Axiovert 135 tv

To collect eggs, gonads were gently washed in sea water to allow their release and fertilized as previously described24 (link). The fertilization success in control samples was approximately 90%. Fertilized eggs (150 eggs/ml) were allowed to develop in a controlled temperature chamber at 18 ± 2 °C and 12:12 light:dark cycle. The development was followed by inverted microscope (Zeiss Axiovert 135 TV) until the pluteus stage, approximately 48 hours post fertilization. Embryos were fixed in 4% formalin and morphological observations were performed to identify eventually abnormal plutei. Embryos were considered normal if they reached the pluteus stage of development, exhibited good body symmetry, showed fully developed skeletal rods and displayed a well differentiated gut. All the morphologies that did not satisfy the above-mentioned criteria were grouped and referred to as abnormal24 (link).

Cells were grown to approximately 80% confluence on coverslips; they were washed with phosphate-buffered saline (PBS), fixed with 4% PFA/PBS for 20 min at room temperature, washed three-times, permeabilized with 0.1% Triton X-100/PBS for 5 min at room temperature. After washing with PBS, nonspecific binding of the antibodies was blocked with 10% FBS/PBS for 30 min at room temperature. They were then stained for 1 h at room temperature with the rabbit anti-FLAG antibody diluted in 5% FBS/PBS, followed by an anti-rabbit AlexaFluor^® 488 (ThermoFisher Scientific) for one hour at room temperature. In the case of HBcAg detection, an AlexaFluor^® 594 conjugated secondary antibody was used for visualization. For specific staining of the ER and the Golgi, AlexaFluor^® 594 Concanavalin A and AlexaFluor^® 647 Lectin HPA (Helix pomatia agglutinin; ThermoFisher Scientific) antibodies were used respectively to stain the cells for a further hour at room temperature. For specific staining of actin and Tubulin, AlexaFluor^® 647 Phalloidin and a mouse a-Tubulin (Sigma-Aldrich) were used, respectively. For Tubulin, a secondary anti-mouse AlexaFluor^® 594 antibody was used for visualization purposes. Images were taken on an Axiovert 135 TV (Zeiss) fluorescent microscope or an EVOS FL Auto Cell Imaging System (ThermoFisher Scientific), as well as a confocal microscope (LSM Pascal, Zeiss).

Viable cells were plated on glass-bottomed dishes (Iwaki, Tokyo Japan) and were observed under an inverted fluorescent microscope (Axiovert 135TV, Carl-Zeiss, Oberkochen, Germany) equipped with Plan-Neofluor 10x, 20x and 40x objectives. Time-lapse micrographs were acquired using a Sensys CCD digital camera (Photometrics, Tucson, AZ, USA) attached to the microscope. For extended image capturing, we used a handmade incubation system that maintains cells under appropriate conditions for culture (37°C, 5% CO₂). HEPES 10 mM (pH 7.2) (final) was added to the culture media to maintain neutral pH during observation. Each dish was photographed up to 2 h at 3 min intervals. The time-lapse images were analyzed using IPLab Spectrum software (Scanalytics, Fairfax, VA, USA.). Cell migration speed was calculated as the distance that the center of gravity of the cell contour moved in each time interval. The shape index (SI) was calculated from the cell contour and indicates the deformation of the shape. The SI of a circle is 1. The formula to obtain SI is as following: [SI] = 4πA/P² (A: area, P: Perimeter) [28 (link)].

