Fibrin fiber manipulations were performed using a combined atomic force/fluorescent microscopy technique [32 (link), 33 ]. The AFM (Topometrix Explorer, Veeco Instruments, Woodbury, NY) rests on a custom-made stage on top of an inverted microscope (Zeiss Axiovert 200, Göttingen, Germany). The cover slide with the fibrin sample is sandwiched between the AFM and the fluorescent microscope. The stage is designed to allow for independent movement of the fibrin sample, objective, and AFM cantilever. Fluorescence images were captured using a Hamamatsu EM-CCD C9100 Camera (Hamamatsu Photonics KK, Japan) and IPLab software (Scanalytics, Fairfax, VA). The AFM cantilever tip (CSC38/AlBS, force constant 0.03–0.08 N/m, MikroMasch, Wilson, OR) was placed between two of the ridges in the striated substrate, next to a fiber for manipulation. The cantilever tip, controlled by nanoManipulator software (3rd Tech, Chapel Hill, NC) was then laterally moved to stretch a single fiber at a rate of 305 nm/s. The elapsed time, tip travel distance, and left-right photodiode signal were recorded by the nanoManipulator software.
Em ccd c9100 camera
Manufactured by Hamamatsu Photonics
Sourced in Japan, United States
The EM-CCD C9100 camera is a high-sensitivity scientific camera designed for low-light imaging applications. It features a back-illuminated electron-multiplying charge-coupled device (EM-CCD) sensor that provides enhanced quantum efficiency and low-noise performance. The camera is capable of capturing images with high spatial resolution and can operate at high frame rates.
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Lab products found in correlation
Axiovert 200, by Zeiss (2 mentions)
Bx51wi dsu confocal microscope, by Olympus (2 mentions)
Topometrix explorer, by Veeco (1 mentions)
Spinning disk confocal head, by PerkinElmer (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
X cite series 120q, by Excelitas (1 mentions)
Stereo investigator software, by MBF Biosciences (1 mentions)
Imspector software, by Miltenyi Biotec (1 mentions)
Szx16, by Olympus (1 mentions)
Ix81 inverted microscope, by Hamamatsu Photonics (1 mentions)
Xcellence software, by Olympus (1 mentions)
Ix81 microscope, by Hamamatsu Photonics (1 mentions)
Stemi sv11, by Zeiss (1 mentions)
12 protocols using em ccd c9100 camera
1
Combined AFM-Fluorescence Fibrin Manipulation
2
Laser-Cutting Protocol for Live Imaging
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Laser-cutting injuries were performed using a spinning disk confocal head (Perkin Elmer) attached to a Zeiss Axio Imager.M2m microscope equipped with a 40×/1.0 NA W Plan-Apochromat dipping lens objective and a Hamamatsu EM-CCD (C9100) camera (Kiehart et al., 2006 ). Micro-Manager software was utilized for time lapse image acquisition and lasercutting (Open Imaging, San Francisco, CA) (Edelstein et al., 2010 (link)). Laser cuts were performed using a Nd:YAG UV laser minilite II (Continuum, San Jose, CA) at a power of 1.3–2.3 μJ (Nova Ophir power meter) with a steering mirror for precise laser incisions on larvae mounted in a mixture of 0.8% low melt agarose and egg water.
3
Immunohistochemical Analysis of xCT and EAAC1 in Mouse Brain
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On PND 15, mice were transcardially perfused with ice-cold 0.9% saline followed by ice-cold 4% paraformaldehyde in phosphate buffer (PB), pH 7.4. Brains were removed, postfixed at 4°C and successively immersed in 20% and 30% sucrose cryoprotection solutions. Sections (22 μm) were collected in 24-well culture plates filled with 0.9% phosphate buffered saline (PBS), pH 7.4. After 3 washes with PBS + 0.3% Triton X-100 (PBST), the sections were incubated overnight at 4°C in rabbit anti-xCT (1:100) or rabbit anti-EAAC1 (1:300) and chicken anti-MAP2 (1:800) primary antibodies with a 2% normal horse serum in PBST solution. After 3 washes in PB solution sections were incubated with anti-rabbit Alexa Fluor 594 and anti-chicken Alexa 488 secondary antibodies (1:300) diluted in PB solution for 2 h. Finally, sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed under the microscope. Photomicrographs were acquired with an Olympus BX51WI DSU confocal microscope (Olympus, Center Valley, PA, USA) coupled to a Hamamatsu EM-CCD C9100 camera (Hamamatsu, Hamamatsu, Japan).
