The lattice light sheet microscope located at the Advanced Imaging Center (AIC) at the Janelia Research Campus of the Howard Hughes Medical Institute (HHMI) (Chen et al., 2014 (link)) was used. HUVECs stably expressing dTomato-2xrGBD and mTurquoise2-CaaX were cultured on fibronectin-coated 5 mm round glass coverslips (Warner Instruments, Catalog # CS-5R) for 2 days. Cells were imaged at 37°C in the presence of 5% CO2 in HEPES buffer (132 mM NaCl2, 20 mM HEPES, 6 mM KCl2, 1 mM MgSO4•7H2O and 1.2 mM K2HPO4•3H2O at pH 7.4), supplemented with 1 mM CaCl2, 0.5% Albuman (Sanquin Reagents, The Netherlands) and 1 g/l D-glucose. Illumination was undertaken using 445 nm and 560 nm diode lasers (MPB Communications), acousto-optic tunable filter (AOTF) transmittance and 100 mW initial box power and an excitation objective (Special Optics, 0.65 NA, 3.74-mm WD). Fluorescence detection was done via a detection objective (Nikon, CFI Apo LWD 25XW, 1.1 NA) and a sCMOS camera (Hamamatsu Orca Flash 4.0 v2). Point-spread functions were measured using 200 nm tetraspeck beads (Invitrogen cat# T7280) for each wavelength. Data was deskewed and deconvolved as described previously (Chen et al., 2014 (link)).
Cs 5r
Manufactured by Warner Instruments
The CS-5R is a compact, single-channel stimulator designed for reliable and precise electrical stimulation. It features an adjustable output voltage range and can be used for a variety of applications in electrophysiology and neuroscience research.
Automatically generated - may contain errors
Lab products found in correlation
Orca flash4.0 v2, by Hamamatsu Photonics (3 mentions)
Cfi apo lwd 25xw, by Hamamatsu Photonics (2 mentions)
T7280, by Thermo Fisher Scientific (2 mentions)
Tetraspeck bead, by Thermo Fisher Scientific (1 mentions)
Trolox, by Santa Cruz Biotechnology (1 mentions)
Tram 34, by Merck Group (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Amira software, by Thermo Fisher Scientific (1 mentions)
Cs 4r, by Warner Instruments (1 mentions)
Projet mjp 2500, by 3D Systems (1 mentions)
Cs 3r, by Warner Instruments (1 mentions)
9 protocols using cs 5r
1
Live-cell Imaging of Endothelial Cytoskeletal Dynamics
2
Lattice Light Sheet Microscopy Workflow
3
Zebrafish Embryo Preparation for LLSM
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To ensure proper development and adequate expression of mem-citrine and before mounting in the stage of the LLSM, the zebrafish embryos were screened using a Leica stereomicroscope for their health and fluorescence brightness. The embryos were then carefully placed in the center of a 5-mm-diameter glass coverslip (CS-5R; Warner Instruments) directly above a drop of rapidly setting 1% (wt/wt; in 1× Danieau buffer) low-melt agarose (UltraPure Low Melting Point Agarose; LMA). During the fluid agarose phase, suspended embryos were gently nudged using a hair-loop to occupy a dorsolateral position; this orientation prevented possible occlusion from the yolk during the imaging at 17–24 h postfertilization of the eyes and the spinal cord. Excessive deterioration of the quality of the light sheet generated by LLSM and reduced light scattering by the agarose was avoided by using Kimmel tissue paper to remove by wicking the extra fluid above the zebrafish before solidification.
