Medium Magnification Research Articles

Screening Mosquito Larvae Under a Fluorescence Binocular Microscope.

Transgenic mosquitoes are used in many aspects of mosquito research, and convenient selection markers are crucial to identifying transgenic individuals even among large numbers of wild types. Visual markers, in the form of fluorescent proteins expressed in larval and adult mosquito tissues, are the most commonly used. This requires observing large numbers of mosquitoes under the fluorescence microscope and recognizing positive individuals expressing the fluorescent genetic marker. As research models, mosquito larvae possess the following two advantages over many other insects, greatly facilitating fluorescence screening: (1) Being aquatic, they can be isolated in a drop of clear water, an ideal medium for live observations under the binocular microscope; and (2) synchronous hatching from many eggs is easily obtained, so that large populations of larvae can be screened in batches of several tens of thousands at a time. Screening at the neonate stage, when larvae are ∼1-mm-long, allows the concentration of hundreds of larvae in a drop of water that fits in the observation field of the microscope at medium magnification. Thus, many individuals can be screened rapidly. We strongly recommend screening larvae at the neonate stage and before any feeding for two reasons, as follows: (1) Food particles can be strongly autofluorescent, thereby dramatically increasing the fluorescence background noise around larvae, and (2) tissue autofluorescence increases during development, notably in the digestive tract, significantly decreasing the specific signal-to-noise ratio. In this protocol, we guide the experimenter step-by-step for a fast and efficient medium-throughput manual screening for fluorescent larvae.

Craniotomy size determines the neurosurgeon - microscope interaction: A proof-of-concept study

PurposeOur study aimed to compare the differences in quantitative parameters, describing the processes of neurosurgeons’ interaction with a microscope when performing traditional and minimally invasive approaches. MethodsWe designed a prospective observational study to assess the quantitative parameters of neurosurgeon-microscope interactions. Eighteen patients with intracranial tumors were enrolled in the research.All cases were divided into 2 groups: patients with deep-seated tumors, which were operated on with craniotomy apertures of standard sizes – 3 to 4.5 cm (standard craniotomy SC group) and patients with deep-seated tumors operated on with minimally invasive burr hole approaches with a diameter of 1.4 cm (BH group).Three video cameras were used to register and analyze surgeon-operating microscope interactions. The interaction of the neurosurgeon with the microscope was described by the following parameters: microscope repositioning; time of work at low, medium, and high magnification; and the number of changes in focal length. All the interaction parameters were measured per minute of the microsurgical operation stage. ResultsNine parameters significantly differed (p value < 0.05) between groups: “total time needed for all microscope positioning adjustments”, “number of microscope positioning adjustments per minute”,”average duration of one microscope position adjustment”,“operating time at high magnification”, “operating time at low magnification”,“proportion of operating time at high magnification”, “proportion of operating time at low magnification”, “total time for interaction between neurosurgeon and microscope”, “proportion of total time for interaction”. Significant P values withstood Benjamini-Hochberg's adjustment for all variables. ConclusionThe results of the study provide the grounds to postulate that there is a direct and significant relationship between the size of the craniotomy and the frequency of microscope repositioning, as well as the degree of intraoperative microscope magnification.

