Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.     

一种基于视网膜主血管方向的视盘定位及提取方法

为分割出眼底图像中的视盘,构建基于眼底图像的计算机辅助诊断系统,提出了一种基于视网膜主血管方向的视盘定位及提取方法。首先,利用Otsu阈值分割眼底图像R通道获取视盘候选区域;然后利用彩色眼底图像的HSV空间的H通道提取视网膜主血管并确定主血管方向;在此基础上,通过在方向图内寻找出对加权匹配滤波器响应值最高的点确定视盘中心位置;最后,利用该位置信息从视盘候选区域中"挑选"出真正的视盘。利用该方法对100幅不同颜色、不同亮度的眼底图像进行视盘分割,得到准确率98%,平均每幅图像处理时间1.3 s。结果表明:该方法稳定可靠,能快速、有效分割出眼底图像中的视盘。     

Interactive graph-cut segmentation for fast creation of finite element models from clinical ct data for hip fracture prediction

In this study, we propose interactive graph cut image segmentation for fast creation of femur finite element (FE) models from clinical computed tomography scans for hip fracture prediction. Using a sample of N = 48 bone scans representing normal, osteopenic and osteoporotic subjects, the proximal femur was segmented using manual (gold standard) and graph cut segmentation. Segmentations were subsequently used to generate FE models to calculate overall stiffness and peak force in a sideways fall simulations. Results show that, comparable FE results can be obtained with the graph cut method, with a reduction from 20 to 2–5 min interaction time. Average differences between segmentation methods of 0.22 mm were not significantly correlated with differences in FE derived stiffness (R² = 0.08, p = 0.05) and weakly correlated to differences in FE derived peak force (R² = 0.16, p = 0.01). We further found that changes in automatically assigned boundary conditions as a consequence of small segmentation differences were significantly correlated with FE derived results. The proposed interactive graph cut segmentation software MITK-GEM is freely available online at https://simtk.org/home/mitk-gem.     

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.     

Computer aided morphometric analysis of oral leukoplakia and oral squamous cell carcinoma

We compared the changes in the cells in the basal layer of normal mucosa, oral leukoplakia with dysplasia and different grades of oral squamous cell carcinoma (OSCC) using computer aided image analysis of tissue sections. We investigated three morphometric parameters: nuclear area (NA), cell area (CA) and their ratio (NA:CA). NA and NA:CA ratio showed a statistically significant increase from dysplasia to increasing grades of OSCC. Nuclear size was useful for differentiating normal tissue, potentially malignant leukoplakia and OSCC.     

前列腺素E1治疗冠状动脉微血管病变的临床研究

目的:本研究旨在探索前列腺素E1(PGE1)对冠状动脉微血管病变(CMVD)治疗的效果及其可能的机制。方法:92例CMVD患者随机分为PGE1治疗组(47例)及常规治疗组(45例)。PGE1治疗组给予PGE1静脉注射,10μg/次,一日一次。常规治疗组给予等剂量生理盐水静脉注射,一日一次。两组治疗时间均为10天。通过治疗前后各组的西雅图心绞痛量表评分、血清血栓调节蛋白(TM)水平、血清超氧化物歧化酶(SOD)水平观察PGE1对CMVD的治疗效果。结果:PGE1治疗组在西雅图心绞痛量表中的5个维度(躯体活动受限程度、心绞痛频率、心绞痛稳定状态、治疗的满意程度、疾病认识)评分均高于常规治疗组(P均0.05)。此外,PGE1治疗组患者血清TM水平较常规治疗组显著下调(PGE1治疗组18.25±7.84 vs常规治疗组23.1±9.11,P0.05),血清SOD水平两组间无统计学差异(PGE1治疗组78.23±18.61 vs常规治疗组71.01±19.1,P=0.07)。结论:PGE1能够缓解CMVD患者的心绞痛状态,提高CMVD患者的生活质量,其机制可能与下调血清TM水平、保护冠状动脉微血管内皮细胞有关。     

Salt‐enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis

Salt‐enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500‐mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH₄)₂SO₄ led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH₄)₂SO₄, the labyrinthopeptin A1 concentration increased up to sevenfold compared to the non‐supplemented control, resulting in 325 mg L^?1 labyrinthopeptin A1 after 10 days of cultivation. The performance of other ammonium‐ and sulfate‐containing salts (e.g., NH₄Cl, K₂SO₄) was much lower than the performance of (NH₄)₂SO₄. A positive correlation between the uptake of glycerol as one of the main carbon sources and nongrowth‐associated labyrinthopeptin productivity was found. The change in the cell morphology of A. namibiensis in conjunction with increased osmolality by the addition of 50 mM (NH₄)₂SO₄, was quantified by image analysis. A. namibiensis always developed a heterogeneous morphology with pellets and loose mycelia present simultaneously. In contrast to the non‐supplemented control, the morphology of (NH₄)₂SO₄‐supplemented cultures was characterized by smaller and circular pellets that were more stable against disintegration in the stationary production phase.     

MyROOT: a method and software for the semiautomatic measurement of primary root length in Arabidopsis seedlings

Root analysis is essential for both academic and agricultural research. Despite the great advances in root phenotyping and imaging, calculating root length is still performed manually and involves considerable amounts of labor and time. To overcome these limitations, we developed MyROOT, a software for the semiautomatic quantification of root growth of seedlings growing directly on agar plates. Our method automatically determines the scale from the image of the plate, and subsequently measures the root length of the individual plants. To this aim, MyROOT combines a bottom‐up root tracking approach with a hypocotyl detection algorithm. At the same time as providing accurate root measurements, MyROOT also significantly minimizes the user intervention required during the process. Using Arabidopsis, we tested MyROOT with seedlings from different growth stages and experimental conditions. When comparing the data obtained from this software with that of manual root measurements, we found a high correlation between both methods (R² = 0.997). When compared with previous developed software with similar features (BRAT and EZ‐Rhizo), MyROOT offered an improved accuracy for root length measurements. Therefore, MyROOT will be of great use to the plant science community by permitting high‐throughput root length measurements while saving both labor and time.     

Optical coherence tomography to detect acute esophageal radiation‐induced damage in mice: A validation study

Radiation therapy for patients with non‐small‐cell lung cancer is hampered by acute radiation‐induced toxicity in the esophagus. This study aims to validate that optical coherence tomography (OCT), a minimally invasive imaging technique with high resolution (~10 μm), is able to visualize and monitor acute radiation‐induced esophageal damage (ARIED) in mice. We compare our findings with histopathology as the gold standard. Irradiated mice receive a single dose of 40 Gy at proximal and distal spots of the esophagus of 10.0 mm in diameter. We scan mice using OCT at two, three, and seven days post‐irradiation. In OCT analysis, we define ARIED as a presence of distorted esophageal layering, change in backscattering signal properties, or change in the esophageal wall thickness. The average esophageal wall thickness is 0.53 mm larger on OCT when ARIED is present based on histopathology. The overall sensitivity and specificity of OCT to detect ARIED compared to histopathology are 94% and 47%, respectively. However, the overall sensitivity of OCT to assess ARIED is 100% seven days post‐irradiation. We validate the capability of OCT to detect ARIED induced by high doses in mice. Nevertheless, clinical studies are required to assess the potential role of OCT to visualize ARIED in humans.