4
Live Cell Imaging of Erythrocyte Invasion
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The sample was imaged in phenol red-free complete medium. For calcium experiments, the imaging medium was supplemented with 5 mM sodium pyruvate, 10 µM Trolox (Santa Cruz 53188-07-1) and 0.25 mM CaCl2. For experiments involving inhibitors, either 100 µg/mL R1 peptide (China Peptides), 1 µg/mL cytochalasin D (Sigma Aldrich C8273), or 10 µM TRAM34 (Sigma Aldrich T6700) were added to the imaging medium. For confocal microscopy, 200 µL of imaging medium and 30 µL of stained erythrocytes per well were loaded to eight-well plate (Ibidi 80826). Then, 5–10 µL of stained schizonts were added to the stained erythrocytes in the first well right before imaging. For LLSM, an acid-washed 5 mm round glass coverslip (Warner Instruments CS-5R) was attached to the bottom of each well before loading 200 µL of phenol red-free RPMI-HEPES and 30 µL of stained erythrocytes to the well. Then, 5–10 µL of stained schizonts were gently added to the top of the coverslip and left to settle on the coverslip for 15 minutes. A tweezer was used to attach the coverslip to the sample stage, then the coverslip was embedded in the microscope bath filled with 8 mL of imaging medium.
5
Lattice Light Sheet Microscopy Workflow
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The lattice light sheet microscope (LLSM) used here is housed in the Advanced Imaging Center (AIC) at the Howard Hughes Medical Institute Janelia research campus. The system is configured and operated as previously described37 . HeLa cells were seeded on 5 mm round glass coverslips and grown for 24–48h (Warner Instruments, Catalog # CS-5R). Samples were illuminated by a 2D optical lattice generated by a spatial light modulator (SLM, Fourth Dimension Displays). The sample is excited by 488 nm or 560 nm diode lasers (MPB Communications) through an excitation objective (Special Optics, 0.65 NA, 3.74-mm WD). Fluorescent emission was collected by detection objective (Nikon, CFI Apo LWD 25XW, 1.1 NA), and detected by a sCMOS camera (Hamamatsu Orca Flash 4.0 v2). Acquired data were deskewed as described37 and deconvolved using an iterative Richardson-Lucy algorithm. Point-spread functions for deconvolution were experimentally measured using 200nm tetraspeck beads adhered to 5 mm glass coverslips (Invitrogen,T7280) for each excitation wavelength.
6
Live Imaging by Lattice Light-Sheet Microscopy
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For live imaging by lattice light-sheet microscopy, the specimens were anesthetized with MS-222. Samples were then suspended in molten 1% low-melting-point agarose containing diluted MS-222 and mounted on #1 5-mm round coverslips (CS-5R, Warner Instruments). Sample and coverslip were stabilized in a custom-made stainless-steel holder and attached to piezo stages (Physik Instrumente). Imaging was conducted at room temperature (21°C) in PBS. Images were acquired via 1.1 NA 25× water-dipping objective (Nikon). Bessel-beam plane illumination microscopy has been previously published (Gao et al., 2014 (link); Planchon et al., 2011 (link)). Briefly, for lattice light-sheet imaging, a collimated 488-nm or 561-nm laser light was passed through a pair of cylindrical lenses to illuminate a stripe across the width of a ferroelectric spatial light modulator (SLM, Forth Dimension Displays). The optical path following the SLM creates a demagnified image of the pattern at the focal plane of a custom-made water-dipping excitation objective (Special Optics, 0.65 NA). The lattice pattern was dithered along the x-axis and swept through the z-axis to create 3D volumes. Deconvolution was performed using Richardson-Lucy iterations and movies were made using the Amira software (FEI).
7
In Vivo Imaging of Mouse Visual Cortex
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To prepare mice for in vivo imaging sessions, we performed surgeries while mice were mounted in a stereotaxic frame under isoflurane anesthesia (1.5-2% isoflurane in O 2 ). To reduce post-operative inflammation and pain, we administered a preoperative dose of carprofen (5 mg/kg; subcutaneous injection into the mouse's lower back), which we repeated once a day for 3 days following the surgery. We created a cranial window by removing a 5mm-diameter skull flap (centered at AP -2.5, ML 2.7) over the right cortical area V1 and surrounding cortical tissue. We covered the exposed cortical surface with a 5-mm-diameter glass coverslip (#1 thickness, 64-0700, CS-5R, Warner Instruments) that was attached within a circular steel annulus (1 mm thick, 5 mm outer diameter, 4.5 mm inner diameter, 50415K22, McMaster) and secured to the cranium using ultraviolet-light curable cyanoacrylate glue (Loctite 4305). Using dental acrylic, we cemented a metal head plate to the skull for headfixation during imaging. In vivo brain imaging studies commenced at least 7 days after surgery.