S3504 A 64-Year-Old Male With Recurrent Brunneroma

Introduction: Brunneroma (also termed Brunner's gland hyperplasia, adenoma, or hamartoma) is a rare benign tumor arising within the alkaline-producing exocrine glands located in the submucosal layer of the duodenum. Most patients with brunneroma are asymptomatic, but others may present with gastric obstruction-like symptoms or upper gastrointestinal bleed. Less than 200 cases have been reported. We present a case of a patient with recurrent Brunneroma. Case Description/Methods: A 64-year-old male was evaluated for intermittent achy abdominal pain in the umbilical region associated with postprandial bloating and early satiety. He had a similar presentation twelve years ago and underwent esophagogastroduodenoscopy (EGD), which showed a 3 cm polypoid mass within the duodenal bulb. The mass was removed endoscopically through snare polypectomy and Roth basket retrieval, leading to the resolution of his symptoms. Given his previous history, an EGD was obtained, which showed a 4 cm tan-pink polypoid mass prolapsing from the duodenal bulb through the pylorus into the stomach (Figure A). Endoscopic ultrasound revealed a mucosal mass on a stalk with a cystic component and no submucosal involvement (Figure B). He was evaluated by an advanced endoscopist and felt the mass was appropriate for endoscopic removal. Therefore, an Endoloop was placed around the mass initially with the base injected with epinephrine and then removed with cautery, placing a clip on the base afterward. Removal of the mass led to the resolution of his symptoms. The pathology report demonstrated Brunner gland hyperplasia, benign metaplastic cysts arising from Brunner gland ducts, and no dysplasia (Figure C; H&E stain medium magnification). Discussion: Brunner's gland is located in the proximal part of the duodenum above the hepatopancreatic sphincter. The mechanism Brunneroma remains unclear. There is currently no consensus regarding surgical versus endoscopic management of Brunneroma. However, there is increasing endoscopic utilization avoiding a more invasive surgical procedure.Figure 1

Grocott Methenamine Silver Staining Is the Optimal Approach to Histological Diagnosis of Pulmonary Cryptococcosis.

BackgroundHistological staining methods for Cryptococcus identification vary in accuracy. This study aimed to investigate the clinical value of Grocott methenamine silver (GMS), periodic acid-Schiff (PAS), and Alcian blue (AB) staining in the diagnosis of pulmonary cryptococcosis (PC).MethodsFrom April 2004 to June 2021, the clinical and pathological data of 152 patients with PC were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The sensitivity and identifiability of GMS, PAS, and AB staining for histological diagnosis were systematically evaluated using statistical methods combined with the microscopic characteristics of PC cases.ResultsStatistical analysis showed that the detection rates of GMS, PAS, and AB staining were 100.0% (152/152), 94.7% (144/152), and 81.6% (124/152), respectively. McNemar’s test showed that the sensitivity of GMS was significantly higher than those of PAS (P = 0.008) and AB stains (P < 0.001). Both PAS and AB stains had obvious non-specific staining, which interfered with the detection of Cryptococcus, and increased diagnostic difficulties. In contrast, in GMS staining, Cryptococcus spores were prominent with a clean background and were clearly observed at low or medium power magnification, with the identifiability significantly better than those of PAS or AB staining.ConclusionGMS staining had sensitivity up to 100%, and identifiability that was better than those of PAS and AB staining. GMS is the best method for histological diagnosis of PC.

Fast determination of sample thickness through scanning moiré fringes in scanning transmission electron microscopy

Sample thickness is an important parameter in transmission electron microscopy (TEM) imaging for interpreting image contrast and understanding the relationship between properties and microstructure. In this study, we introduce a method for sample thickness determination in scanning TEM (STEM) mode based on scanning moiré fringes (SMFs). Focal-series SMF imaging is used and sample thickness can be determined in situ at a medium magnification range, with beam damage and contamination avoided to a large extent. It provides a fast and convenient approach for determining sample thickness in TEM imaging, which is particularly useful for beam-sensitive materials.

Significance of Ki67, BCL 2 Immunoexpression in Urothelial Carcinoma and their Association with Grading and Staging: A Descriptive Cross-sectional Study

