Cells were grown in filtered minimal medium (EMM) containing glutamate and implemented in amino acids and bases. Around 5.106 to 1.107 cells from an exponential culture were centrifuged at low speed (1,500 rpm for 1 min) and then resuspended in 1 ml of fresh filtered media. A drop of 1 µl was dropped onto a microscopy agarose slide containing a layer of 1.4% agarose dissolved in filtered media. Cells were observed with a LEICA DMRXA microscope equipped of an oil immersion 100× objective, with a numerical aperture of 1.4 and coupled to a COOLSNAP HQ camera (Roper Scientific, USA). The filters used were a FITC filter to collect GFP signal, CFP for CFP signal, and YFP for YFP signal. Images were taken with the Z-stack (3D) parameterized at 15 slices and were analyzed using METAMORPH (Roper Scientific, USA) and Image J software.
Metamorph
Manufactured by Teledyne
Sourced in France
Metamorph is a versatile imaging and analysis software platform developed by Teledyne. It provides a comprehensive suite of tools for acquiring, processing, and analyzing digital images from a variety of microscopy and imaging techniques. The core function of Metamorph is to facilitate the capture, storage, and analysis of high-quality images in a user-friendly and efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
Coolsnap hq camera, by Teledyne (1 mentions)
Tcs sp2 aobs confocal laser scanning imaging system, by Leica (1 mentions)
Autoquant x3, by Media Cybernetics (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Ccd camera, by Teledyne (1 mentions)
Dmi6000 fluorescence microscope, by Leica (1 mentions)
7 protocols using metamorph
1
Microscopic Imaging of Fission Yeast
2
Cortical Neuron Volume Dynamics
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Transverse brain slices from P2 mice were labeled with Cell Tracker green to visualize living cells and cultured under a fluorescent inverted Leica DM microscope under controlled temperatures. Slices were then perfused with aCSF, 400 μmol/L glutamate alone, or in the presence of 1 mmol/L MgSO4. Z‐series images of cell tracker green‐positive cells were acquired every 10 min and saved in TIFF format using a computer‐assisted image analysis station (Metamorph; Roper Scientific, Lisses, France). Then, regions of interest (ROIs) corresponding to fluorescent cell bodies present in cortical layers V and VI were integrated to compute cell volume using the Mercator Software (Explora Nova, La Rochelle, France).
3
Quantifying Placental Protrusions and Membrane Thickness
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Anonymized Cresyl violet stained slices from control and alcohol-exposed placentae were used for blind quantification of the protrusion number and the protrusion length. Practically, z-series of images were acquired and saved in TIFF format with a confocal laser scanning microscope (Leica DMI 6000B microscope and a Leica TCS SP2 AOBS confocal laser scanning imaging system (Leica Microsystems AG). Afterwards, acquired images were deconvoluted using AutoQuant X3 software (Media Cybernetics Inc., Rockville, MD, USA) and loaded into IMARIS imaging software (Bitplane, Zurich, Switzerland). Image segmentation was used to discriminate protrusions within the labyrinth zone for 3D reconstruction and quantification of protrusion density and protrusion length. For quantification of the Reichert’s membrane thichness and the density of round shape giant trophoblasts, anonymized toluidin blue stained semithin sections were used. Acquired TIFF format images were opened in the computer-assisted image analysis station Metamorph (Roper Scientific, Evry, France). After calibration of the objective used for acquisitions, thickness and cell density were quantified using the integrated morphometric analysis tools. Because of anatomic specificities between mouse and human placentae (hemotrichorial versus hemomonochorial), morphometric criteria quantified were different between the two species.
4
Quantifying Synaptic Markers in Brain Regions
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We determined regions of interest in the molecular layer and in the hilus. Quantification of VGAT and VGLUT1 was performed using MetaMorph software (Roper Scientific). Images were first flatten background filtered (kernel size, 3 × 3 × 2) to enhance cluster outlines, and an intensity threshold was applied automatically and confirmed visually to select appropriate clusters and avoid their coalescence. The total number of VGAT or VGLUT1 clusters in each region of interest (total number per 581 μm2) was quantified.
5
Real-time Monitoring of Circadian Oscillations
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For real-time monitoring of intracellular fluorescence circadian oscillations, 4 × 104 Alu-egfp or IRAlu-egfp GH4C1 cells were seeded on glass-bottom 24 well plates (coated with poly- ornithine). The next day, the medium was changed with a growth medium containing 100 nM forskolin to synchronize the cellular circadian oscillators. 20 min later, the medium was changed again with a DMEM without phenol red medium containing 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 100 mM pyruvate. The next day, plates were transferred to an inverted microscope (Axiovert-200, Zeiss) equipped with an environmental chamber, the temperature of which was set to 37°C and CO2 regulated to 5%. Egfp was excited through a 475/40 band-pass filter. An EMCCD camera (Rolera EM-C2, Q-Imaging) was used to collect emitted fluorescence at 530/50 nm. Time-lapse recordings of multiple regions in each well were realized over 96 hr with 1 picture every 30 min (Metamorph, Roper Scientific), using a 40X objective. The data were processed for tracking and measuring fluorescence fluctuations of individual cells with the CGE (Circadian Gene Expression) Plugging of ImageJ (Sage et al., 2010 (link)). Cells retained for analysis fulfilled two criteria: they were detected for at least 48 hr and their mean fluorescent level was 10% higher than mean level of the background.
6
Visualizing Brain Microvascular Network
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An immunohistochemical study targeting the endothelial cell marker CD31 was carried out to visualize the brain microvascular network on histological sections from control and alcohol-exposed animals. Immunolabelings were analyzed under a DMI 6000 fluorescence microscope (Leica) equipped with a CCD camera (Roper Scientific, Lisses, France). For vascular network measurements, a morphometric approach was employed using the software Metamorph (Roper Scientific) [22 (link)]. In particular, quantification of the angular orientation was performed in the fronto-parietal cortex on two slices per animal and five to seven mice from four different litters per group.
7
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was observed using the ratiometric probe JC-1. In healthy cells, the intact membrane potential allows the lipophilic dye JC-1 to enter the mitochondria where it accumulates and aggregates, producing an intense orange signal. In cells where the mitochondrial membrane potential has collapsed, the JC-1 probe remains unaggregated in the cytosol, where it is labeled green.49 (link) Acute E15 GE slices were treated for 10 h at 37 °C in the absence or presence of 100 μM ketamine, incubated for 30 min with 3 μl/ml JC-1 and finally washed twice for 5 min with phosphate buffer saline (PBS) at the same temperature. Fluorescence was visualized immediately without prior fixation at 485 and 530 nm excitation and emission wavelengths for green and 550 and 590 nm excitation and emission wavelengths for orange fluorescence, respectively. Green and orange signals were acquired and saved in TIFF format using a computer-assisted image analysis station (Metamorph, Roper Scientific, Evry, France).
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