4
Fluorescence Microscopy Imaging Protocol
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We used an inverted fluorescence microscope (Axiovert 200 or Observer D, Zeiss, Göttingen, Germany) in these experiments. Fluorescence images were taken with a 40x lens (NA of 0.7), with an excitation source from a short arc mercury lamp (Osram HBO 103W/2, Atlanta Light Bulbs Co., Atlanta, GA). The exposure time was 200 ms. The field of view (image size on computer) was 180 μm × 180 μm. In order to get the same photobleaching for all the fibers in a particular image, fluorescence images of each area were taken only once, under the same conditions. Fluorescence images were collected with a Hamamatsu EM-CCD C9100 camera (Hamamatsu Photonics, KK, Japan) with IPLab software (Scanalytics, Fairfax, VA).
5
Time-lapse Imaging of Cell Biomass
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QPI of MCF-7, BT-474, and HeLa cells was performed as described in Yu et al.74 (link). Fluorescence images were obtained with an EM-CCD C9100 camera (Hamamatsu Photonics) and an X-Cite Series 120 Q (Lumen Dynamics) source. Image collection occurred every 5 min for 12 h at 14–16 imaging locations containing cells plated with sufficient spacing to enable automated image processing and biomass segmentation.
6
Confocal Microscopy for Vibratome Sections
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For all imaging experiments on (non-cleared) vibratome sections an Olympus BX51WI DSU confocal microscope (Olympus, Center Valley, PA, USA) coupled to a Hamamatsu EM-CCD C9100 camera (Hamamatsu Photonics K. K., Hamamatsu, Japan) was used. The system was equipped with a motorized stage and a LEP MAC 5000 Controller System (Ludl electronic products, Hawthorne, NY, USA). Images were taken with either, an Olympus PlanApo 2x/0.08 NA, 4x/0.16 NA or UPlanSApo 10x/0.40 NA objective (Olympus, Center Valley, PA, USA). Excitation and emission characteristics for all dyes are given in Supplementary Table 2 . For acquisition the Stereo Investigator software (MBF Bioscience, Williston, Vermont, USA) was used.
7
High-resolution Imaging of Acute Brain Slices
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High‐resolution videos were obtained using an Olympus microscope with a water immersion objective (20×; NA 0.95). One movie (per condition) was acquired on a TrimscopeTM (LaVision Biotec) using a Ti‐sapphire laser tuned to 820 nm. Images were captured using a Hamamatsu C9100 EM‐CCD camera at 7.65 Hz for a total of 4,000 frames on an area of 450µmX525µm. During data acquisition, acute postnatal mouse brain slices were continuously perfused with oxygenated standard ACSF to approximately 37°C at 1 ml/min. After baseline recordings were obtained, drugs were washed into the bath for 10 min prior to reimaging under the new condition. At the end of each movie, a Z‐stack ± 20 µm (in steps of 1 µm) of the focus plane was acquired for cell body detection. All data collection was done with ImSpector Software (LaVision BioTec).
8
Fluorescence Imaging of Electroporated Cells
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After nsEP exposures, dishes were kept covered for 15 minutes, and then washed 5 times with PBS to remove all YP. Images of electropermeabilized cells were acquired using an Olympus SZX16 fluorescence stereo microscope (Olympus America, Hamden, CT) equipped with a Hamamatsu C9100 EM-CCD camera (Hamamatsu, Shizuoka Prefecture, Japan) and a 0.8x, 0.12 NA objective. YP emission was detected using an X-Cite Series 120Q fluorescence light source (Excelitas Technologies Corporation, Waltham, MA) and a GFP filter (ex. 460–490 nm/em. 510 nm longpass).
Images were quantified using MetaMorph 7.8.13 software (Molecular Devices, Foster City, CA). The background-corrected YP fluorescence was measured within 16 regions of interest along a long drawn between electrodes 2 and 3 (Fig.2A ) and plotted against distance from the center of the gap between the electrodes.
Images were quantified using MetaMorph 7.8.13 software (Molecular Devices, Foster City, CA). The background-corrected YP fluorescence was measured within 16 regions of interest along a long drawn between electrodes 2 and 3 (Fig.
9
Visualizing Myoblast Cytoskeleton Dynamics
10
Multicolor TIRF Microscopy Imaging
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Images were acquired through an APO N TIRF 60 × /1.49 NA oil objective on an Olympus IX81. Excitation by 488 nm (Spectra Physics) and 561 nm (Cobolt) lasers was combined using an LMM5 laser merge module and delivered to a Spectral Diskovery TIRF (Oxford Instruments) with an identical penetration depth set to 150 nm for all wavelengths. Image acquisition by a C9100 EM-CCD camera (Hamamatsu) was handled by MetaMorph (v7.8.3).
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