Introduction: Urinary bladder is the most common site of malignancy in the urinary tract and the most common type of cancer of urothelial origin is urothelial cell carcinoma. This subtype constitutes more than 90% of bladder cancer. Histological grade of tumour is one of the most important predictive parameter for prognosis and biological behavior of urothelial carcinoma. As some crucial discordances may arise between pathologists because of subjectivity, so immunohistochemistry can be used for accurate diagnosis and prediction of prognosis. Aim: To study the immununohistochemical expression of B-Cell Lymphoma 2 (BCL 2) and Ki(Kiel)-67 markers and their association with tumour grade and stage in urothelial carcinomas. Materials and Methods: A descriptive cross-sectional study was conducted in the Department of Pathology of a tertiary care R G Kar Medical College and Hospital, Kolkata, West Bengal, India. West Bengal, India, on a total of 75 cases from January 2018 to June 2019. The study population comprised of cases of urothelial lesions as diagnosed by cystoscopy, who underwent standardized operative procedures. Relevant sections were taken from different parts of the fixed specimens. Histopathological Examination (HPE) was done to confirm the presence or absence of non-invasive and invasive carcinoma, and the depth of invasive tumour cells for pathological staging. Evaluation of the level of cytological and architectural disorder at low and medium magnification (100X and 200X) were done for grading. Cytological disorder were defined as abnormalities in nuclear size, shape, and chromatin, while architectural disorder were defined as abnormalities in the orientation of the cells in relation to each other and to the basement membrane of the papillae. Immunohistochemical analysis was done for Ki67 and BCL 2 expression in paraffin embedded sections in all the cases. In this study, chi-square test was done with International Business Management(IBM) Statistical Package for Social Sciences (SPSS), as per which, the association between two variables is statistically significant if p value is &lt;0.05. Results: Out of total 75 cases(mean age: 67.3 years) analysed, 58 cases (77.3%) had Invasive type of bladder neoplasm, followed by low grade papillary urothelial carcinoma 10 (13.3%), and papillary urothelial neoplasm of low malignant potential 07 (9.3%). Ki67 expression in urothelial carcinoma has more significance because 59 cases (78.7%) showed high level expression (&gt;20%) out of 75 cases, whereas only 12 (16%) cases show high level expression (4+) of BCL 2 marker. Conclusion: Ki67 overexpression is seen more commonly, than BCL 2, in high grade tumours as well as in advanced stage. So Ki67 may be used as a marker to predict aggressive behavior and to differentiate low grade and high grade tumours also.

Retracted Rotator Cuff Repairs Heal With Disorganized Fibrogenesis Without Affecting Biomechanical Properties: A Comparative Animal Model Study

To determine the differences in the scar tissue formation during the healing of the repaired retracted cuff tear from that of the nonretracted tear. Eighteen right rabbit shoulders received a 1-cm transverse cuff incision over the footprint to simulate "nonretracted cuff tears" before the transosseous repairs (group A). A 1-cm tendinous portion was excised from 18 left shoulders to create defects to simulate "retracted cuff tears" before repairing the defects (group B). At week 12 postrepair, 20 and 16 shoulders underwent histologic and biomechanical analyses, respectively. Eight shoulders were used as a control group for biomechanical analyses. All specimens showed good healing and continuity of the repaired tendons. At low magnification, fibrous tissue firmly held the tendon-to-bone junctions in group A; however, all specimens in group B showed medially retracted tendons with fibrous tissue continuity between the tendon stumps and footprints. At medium magnification, more irregular collagen fiber orientation was observed in group B. Polarized light microscopy showed fibrous tissue continuity with medially retracted tendons in group B. When we quantified collagen fiber orientation using ImageJ software, group B had inferior grayscale measurements when compared with group A (P= .001). At week 12, no statistical differences existed in mean loads-to-failure at the repair sites between the groups (P= .783). In the nonretracted cuff tears, fibrous tissue bound the tendon-to-bone junction with healing. After the healing of the retracted cuff tears, continuity of nontendinous tissue was observed adjacent to the medially retracted tendon, which comprised more disorganized immature fibrous tissue than that in the nonretracted cuff tears. Unlike the healing of nonretracted rotator cuff tear, repairing of the "retracted" tendon end of cuff tear still resulted in retraction of the tendon back to its original position but being held down with fibrous tissue to the footprint.

