Four wells from each day (5, 10, 14, and 20) and culture type (cocultures of ECs with FBs, monocultures of ECs or FBs, respectively) were examined with a light microscope (Zeiss MicroImaging GmbH, Jena, Germany) and photographed with a digital camera (ColorView, Olympus, Münster, Germany). PC-based laboratory imaging programs (analysis docu, Version 5.2, Olympus, Münster, Germany and NIS-Elements AR, Version 4.5, Nikon, Düsseldorf, Germany) were used for processing. For measurements of capillary-like structures, visual fields were standardized by taking 7 images of each cell culture at the same magnification (5×) with a total area of inspection of 40.6 mm2. The morphometric features of capillary-like structures that were evaluated included: average number of tubes per mm2, average length of tubes (10 tubes per field of view), average diameter of tubes (10 tubes per field of view), average number of tubes with branches, including the percentage proportion of branching and the average length of branches (10 branches per field of view), and branching distribution pattern (single-, double-, triple- and multibranched). Branches were defined as projections of capillary-like structures, which were detectable at a magnification of 5×. The percentage proportion of branching was defined by the number of branched tubes in relation to the total number of tubes that were counted.
Colorview
Manufactured by Olympus
Sourced in Germany
ColorView is a high-performance digital camera system designed for microscopy applications. It features a precise color reproduction and high-resolution image capture to provide detailed visual documentation of microscopic samples.
Automatically generated - may contain errors
6 protocols using colorview
1
Quantifying Capillary-like Structure Morphometrics
2
Fluorescence Microscopy of Cryosections
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Cryosections and cells were examined using a light microscope (BX-53, Olympus LRI Instruments AB, Lund, Sweden) equipped with a mercury lamp and filters for fluorescence (U-MWG, U-MWB, U-MWU). Images were taken in 10x, 20x or 40x magnification using an Olympus Color View digital camera and captured using cellSens Dimension imaging software (Olympus).
3
Histologic Response Grading for Tumor Samples
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Tumor fragments fixed in formaldehyde were embedded in paraffin blocks and 4-μm sections were cut for hematoxylin and eosin (H&E) and IHC. Histologic response (HR) was graded by assessing the magnitude of necrosis, myxoid degeneration, or fibrosis using the following grading scheme: 1—minimal (0-10%), 2—low (> 10% and ≤ 50%), 3—moderate (> 50% and ≤ 90%), and 4—high (> 90%) grade [19,20] . In addition, we have also added to the grading system the area with myxoid degeneration, which is characterized by the replacement of viable tumor tissue by an amorphous matrix with low cellularity as a pattern of response to TKI treatment observed [21] (link). Mitotic figures and apoptotic cells were counted in 10 high power fields (0.45-mm field diameter) at × 400 magnification on H&E staining. Cl-PARP, a marker for apoptotic activity, was assessed by counting positive cells in 10 high power fields. Additionally, the antibody Ki67 was used as a measure for proliferative activity and was scored as the average of Ki67-positive tumor cells on five digital microscopic pictures taken at × 400 magnification. Microscopy was performed using the Olympus CH-300 microscope; pictures were taken with digital camera Color View (all Olympus, Tokyo, Japan).
4
Quantifying Immunolabeled ECM Components in Cell Cultures
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The assessment of immunolabeled ECM components including the three proteins laminin, collagen III, and fibronectin, was conducted in cocultures and in monocultures after 5, 10, 14, and 20 days in vitro. Ten images were taken with a 20× objective from each cell culture type (Zeiss MicroImaging GmbH, Jena, Germany and ColorView, Olympus, Münster, Germany). The quantification of the fixed, immunostained ECM proteins was evaluated using a quantitative multiphase analysis for determining phase composition (analySIS docu, Version 5.2, Olympus, Münster, Germany) with a corresponding pixel intensity profile that was calibrated manually. We defined the color intensity ranges (negative, moderate, and high) for the separate phases. The proportion of positively labeled ECM (regions that displayed a distinct color change) was expressed as a percentage of the total area. The total amount of ECM was defined as the percentage of all positively labeled areas of one slide (moderate and high intensity). The amount of the individual ECM proteins (laminin, collagen III, and fibronectin) was determined as the proportion of the total response. The ECM production was measured through the pixel intensity profile, and the percentage of positively labeled pixels mirrored the quantity of immunolabeled ECM.
5
Bee Larvae Size Measurement Protocol
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Head capsule width and fresh body weight of the hatched bee larvae (age 22–24 days) were taken as size measures.
The head capsule width was measured on the widest point with a binocular microscope (Olympus SZX16, ColorView, Olympus) with a caliper (1/50 mm nonius) according to Vogelweith et al. [72 (link)]. Referring to Bosch and Vicens [73 (link)], the head width constitutes the best estimators of adult weight and provision weight in both sexes besides wing lengths. No pollen for the provision weight was collected due to infestation with a parasitic moth in August, when the bees were in the pupal stage. The analyzed developmental stages mentioned in the following include the larvae, pharate referring to the individual enclosed in the cocoon, and the imago referring to the hatched individual.
The head capsule width was measured on the widest point with a binocular microscope (Olympus SZX16, ColorView, Olympus) with a caliper (1/50 mm nonius) according to Vogelweith et al. [72 (link)]. Referring to Bosch and Vicens [73 (link)], the head width constitutes the best estimators of adult weight and provision weight in both sexes besides wing lengths. No pollen for the provision weight was collected due to infestation with a parasitic moth in August, when the bees were in the pupal stage. The analyzed developmental stages mentioned in the following include the larvae, pharate referring to the individual enclosed in the cocoon, and the imago referring to the hatched individual.
6
Lipid Droplet Flocculation in Mimicked MFG Emulsions
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The flocculation of lipid droplets of the mimicking MFG emulsions were observed based on the method of Phan et al [19] . The emulsion sample was first diluted 10-fold in ultrapure water, then placed 5 μL of sample carefully on a glass slide, and covered with a coverslip. Finally, samples were observed under 40× magnification by using the Leitz Diaplan microscope (Wetzlar, Germany). Image of sample was recorded with the camera (built-in Olympus Color View) and analyzed using Olympus Belgium NV cell D software (Aartselaar, Belgium).
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