Fluorescence imaging was done by using a Nikon eclipse 90i microscope equipped with a Nikon Plan Apo 20x, 40x, 60x, and 100x oil objective (N.A. = 1.40), and a Photometrics Coolsnap ES2 camera or a Hamamatsu Orca Flash LT + CMOS camera. L4 animals were transferred to a fresh NGM plate seeded with OP50 bacterial lawn and were stored in a 20 °C incubator for 24 h prior to imaging. Adult animals were paralyzed with 30 mg/ml 2, 3- Butanedione monoxime (BDM, Sigma) in M9 buffer, and then mounted on 2% agarose imaging pad. Metamorph 7.0 software (Universal Imaging) was used to capture image stacks and to obtain maximum intensity projections. All images were captured from left or right laterally positioned animals facing up. Fluorescence imaging of AVL and hmc were captured from the neck region where the terminal bulb of the pharynx is located. To analyze the ablation of hmc in N2 and hlh-8, GFP was expressed in both AVL and hmc (Pnmur-3::GFP) and the neck region was imaged in adult animals.
Metamorph 7
Manufactured by Universal Imaging
Sourced in United States, Germany
Metamorph 7.1 is a versatile software platform for automated imaging and analysis. It provides a suite of tools for image acquisition, processing, and analysis to support a wide range of microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Plan apo 20x, by Nikon (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Mowiol, by Merck Group (1 mentions)
Axiophot system, by Zeiss (1 mentions)
1 4 diazobicyclo 2.2.2 octane, by Merck Group (1 mentions)
Plan apo 100x, by Olympus (1 mentions)
Igor pro, by Wavemetrics (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Te2000 u microscope, by Nikon (1 mentions)
Tcs sp8 upright, by Leica (1 mentions)
Coolsnap hq2 camera, by Prior Scientific (1 mentions)
Oil immersion objective, by Leica (1 mentions)
Axiovert 200m inverted, by Zeiss (1 mentions)
Plan apochromat objective, by Zeiss (1 mentions)
Coolsnaphq2, by Leica (1 mentions)
Coolsnap hq, by Teledyne (1 mentions)
Te2000 u, by Nikon (1 mentions)
17 protocols using metamorph 7
1
Fluorescence Imaging of C. elegans
2
Fluorescence Microscopy of Cytoskeleton
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ZF4 or HeLa cells grown on glass coverslips were washed with PBS, fixed with 4% formaldehyde in PBS and permeabilized with 0.15% (v/v) Triton X-100 in PBS for 10 min at room temperature. Subsequently, the cells were incubated with TRITC-conjugated phalloidin and washed again with PBS. Cells were embedded with Mowiol supplemented with 1,4-diazobicyclo[2.2.2]octane (Sigma) and analysed by fluorescence microscopy using an Axiophot system (Zeiss, Oberkochen, Germany) processed by using the Metamorph 7.0 software (Universal Imaging, Downingtown, PA).
3
Quantitative Imaging of Coelomocyte Fluorescence
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Quantitative imaging of coelomocyte fluorescence was performed as previously described [11 (link)] using a Zeiss Axioskop equipped with an Olympus PlanAPO 100x (NA = 1.4) objective and a CoolSNAP HQ CCD camera (Photometrics). Worms were immobilized with 30 mg/ml BDM (Sigma). The anterior coelomocytes were imaged in L4/A lethargus (determined by absence of pharyngeal pumping), and 1 day-old adult animals. Image stacks were captured and maximum intensity projections were obtained using Metamorph 7.1 software (Universal Imaging). YFP fluorescence was normalized to the absolute mean fluorescence of 0.5 mm FluoSphere beads (Molecular Probes). Statistical significance was determined using Kolmogorov-Smirnov test.
4
Quantitative Analysis of Synaptic Protein Localization
5
Visualizing Hippocampal Neuron Morphology
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Hippocampal neurons were fixed and permeabilized as previously described (Raynaud et al., 2013 (link)). Untreated or shRNA-GFP–expressing neurons were visualized using a Zeiss Axioimager Z1 microscope. For brain slices, we used a Zeiss LMS780 confocal microscope with a 63×/1.4 numerical aperture (NA) oil objective or a Leica wide-field DM6000 microscope with a 100×/1.4 NA oil objective. Images were recorded with a charge-coupled device HQ2 camera (Roper Scientific, Martiensried, Germany) controlled by MetaMorph 7.1 (Universal Imaging).
Morphometric analyses were performed in different fields from at least three different cultures using ImageJ software (National Institutes of Health, Bethesda, MD). Spines were defined as dendritic protrusions with a neck and a head. Dendrites were randomly selected and spines manually counted over a 50-μm length of dendrite. Data were then expressed as density of spines/10-μm length of dendrite. The same spines were used to measure the area of their head. Each spine was manually traced and the surface calculated using ImageJ. All experiments were conducted in a double-blinded manner, and dendritic spines were analyzed from at least 20 neurons from at least three independent cultures (n = 3 or 4 experiments, as indicated).