S1780 An Unexpected Outcome of Incidental Rectal Nodule

INTRODUCTION: Well differentiated neuroendocrine tumors (NETs) most commonly originate in the gastrointestinal (GI) tract. Generally speaking, NETs are relatively rare tumors, with an age-adjusted incidence for non-pancreatic primaries of 4.7 per 100,000. Within the GI tract, the most common site for NETs is the small intestine, followed by rectum, appendix colon, and stomach. CASE DESCRIPTION/METHODS: We present a case of a 39-year-old female with no significant past medical history referred by an outside gastroenterologist for endoscopic ultrasound (EUS) and possible resection of rectal subepithelial lesion (SEL) identified on colonoscopy performed for evaluation of diarrhea. On EUS the SEL was found to be well-circumscribed, hypoechoic, homogeneous, arising from muscularis mucosa and measuring approximately 3 × 5 mm (Figure 1a). The SEL was then removed by band-assisted endoscopic mucosal resection (EMR) utilizing Boston Scientific Captivator EMR device (Figure 1b). Pathologic review revealed that the SEL was a well-differentiated NET (Figure 2a) with immunohistochemical staining positive for synaptophysin (Figure 2b) and negative for chromogranin. Ki-67 was positive in <1% of tumor cells (Figure 2c). The findings were compatible with a carcinoid tumor and the resection margins were noted to be negative. The patient subsequently underwent a dotatate PET-CT scan that showed dotatate avid implants within the pelvis as well as small dotatate avid retroperitoneal lymph nodes consistent with metastatic carcinoid (Figure 3). Currently she remains asymptomatic and under observation by oncology without administration of systemic therapy. DISCUSSION: Rectal NETs are usually asymptomatic and found incidentally on endoscopy that is being performed for another reason. Most rectal NETs are localized at the time of diagnosis, however, the size of the tumor correlates with the likelihood of metastatic disease. Tumors that are smaller than 1.0 cm are infrequently metastatic. Approximately 6 percent of tumors between 1 and 1.9 cm are metastatic and 24 percent of those over 2 cm metastasize to the liver. Other features that portend a poor prognosis include deep invasion into the muscularis propria or deeper, lymphovascular invasion and high mitotic rate (≥2 per 50 high-power fields). Treatment for neuroendocrine tumors range from observation in asymptomatic patients to surgical resection to systemic therapy with somatostatin analogs. The treatment of choice is tailored to each individual patient.Figure 1.: (a) EUS image of subepithelial rectal lesion; (b) endoscopy image showing band application prior to endoscopic mucosal resection (EMR).Figure 2.: (a) Pathology slide displaying medium magnification of rectal neuroendocrine tumor; (b) Pathology slide of rectal neuroendocrine tumor with synaptophysin stain; (c ) Pathology slide of rectal neuroendocrine tumor with Ki-67 stain.Figure 3.: Imaging displaying dotatate avid implants in pelvis.

S2031 Esophageal Polyps Associated With Barrett’s Esophagus: Is This an Adenocarcinoma or Not?

INTRODUCTION: Barrett’s esophagus (BE) appears usually as a flat salmon-colored lesion. Esophageal adenocarcinomas associated with BE, traditionally appearing as an ulcer or a mass, seem to originate via the BE-dysplasia-carcinoma sequence. There is only a handful of reported cases of polypoid esophageal adenocarcinomas associated with BE. We describe a compelling case of incidental finding of BE with multiple esophageal polyps that turned out to be invasive adenocarcinoma. CASE DESCRIPTION/METHODS: A 60-year-old male with a past medical history of coronary artery disease and atrial fibrillation underwent laparoscopic cholecystectomy for chronic abdominal discomfort. His postoperative course was complicated by biliary leak; hence ERCP was performed and successful biliary stent placement was achieved. Two months later, an upper endoscopy was being performed for biliary stent removal when multiple 4 to 15 mm pedunculated and sessile polyps were identified in the distal esophagus (Figure 1). The upper endoscopy also revealed mucosal changes consistent with long-segment BE, C8-M10 per Prague criteria, a small hiatal hernia and multiple small gastric polyps. All large esophageal polyps were successfully resected with cold snare, after Eleview injection. The exception was a 10 mm polyp that was not liftable. Pathology reading of the resected polyps demonstrated low- and high-grade dysplasia along with intramucosal carcinoma (Figure 2). While the unresectable polyp revealed a moderately to poorly differentiated adenocarcinoma with HER2 positivity on FISH analysis. This was followed by an endoscopic ultrasound, which showed a 2 cm mass with irregular borders infiltrating the submucosa plus peritumoral lymph node involvement (Figure 3). The patient was referred to a multidisciplinary team of GI oncologists as well as a foregut surgeon for further management. DISCUSSION: Polypoid lesions of the esophagus are considered rare. To this date, there is no clear consensus on their management. We did a literature review, and we found 27 reported cases of polypoid dysplasia of the esophagus, 10 of them were having multiple lesions. The table is showing their characteristics and outcome. Through this case, we emphasize the association between these polypoid lesions and the adenocarcinoma of the esophagus. Hence, when esophageal polyps associated with BE are encountered, we are recommending their resection associated to multiple biopsies.Figure 1.: Upper Gastrointestinal endoscopy: Multiple polypoid lesions in the lower third of the esophagus, no signs of bleeding.Figure 2.: Image A, Esophageal Biopsy, High Magnification: Intestinal metaplasia with nuclear enlargement, loss of nuclear polarization, occasional prominent nucleoli, and increased apoptosis, as well as crypt fusion and cribriforming; changes consistent with high-grade dysplasia. Image B: Sheets of infiltrating malignant cells at medium magnification infiltrating the lamina propria.Figure 3.: Upper Endoscopic Ultrasonography: Hypoechoic mass in the lower third of the esophagus, the lesion is non circumferential, the borders are irregular, 5 mm in thickness with submucosal invasion.