The basic dSTORM-system is realized with a standard inverted microscope stand (AxioVert 135 TV, Zeiss, Germany) equipped with a nosepiece-stage (IX2-NPS, Olympus, Japan) to keep drift low. A 637 nm diode laser (P = 150 mW, OBIS, Coherent, USA) is focused to the back-focal plane of the microscope objective lens (ZEISS 100×, NA = 1.46) to realize a homogenous illumination in the sample plane. Using an adjustable mirror, it is also possible to change the laser position in the back-focal plane. This enables background reduced total internal reflection (TIRF) illumination. The microscope can potentially be replaced by a low-price customized optical setup also relying on cheap lasers [12 (link)], making it available for about ≪ 10k$.
An emCCD camera (iXon3 DU-897, Andor, UK, Table 1) can be used to image the sample in widefield and STORM-mode during normal operation (dSTORM reconstruction result Fig 6). For imaging via the cellphone, the beam-path is switched from the camera port to the eyepiece, where a common 10× monocular eyepiece is equipped with a custom-made 3D-printed cellphone adapter [48 ]. The cellphone (P9 EVA-L09, Huawei, China, Table 1) is placed with its camera lens in the Ramsden disk of the eyepiece (see Fig 1b), since an eyepiece images the intermediate image produced by the tube lens of the Axiovert body to infinity.

Cultures were stained with a calcium sensitive fluorescent dye (Fluo4, Invitrogen) and the calcium transients [17] (link) imaged during application of magnetic pulses. The culture was placed in recording solution [17] (link) that filled a near-spherical glass ball (Figure 2b), approximately 60 mm in diameter, whose bottom was flattened to create a circular base approximately 30 mm in diameter on which the coverslip lay. At the top of the sphere a slot was opened through which the coverslip and fluid could be inserted and at the base of the sphere a viewing hole 13 mm in diameter was made, slightly off-center and near the circumference of the base, which was then sealed with an optically transparent glass coverslip. The glass sphere was placed inside the cross coil, with the flattened base positioned over the lower pole (Figure 2c and Video S1). The magnet and sphere were placed in an inverted microscope (Zeiss Axiovert 135TV), with the objective positioned under the viewing hole.

IAR on polyvinyl chloride (PVC) were determined essentially as previously described (81 (link)). A CoverWell perfusion chamber (19 × 6 × 0.5 mm; Invitrogen) was placed on top of a dry, uncoated PVC microscopy slide (treated in 70% EtOH-1% HCl ON and washed with ddH₂O) and sealed with silicone lubricant. ON cultures (TSB, 37°C for 22 ± 2 h, 225 rpm) were diluted in citric acid-Na₂HPO₄ buffer (pH 6.6) to an OD₆₀₀ of 0.100 ± 0.005 and pumped through the chamber at a pressure of 0.0505 Pa. Three separate vistas in the middle of the channel (along the x axis) were taken every 5 min for 30 min, using a 40× objective (Zeiss Fluar 40×/NA 1.3, oil immersion) and an inverted microscope with an automated stage (Zeiss Axiovert 135 TV). Cells were counted for each vista over time, and the median number of cells attached was determined for each time point. The initial adhesion rate was defined as the slope of the linear regression through the median number of attached cells over time [with a y intercept of 0 and units of cells per (minute × square centimeter)]. Measurements were done at least in biological duplicate.

Slices containing blood vessels were selected and pre‐incubated with DMSO, Ketanserin (+)‐tartrate salt (Sigma Aldrich, # S006‐50MG), Riociguat or BI 703704 at 90% O₂, shaking conditions and 37°C for 30 min before they were placed under the microscope (Zeiss, Axiovert 135TV). Slices were immobilized with a platinum ring before the start of the experiment. 1 mM serotonin (Sigma Aldrich, # 14927–25 mg) was added to the slices to induce contraction and pictures of the blood vessel were taken every 10 s for 7 min using Axiovison software. Endothelin‐1 (ET‐1) (Sigma Aldrich, # E7764‐1 mg) at a final concentration of 10 mM was added to the slice as a positive control for the contractibility of the blood vessel. Only blood vessels that responded to ET‐1 treatment were included in the final analysis. Blood vessel area was analyzed using a proprietary application based on a commercially available machine vision software library (Halcon 13.0.2, MVTec Software GmbH). The area of the unstimulated vessel was set as 100% and used for normalization.