Morphometric analyses were performed in different fields from at least three different cultures using ImageJ software (National Institutes of Health, Bethesda, MD). Spines were defined as dendritic protrusions with a neck and a head. Dendrites were randomly selected and spines manually counted over a 50-μm length of dendrite. Data were then expressed as density of spines/10-μm length of dendrite. The same spines were used to measure the area of their head. Each spine was manually traced and the surface calculated using ImageJ. All experiments were conducted in a double-blinded manner, and dendritic spines were analyzed from at least 20 neurons from at least three independent cultures (n = 3 or 4 experiments, as indicated).
6
Confocal and Spinning-Disc Microscopy Protocol
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Confocal microscopy was carried out with a TCS-SP8 upright confocal laser scanning microscope equipped with 63× oil immersion objective (NA = 1.4; Leica, Microsystems). Alexa 488 and GFP were excited at 488 nm and observed in a 505–540 nm window. Alexa 594 was excited at 594 nm and observed in a 600–630 nm window. Alexa 647 was excited at 633 nm and observed in a 650–700 nm window. For dual color acquisition, images were sequentially acquired in line scan mode (average line = 2). Overlays were performed with post acquisition Leica Confocal Software functions to obtain the presented snapshots. Golgi exit of granules was monitored by time-lapse fluorescence microscopy using a spinning-disc confocal microscope. This microscopy was carried out with a Yokogawa CSU-22 spinning-disc head on a Nikon TE-2000 U microscope equipped with a 100× NA 1.4 oil immersion objective and a Coolsnap HQ2 camera, a NanoScanZ piezo focusing stage (Prior Scientific) and a motorized scanning stage (Marzhauser). This microscope was steered with Metamorph 7.1 (Universal Imaging Corporation). The fast scan mode at 512 × 512 pixel resolution was used. Video sequences were acquired at 5 frames s−1 (100–200 ms exposures).
7
Anchorage-Independent Growth Assay
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To determine anchorage-independent cell growth, 1 × 104 cells (SNUOT-Rb1) were suspended in 3 mL of media containing 0.3% agar with 10% FBS and applied onto pre-solidified 0.6% agar (3 mL) with no FBS in 60-mm dishes. Each experiment was performed triplicate. After 3 weeks of incubation, colonies on soft agar were observed under a phase-contrast microscope. The colony size and number were determined with the Metamorph 7.1 program (Universal Imaging) using the photographs.
8
Pyramidal Neuron 3D Imaging Protocol
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Pyramidal neurons were selected for image acquisition based on their pyramidal or pear-shaped somata with 3–4 primary dendrites. Three-dimensional fluorescence images (16-bit, 512 × 512) were acquired using MetaMorph 7.0 (Universal Imaging) in conjunction with a Yokogawa CSU 10 spinning disk confocal (Perkin Elmer) fitted on a Zeiss Axiovert 200M inverted microscope. The excitation light of a Krypton/Argon ion laser (643-RYB-A02; Melles Griot) was selected by 488/10 nm, 568/10 nm or 647/10 nm filters (Sutter Lambda filter changer), reflected and then focused through a 63× 1.4 numerical aperture (NA) oil immersion Plan-Apochromat objective lens (Carl Zeiss MicroImaging, Inc.) or a 10× 0.45 NA Plan-Apochromat objective lens. Detection of the fluorescence emission, after passing a 525/50 nm bandpass filter for Alexa 488, a 607/45 nm bandpass filter for Alexa 568 or a 700/75 nm filter for Alexa 647, was obtained using a Cascade 512B camera (Roper). For between sample comparison, all images were acquired with the same settings without knowledge of the experimental condition during image acquisition. To acquire image stacks that could be deconvolved for further analysis, images were sampled using the Nyquist criterion.
9
In Vitro Oocyte Maturation Assay
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Oocyte in vitro maturation assays were performed as previously described (Brunet et al., 2008 (link)). Briefly, oocytes were collected from ovaries of 10- or 26-week-old hmmr+/+ and hmmrm/m mice and placed in M2 medium pre-warmed to 37°C and supplemented with 4 mg/ml BSA and 1 mM milrinone. For video microscopy of oocyte meiotic maturation, oocytes were transferred to a Ludin Chamber containing M2 medium with 4 mg/ml BSA. Time-lapse images were acquired using a Photometrics CCD camera (CoolSnap HQ2) mounted on a Leica HC PL APO 20×/0.7 NA objective enclosed in a thermostatic chamber (Life Imaging Service). Images were taken every 15 min for 18–20 h at 20× magnification. Metamorph 7.0 (Universal Imaging) and ImageJ (NIH) software were used for image analysis.
10
Sperm Migration in Oviduct Imaging
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