Optimizing Visual Performance With Digitally Assisted Vitreoretinal Surgery.

Evaluate depth of field (DOF) and lateral resolution (LR) on digitally assisted vitreoretinal surgery (DAVS) with different camera aperture values and TV viewing distances to determine maximal optical performance. Five retinal surgeons objectively evaluated DOF and LR at low (10×), medium (15×), and high (20×) magnification on a conventional microscope (CM) and DAVS. DAVS was evaluated at different camera apertures (30%, 50%, and 75%) and TV viewing distances (1.2 meters [m], 1.5 m, and 2.0 m). A comparison between CM and optimized DAVS values was carried out. On DAVS, DOF significantly increased as camera aperture size was reduced (75% to 50%, P = .022; 75% to 30%, P ≤ .001; and 50% to 30%, P = .001) and as microscope magnification was reduced (high to medium, P = .009; high to low, P ≤ .001; and medium to low, P = .002). LR significantly increased as microscope magnification was increased (low to medium, P = .010; low to high, P ≤ .001; and medium to high, P = .015) and as TV display distance decreased (2.0 m to 1.5 m, P = .019; 2.0 m to 1.2 m, P = .009; and 1.5 m to1.2 m, P = .185). When comparing optimized DAVS against CM, DOF was significantly larger on DAVS (P = .019). LR on DAVS outperformed the CM, but statistical significant was not reached (P = .185). The authors' study is the first to evaluate different aperture, magnification, and TV distance settings, and when optimized, DAVS can significantly outperform a CM with respect to DOF and LR. Optimized visual performance for DAVS is obtained with a 30% camera aperture and a TV viewing distance no further than 1.5 m. Maximal microscope magnification to exploit LR during macular surgery and low-to-medium magnification to increase DOF for overall vitrectomy surgery is recommended. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:S15-S21.].

Characterisation of the multi-scale fabric features of high plasticity clays

An investigation of the microstructural features of a high-plasticity clay, in both its natural conditions and reconstituted in the laboratory, is described. Scanning electron microscopy is used here to characterise the fabric at different magnifications, while image processing of the micrographs delivers a quantitative assessment of the fabric orientation. The results of energy-dispersive X-ray spectroscopy and swelling tests, as reported in a previous study by the authors, are used to characterise the bonding nature and strength, as well as mercury intrusion porosimetry to investigate clay porosimetry. Despite their identical composition, the natural and the reconstituted clay have experienced different deposition and loading history, generating different microstructural features that are shown to underlie their differences in state. For both clays, one-dimensional (1D) compression to medium–high pressures is seen to determine a well-oriented medium magnification fabric. However, larger-scale observations and the corresponding image processing results reveal non-uniform local fabric features, hence making fabric characterisation dependent on the scale of analysis and bringing about the issue of identifying the clay micro-scale representative element volume relating to the clay macro-behaviour. The micro-REV is identified for the clays under study and its connection with the macro-behaviour characterised. The microstructural evolution induced by 1D compression to very high pressures is shown to concern mainly the clay porosity and porosimetry, the fabric orientation being steady, thus explaining the isotropic hardening observed in laboratory tests.

