To assess changes in renal microcirculation after CLP, intravital video microscopy was performed as previously described (20 (link), 23 (link)). Mice were anesthetized with isoflurane and FITC-labeled dextran (2 μmol/kg in 3 ml/kg normal saline) was injected via the penile vein to visualize microvascular flow. After 10 minutes, the left kidney was exposed by a flank incision and placed on a glass stage above an inverted Zeiss Axiovert 200M fluorescent microscope with an Axiocam HSm camera (Zeiss, Jena, Germany). Ten second videos were acquired at ~30 frames per second from five randomly selected, non-overlapping fields for each animal. Core body temperature was maintained at 35-37°C with a heating lamp. Roughly 150 capillaries from each animal were selected for analysis and categorized into three categories based on the degree of perfusion as follows: “continuous flow” where red blood cell movement was uninterrupted throughout the video; “intermittent flow” where red blood cell movement stopped or reversed during the course of the video; or “no flow” where no red blood cell movement was observed throughout the video. Data are shown as percentage (mean ± S.E.M.) of vessels in each category.
Axiocam hsm camera
Manufactured by Zeiss
Sourced in Germany
The Axiocam HSm is a high-speed camera designed for microscopy applications. It features a CMOS sensor with a resolution of up to 2.3 megapixels and can capture images at up to 100 frames per second. The camera is designed for integration with Zeiss microscope systems.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m fluorescent microscope, by Zeiss (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Dm5000 microscope, by Leica (1 mentions)
Dmi6000 microscope, by Leica (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Stereo discovery v12, by Zeiss (1 mentions)
Application suite x software, by Leica (1 mentions)
Axio observer, by Zeiss (1 mentions)
Axioskop 2 mot, by Zeiss (1 mentions)
Orca flash 4, by Zeiss (1 mentions)
Dfc300fx digital, by Leica (1 mentions)
Axio observer a1 microscope, by Zeiss (1 mentions)
Axiovision, by Zeiss (1 mentions)
Ec plan neofluor, by Zeiss (1 mentions)
Axioskop2 mot plus microscope, by Zeiss (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Fluosphere, by Thermo Fisher Scientific (1 mentions)
Rhodamine 6g, by Merck Group (1 mentions)
Fecl3, by Merck Group (1 mentions)
Imager a2, by Zeiss (1 mentions)
7 protocols using axiocam hsm camera
1
Intravital Imaging of Renal Microcirculation
2
Microscopy and Image Analysis Methods
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WISH, SISH, histological stainings and IHC on sections of all mouse experiments were documented with a Leica DM5000 microscope equipped with a Leica DFC300FX digital camera and the used software was Leica FireCam (Version 1.9.1). Immunofluorescence staining of mouse tissue sections were photographed with the Leica DMI6000 microscope equipped with a Leica DFC350FXR2 camera and the Leica Application Suite X software. Images were processed in Adobe Photoshop CS4 or CC, or FIJI ImageJ version 2.0.0, figures assembled using Adobe Illustrator CS4, CS6 or CC. Xenopus WISH was imaged with a Zeiss SteREO Discovery.V12 or if sections were made with a Zeiss Axioskop 2 mot plus each equipped with an AxioCam HRc in combination with AxioVision 4.7. Fluorescence imaging of Xenopus was performed on a Zeiss AxioObserver with a LSM700 and Zeiss ZEN black software. CBF was documented with a Zeiss Axioskop 2 mot plus equipped with a high-speed Hamamatsu video camera Orca flash 4.0 and Zeiss ZEN blue. CGF analysis was done with a Zeiss Axioskop 2 mot plus combined with a Zeiss AxioCam HSm camera and Zeiss AxioVision 4.7. CBF and CGF was assessed using the ImageJ plugin Particle Tracker56 (link). All trajectories were further analysed using a custom-made program written in RStudio which calculated the velocity of each particle track57 (link).
