evaluation of lymph nodes from breast cancer patients.

Oral cancer is a significant health problem in the USA and throughout the world. Most oral cancer patients are diagnosed at a late stage, when treatment is less successful and treatment-associated morbidity is more severe. A number of new diagnostic aids to conventional oral examination have recently been introduced to assist in the early detection of oral neoplasia. In particular, autofluorescence imaging has emerged as a promising adjunctive technique to improve early identification of oral premalignant lesions. Direct visual inspection of tissue autofluorescence has shown encouraging results in high-prevalence populations, but the technique requires subjective interpretation and depends on the visual recognition skills of the examiner. Capturing and analyzing digital fluorescence images can reduce subjectivity and potentially improve sensitivity of detection of precancerous changes. Recent studies of wide-field autofluorescence imaging in low-prevalence populations suggest that benign lesions such as inflammation may give rise to false-positive results. High-resolution fluorescence imaging is a new modality that can be used in conjunction with wide-field imaging to improve specificity by imaging subcellular detail of neoplastic tissues. The combination of wide-field and high-resolution fluorescence imaging systems with automated image analysis should be investigated to maximize overall diagnostic performance for early detection of oral neoplasia.

Worldwide incidence and mortality rates due to cancer continue to rise, with the burden of disease increasingly shifting to developing countries. Several optical diagnostic methods are under development to enable earlier detection of cancer, however, these are primarily intended for use in healthcare facilities in industrialized countries. Using knowledge gained from early clinical studies with large-scale prototype systems, we have designed and tested low-cost, portable versions of these instruments. We propose that these systems may be used for early diagnosis and screening in developing countries, and that pilot clinical studies are warranted in these low-resource settings.

Background & Aims High-resolution microendoscopy is an optical imaging technique with the potential to improve the accuracy of endoscopic screening for esophageal squamous neoplasia. Although these microscopic images can be interpreted readily by trained personnel, quantitative image analysis software could facilitate the use of this technology in low-resource settings. In this study, we developed and evaluated quantitative image analysis criteria for the evaluation of neoplastic and non-neoplastic squamous esophageal mucosa. Methods We performed an image analysis of 177 patients undergoing standard upper endoscopy for screening or surveillance of esophageal squamous neoplasia, using high-resolution microendoscopy, at 2 hospitals in China and at 1 hospital in the United States from May 2010 to October 2012. Biopsy specimens were collected from imaged sites (n = 375), and a consensus diagnosis was provided by 2 expert gastrointestinal pathologists and used as the standard. Results Quantitative information from the high-resolution images was used to develop an algorithm to identify high-grade squamous dysplasia or invasive squamous cell cancer, based on histopathology findings. Optimal performance was obtained using the mean nuclear area as the basis for classification, resulting in sensitivities and specificities of 93% and 92% in the training set, 87% and 97% in the test set, and 84% and 95% in an independent validation set, respectively. Conclusions High-resolution microendoscopy with quantitative image analysis can aid in the identification of esophageal squamous neoplasia. Use of software-based image guides may overcome issues of training and expertise in low-resource settings, allowing for widespread use of these optical biopsy technologies.

This paper describes a warpage study on LOC-TSOP memory devices. The main objectives of this study are to evaluate the impact of the mold compound on the package warpage with different sized dies. It was found that the balance of the bending between at the edge side region and at the die attach region controls the package warpage. The package tends to bend convex upward at the edge side region and concave downward at the die attach region. The various mold compounds were prepared to study the effect of the components, particularly resin and filler. It has shown that the optimum mold compound can be changed according to the inner structure of the LOC-TSOP. The effect of post mold cure on package warpage was also examined based on the cure rate.

OBJECTIVE When the pedicle screw insertion technique is failed or not applicable, C7 intralaminar screw insertion method has been used as an alternative or salvage fixation method recently. However, profound understanding of anatomy is required for safe application of the bilaterally crossing laminar screw at C7 in clinic. In this cadaveric study, we evaluated the anatomic feasibility of the bilateral crossing intralaminar screw insertion and especially focused on determination of proper screw entry point. METHODS The C7 vertebrae from 18 adult specimens were studied. Morphometric measurements of the mid-laminar height, the minimum laminar thickness, the maximal screw length, and spino-laminar angle were performed and cross-sectioned vertically at the screw entry point (spino-laminar junction). The sectioned surface was equally divided into 3 parts and maximal thickness and surface area of the parts were measured. All measurements were obtained bilaterally. RESULTS The mean mid-laminar height was 13.7 mm, mean minimal laminar thickness was 6.6 mm, mean maximal screw length was 24.6 mm, and mean spinolaminar angle was 50.8±4.7°. Based on the measured laminar thickness, the feasibility of 3.5 mm diameter intralaminar screw application was 83.3% (30 sides laminae out of total 36) when assuming a tolerance of 1 mm on each side. Cross-sectional measurement results showed that the mean maximal thickness of upper, middle, and lower thirds was 5.0 mm, 7.5 mm, and 7.3 mm, respectively, and mean surface area for each part was 21.2 mm(2), 46.8 mm(2), and 34.7 mm(2), respectively. Fourteen (38.9%) sides of laminae would be feasible for 3.5 mm intralaminar screw insertion when upper thirds of C7 spino-laminar junction is the screw entry point. In case of middle and lower thirds of C7 spino-laminar junction, 32 (88.9%) and 28 (77.8%) sides of laminae were feasible for 3.5 mm screw insertion, respectively. CONCLUSION The vertical cross-sectioned area of middle thirds at C7 spinolaminar junction was the largest area and 3.5 mm screw can be accommodated with 77.8% of feasibility when lower thirds were the screw entry point. Thus, selection of middle and lower thirds for each side of screw entry point in spino-laminar junction would be the safest way to place bilateral crossing laminar screw within the entire lamina. This anatomic study result will help surgeons to place the screw safely and accurately.

Background: Laparoscopic and natural orifice transluminal endoscopic surgery techniques can diagnose peritoneal findings that suggest tumor cell dissemination. However, they have not been incorporated into routine practice, mainly owing to their complexity. To develop a minimally invasive endoscopic technique for the diagnosis of peritoneal findings, we conducted feasibility study using an acute swine model. Materials and Methods: This study involved six domestic pigs. Trans-gastric access to the peritoneal cavity was performed utilizing an endoscopic ultrasound fine needle aspiration (EUS-FNA) technique. After dilation of the needle hole with a biliary dilatation catheter and balloon, a small-caliber scope was inserted into the peritoneal cavity. Peritoneal images were obtained with the scope and a high-resolution microendoscope (HRME). Main outcome measurements were technical feasibility and time needed to access the peritoneal cavity. Results: Direct visualization of the peritoneum was successful in all six pigs and gained access to the gross appearance of the peritoneal cavity. HRME imaging with topical contrast agent also obtained reasonable quality images representing nuclei of the peritoneal mesothelium. Average operation time from the initiation of EUS-FNA to acquiring peritoneal images was 26.5 min (range 15-40 min). Autopsy found no damage to the adjacent organs, and stomach wall defects were tightly closed with hemostasis clips. Conclusion: EUS-assisted direct peritoneal visualization with small-caliber scope is technically feasible. HRME may assist in the diagnosis of findings on the peritoneum.