S69 From outside: MU characteristics by stimulation methods during acute and chronic denervation

Introduction We can observe different characteristics of the motor units and their compound activity by stimulating the nerve directed to the relevant muscle and looking through different magnifications of our electrophysiological microscope. Methods Low magnification: CMAP, medium magnification: estimating the number of motor units innervating the muscle (MUNE), higher magnification: CMAPscan and F waves. Results CMAP: The most simple, easy and inclusive of all. Gives an overall impression about the condition of the muscle. However it is not specific, has wide normal ranges and tends to veil the abnormality by reorganizational changes of the constituent MUs. MUNE methods: To unveil the amount of MU loss caused by the lesion, but more time consuming. Each method has its own drawbacks. Incorrect results can be caused by the reinnervation process itself. May behave differently in chronic and acute denervation. CMAP scan: To observe the general appearance of the constituent MUs, especially those enlarged due to the denervation-reinnervation process. Can be used for qualitative observation or for follow-up with quantitative parameters. F-wave characteristics: To observe the components of CMAP through a narrow window. We can deduce information about the MUs and the physiological conditions acting on them, especially by taking their less frequently used parameters into consideration, e.g. persistence, amplitude and repeater Fs. Conclusion We can extract more information about the pathophysiology of MUs and their compound function by looking concurrently through the different magnifications provided by our routine neurophysiological device.

Strain mapping in a nanoscale-triangular SiGe pattern by dark-field electron holography with medium magnification mode.

Recent years have seen a great deal of progress in the development of transmission electron microscopy-based techniques for strain measurement. Dark-field electron holography (DFEH) is a new technique offering configuration of the off-axis principle. Using this technique with medium magnification (Holo-M), we carried out strain measurements in nanoscale-triangular SiGe/(001) Si with (004), (2-20) and (-111) diffraction spots. The reconstruction of holograms and interpretation of strain maps in term of strain precision were discussed and the strain distributions in the SiGe/(001) Si patterns were visualized. Based on linear anisotropic elastic theory for strain simulation, the simulated results obtained by the finite element method compared with the experimental results acquired byDFEH. The strain values were found to be 0.9-1.0%, 1.1-1.2% and 1.0-1.1%, for the (004), (2-20) and (-111) diffracted beams, respectively, and the strain precisions were determined to be ~2.1×10-3, 3.2×10-3 and 9.1×10-3 for the corresponding diffraction spots. As a result, DFEH is highlighted as a powerful technique for strain measurement, offering high-strain precision, high-spatial resolution and a large field of view.

RNA in-situ hybridization is a practical and effective method for determining HPV status of oropharyngeal squamous cell carcinoma including discordant cases that are p16 positive by immunohistochemistry but HPV negative by DNA in-situ hybridization

Evaluation of human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC) has become increasingly important for prognostication and clinical trial enrollment. This assessment is confounded in OPSCCs that are p16 positive by immunohistochemistry (IHC) but HPV negative by DNA in situ hybridization (DISH). This study evaluates whether E6/E7 mRNA in situ hybridization (RISH) can detect transcriptionally active HPV in these problematic cases. Eighty-two head and neck squamous cell carcinoma cases that had previously undergone p16 IHC and HPV DISH were evaluated with two RISH platforms and a second-generation DISH probe. The study included 21 p16+/DISH+ concordant cases, 19 p16-/DISH- concordant cases, and 42 p16+/DISH- discordant cases. RISH identified E6/E7 mRNA in 37 (88%) p16+/DISH- cases, 21 (100%) p16+/DISH+ cases, and 0 (0%) p16-/DISH- cases. RISH signals were clearly visible at low to medium magnification in 97% of positive cases, facilitating almost-perfect inter-observer reproducibility. The performance of the manual and automated RISH platforms were equivalent (kappa=0.915). Only 29% of carcinomas that demonstrated E6/E7 mRNA transcriptional activity were positive using the 2nd generation DISH probe. HPV RISH is a highly sensitive and specific platform that can clarify the HPV status of those perplexing OPSCCs that are p16 positive by IHC but HPV negative by DISH. Moreover, it is easy to interpret, readily adaptable to the clinical laboratory, and provides direct evidence of HPV transcriptional activity. E6/E7 RISH should be considered as a first-line platform for determination of HPV status in OPSCCs.