3
Bacterial Morphology during Phage Infection
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To observe bacterial morphology during phage infection, a 5 μL sample of the infected culture was placed on a glass slide and then covered with a coverslip. Cells were imaged using an EC Plan-Neofluor 100 × objective installed on a Zeiss Axio Observer A1 microscope equipped an Axiocam HSM camera. All images were edited using the AxioVision software package (Zeiss).
4
Quantifying Cytoplasmic Fluorescence Dynamics
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The cells were imaged with a Zeiss Observer.Z1 wide field microscope. PIC was excited by using an LED with a wavelength of 530 nm. Emission light was detected using the AHF Analysentechnik beam splitter Dual 532/640 ET, the AHF Analysentechnik detector cube H 643 and a Zeiss AxioCam HSm camera. To control the temperature of the samples the Brook Industries Z‐HC‐K‐3112 temperature stage was used. Images were recorded every 2.5 minutes (100 ms exposure time) for 60 min and analyzed using ImageJ. ROIs (Regions of Interest) were set to analyze the average fluorescence intensity in the cytoplasm. A sample size of at least 100 cells was measured and analyzed and the errors determined as standard deviation (s.d.).
5
High-Speed Video Analysis of Ciliary Beating
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Videos were recorded at stage 32 embryos using a Zeiss Axioskop 2 mot plus microscope. Embryos were mounted on a slide containing a chamber constructed from duct tape. The most ventral part of the belly was recorded with a high-speed Hamamatsu video camera Orca flash 4.0 at 800 frames per second (fps) for 1 s to analyse ciliary beating. Ciliary flow was analysed using 1 μm fluorescent beads (Invitrogen FluoSpheres; 1:2000). Beads were added to the culture medium (0.1 × MBSH) and specimens were imaged using a Zeiss Axiocam HSm camera at 175 fps. Evaluation of CBF and bead transport was previously reported in33 (link).
6
In Vivo Arterial Injury Assessment
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After anesthesia, the carotid arteries of the animals were isolated and dissected as described above. After dissection, the substances under study were administered intravenously through the retro-orbital plexus, and 5 min later, rhodamine 6G (Sigma-Aldrich, Saint Louis, MO, USA) (1 mg/kg) was also administered via retro-orbital injection in the opposite optic cavity to label mitochondria from platelets and leukocytes. For the aspirin control, aspirin diluted in acidic medium was administered by gavage 30 min before the dissection procedure. The arterial injury was induced after 5 min by applying, for 2 min, a piece of filter paper (2 × 1 mm) saturated with 15% FeCl3 (Sigma-Aldrich, Saint Louis, MO, USA). The injured artery was then monitored for 12 min (defined as the maximum fluorescence time) using an intravital fluorescence microscope (Carl Zeiss Imager.A2) coupled with an Axiocam HSm camera (Carl Zeiss, Jena, Germany). After obtaining images, the animals were euthanized.
7
Quantifying Bacterial Motility Dynamics
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Bacterial movement was captured using a Zeiss AxioObserver Z1 inverted microscope equipped with an AxioCam Hsm camera and a 63 × oil immersion objective at a spatial resolution of 0.31 μm/pixel and temporal resolution of 30 fps. A minimum of 80 bacteria were tracked in each experiment and three independent set of experiments were carried out for each strain. All experiments were conducted at 37 °C. The recorded images were analyzed in ImageJ using the Manual Tracking plug-in tool (NIH, Bethesda, MD). The instantaneous speeds were calculated by dividing the distances travelled during each time increment by the time increment. The average swimming speed was determined through averaging the instantaneous swimming speeds over independent experiments. The percentage of motile bacteria in the total population was quantified using an image processing algorithm developed in MATLAB, which detects the stationary (non-motile) subpopulation. The position of each bacterium was tracked at 30 s intervals, and bacteria with unchanged coordinates over two consecutive images were deemed non-motile. A total of 18 images (containing approximately 1500 bacteria on average) were analyzed for each experiment, and three independent set of experiments were carried out for each strain.
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