Compressed sensing traction force microscopy

Adherent cells exert traction forces on their substrate, and these forces play important roles in biological functions such as mechanosensing, cell differentiation and cancer invasion. The method of choice to assess these active forces is traction force microscopy (TFM). Despite recent advances, TFM remains highly sensitive to measurement noise and exhibits limited spatial resolution. To improve the resolution and noise robustness of TFM, here we adapt techniques from compressed sensing (CS) to the reconstruction of the traction field from the substrate displacement field. CS enables the recovery of sparse signals at higher resolution from lower resolution data. Focal adhesions (FAs) of adherent cells are spatially sparse implying that traction fields are also sparse. Here we show, by simulation and by experiment, that the CS approach enables circumventing the Nyquist–Shannon sampling theorem to faithfully reconstruct the traction field at a higher resolution than that of the displacement field. This allows reaching state-of-the-art resolution using only a medium magnification objective. We also find that CS improves reconstruction quality in the presence of noise. Statement of SignificanceA great scientific advance of the past decade is the recognition that physical forces determine an increasing list of biological processes. Traction force microscopy which measures the forces that cells exert on their surroundings has seen significant recent improvements, however the technique remains sensitive to measurement noise and severely limited in spatial resolution. We exploit the fact that the force fields are sparse to boost the spatial resolution and noise robustness by applying ideas from compressed sensing. The novel method allows high resolution on a larger field of view. This may in turn allow better understanding of the cell forces at the multicellular level, which are known to be important in wound healing and cancer invasion.

Pattern image enhancement by extended depth of field

Most optical defect localization techniques such as dynamic laser stimulation or photon emission microscopy require a pattern image of the device to be taken. The main purpose is for device navigation, but it also enables the analyst to identify the location of the monitored activity by superimposing it onto the pattern image. The defect localization workflow usually starts at low or medium magnification. At these scales, several factors can lead to a lack of orthogonality of the sample with the optical axis of the system. Therefore, images can be locally out of focus and poorly resolved. In this paper, a method based on Depth of Field Extension is suggested to correct the pattern image.

Accurate Blood Flow Measurements: Are Artificial Tracers Necessary?

Imaging-based blood flow measurement techniques, such as particle image velocimetry, have become an important tool in cardiovascular research. They provide quantitative information about blood flow, which benefits applications ranging from developmental biology to tumor perfusion studies. Studies using these methods can be classified based on whether they use artificial tracers or red blood cells to visualize the fluid motion. We here present the first direct comparison in vivo of both methods. For high magnification cases, the experiments using red blood cells strongly underestimate the flow (up to 50% in the present case), as compared to the tracer results. For medium magnification cases, the results from both methods are indistinguishable as they give the same underestimation of the real velocities (approximately 33%, based on in vitro reference measurements). These results suggest that flow characteristics reported in literature cannot be compared without a careful evaluation of the imaging characteristics. A method to predict the expected flow averaging behavior for a particular facility is presented.

14. Three famous diseases: addison, hodgkin and bright

This is a display of the gross pictures of three specimens from original cases from which each of these men described the diseases that are now named after them. Also displayed are digital images taken at low, medium and high magnification of microscopic sections taken from these cases. The specimens are now 180 years old. At the time there were no facilities for microscopic examination. In 1960 the specimens were taken from their original display jars and re-mounted in clear solutions. At that time a block of tissue was taken from each of them and paraffin sections were made. A store of unstained sections was kept for use in later years. The author was able to obtain samples of each of these unstained sections in 2010 and these microscopic sections were stained by Stephen Riley and Barry Madigan, and then photographed. This is a unique presentation and gives pathologists an opportunity of seeing what they think the diagnoses really are, and how close were these cases to diseases that are called by their names today. My diagnoses for the cases are as follows. Addison All the cases displayed would be consistent with a diagnosis of chronic tuberculous inflammation which was the diagnosis that Addison made himself. A ZN stain was performed on each case and all three were negative, which is not surprising in view of the chronic inflammatory reaction that is present. Hodgkin Cases 1 and 2 are consistent with a diagnosis of Hodgkin’s disease with numerous RS cells being seen. These could both be mixed cellularity classical Hodgkin lymphoma. Case 3 appears to be a diffuse B cell lymphoma of uncertain type. In spite of prolonged attempts at antigen retrieval, CD20 immu-nostain on Case 3 was entirely negative and CD30 on Cases 1 and 2 were also negative. Bright Case 1: some form of acute diffuse proliferative glomer-ulonephritis. Case 2: amyloidosis; Congo red stain is positive. Case 3: possibly diabetic glomerulosclerosis; the Congo red stain is totally negative.

In Situ Mitochondrial Ca2+ Buffering Differences of Intact Neurons and Astrocytes from Cortex and Striatum

The striatum is particularly vulnerable to neurological disorders, such as Huntington disease. Previous studies, with nonsynaptic mitochondria isolated from cortical and striatal homogenates, suggest that striatal mitochondria are highly vulnerable to Ca(2+) loads, possibly influencing striatal vulnerability. However, whether and how neuronal and glial mitochondria from cortex and striatum differ in Ca(2+) vulnerability remains unknown. We test this hypothesis using a novel strategy allowing comparisons of mitochondrial Ca(2+) buffering capacity in cortical and striatal neuron-astrocyte co-cultures. We provide original evidence that mitochondria not only in neurons but also in astrocytes from striatal origin exhibit a decreased Ca(2+) buffering capacity when compared with cortical counterparts. The decreased mitochondrial Ca(2+) buffering capacity in striatal versus cortical astrocytes does not stem from variation in mitochondrial concentration or in the rate of intracellular Ca(2+) elevation, being mechanistically linked to an increased propensity to undergo cyclosporin A (CsA)-sensitive permeability transition. Indeed, 1 microm CsA selectively increased the mitochondrial Ca(2+) buffering capacity of striatal astrocytes, without modifying that of neurons or cortical astrocytes. Neither thapsigargin nor FK506 modified mitochondrial Ca(2+) buffering differences between cell types, excluding a predominant contribution of endoplasmic reticulum or calcineurin. These results provide additional insight into the mechanisms of striatal vulnerability, showing that the increased Ca(2+) vulnerability of striatal versus cortical mitochondria resides in both intact neurons and astrocytes, thus positioning the striatum at greater risk for disturbed neuron-astrocyte interactions. Also, the selective effect of CsA over striatal astrocytes suggests that in vivo neuronal sheltering with this compound may indirectly result from astrocytic protection.

Optimisation of Magnification Levels for near Infrared Chemical Imaging of Blending of Pharmaceutical Powders

A critical parameter in the evaluation of pharmaceutical dosage forms by hyperspectral imaging is the level of magnification. If the magnification (as set by the optical objective) is inadequate to resolve the relevant features, then the value of the imaging is diminished; if the magnification level is greater than is required, then the field of view is unnecessarily reduced. The purpose of this study was to determine an optimum magnification level for the study of powder mixing. Relevant features in this system include distribution of individual components within samples and the overall content of a given sample. In the present study, three magnification levels of near infrared (NIR) chemical imaging objectives were evaluated for their effects on imaging a blend of pharmaceutical materials (powders). High, medium and low objective magnification levels were investigated by comparing the resulting blend surface images of a two-component (salicylic acid and lactose) pharmaceutical powder mixture. Multiple images from high and medium magnification were concatenated so that an equivalent field of view was obtained for all magnification levels. Univariate images, principal component analysis score images, partial least squares predicted images and spectra extracted from different intensity regions in the area images were analysed qualitatively and quantitatively for comparison. A series of images spanning a strip across the centre of the circular field were collected at each magnification level and compared with respect to surface features elucidated and area of blend surface imaged. Analyses of images indicate that the three magnification levels delineate the component distribution for this particular powder system similarly. Images obtained at the low magnification level demonstrated adequate resolution and provided the broadest view of the blend surface. It is concluded that the low optical magnification level was adequate for the system being studied and is the preferred mode for pharmaceutical powder blend image data collection for this system.