8
Collagen Matrix for Cell Mechanobiology
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Precooled HeLa cells were mixed on ice with a collagen solution containing 20 mM Hepes, 20 µg/ml fibronectin, 0.3% sodium bicarbonate, and 1 M NaOH to neutralize 0.02 N collagen in acetic acid to obtain a final collagen concentration of 2 mg/ml and cell density of 5–8 × 105 cells/ml (∼200 Pa; Mason et al., 2013 (link)). For stiff collagen matrix (∼800 Pa), collagen stock solution was preincubated for 30 min on ice with ribose (200 mM) and thereafter used for preparation. For visualization of collagen fibers, collagen was stained with Cy5 in 0.1 M sodium bicarbonate and dialyzed overnight in 0.1% acetic acid. For measurement of forces exerted on the collagen matrix, beads were embedded into the 3D matrix. Briefly, AF647-carboxylate–modified microbeads (0.2-µm diameter; Molecular Probes) were sedimented at 3,000 g for 20 min, resuspended in medium by vortexing, and sonicated for 60 min. Beads were mixed at 1.8 × 109 beads/ml final concentration with collagen-cell solution on ice to prevent polymerization. 10-20 µl of collagen-cell mix (with or without beads) were pipetted on collagen-coated 5-mm round glass coverslips (CS-5R, Warner Instruments), left to polymerize at 37°C for 30 min, and imaged within 36 h.
9
Customized Headpost and Cranial Window Implants
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Headpost implant design was adapted from Ghanbari et al. 55 , and consists of a custom-made Titanium or Stainless-steel head-plate, a 3D-printed frame, and three 0-80 screws to hold the frame to the head-plate.
We fabricated the head-plate with Titanium or Stainless-steel plate (McMaster-Carr) using a waterjet system (OMax), and 3D printed the frame using a ProJet MJP 2500 (3D Systems). Our design files can be found online (https://github.com/ckemere/TreadmillTracker/tree/master/UMinnHeadposts). We assembled the headpost implant after tapping the 3D printed frame with 0-80 tap and securing the headplate over the frame with three screws. The entire headpost is then stored in 70% Ethanol prior to surgery.
Cranial window fabrication procedure was adapted from Goldey et al. 56 . Windows were made of 2 stacked round coverslips (Warner Instruments # CS-3R, CS-4R, CS-5R) of different diameters. To fabricate the stacked windows, a 3 mm (or 4 mm) round coverslip was epoxied to a 4 mm (or 5 mm) cover slip using an optical adhesive (Norland Products Inc. e.g. # NOA 61, 71, 84) and cured using longwavelength UV light. To accommodate the large 5 mm stacked window, we cut off the right side of the 3D-printed frame to allow for extra space for the C&B Metabond to bind to the skull outside of the stacked cranial window. Fabricated stacked windows were stored in 70% ethanol prior to surgery.
We fabricated the head-plate with Titanium or Stainless-steel plate (McMaster-Carr) using a waterjet system (OMax), and 3D printed the frame using a ProJet MJP 2500 (3D Systems). Our design files can be found online (https://github.com/ckemere/TreadmillTracker/tree/master/UMinnHeadposts). We assembled the headpost implant after tapping the 3D printed frame with 0-80 tap and securing the headplate over the frame with three screws. The entire headpost is then stored in 70% Ethanol prior to surgery.
Cranial window fabrication procedure was adapted from Goldey et al. 56 . Windows were made of 2 stacked round coverslips (Warner Instruments # CS-3R, CS-4R, CS-5R) of different diameters. To fabricate the stacked windows, a 3 mm (or 4 mm) round coverslip was epoxied to a 4 mm (or 5 mm) cover slip using an optical adhesive (Norland Products Inc. e.g. # NOA 61, 71, 84) and cured using longwavelength UV light. To accommodate the large 5 mm stacked window, we cut off the right side of the 3D-printed frame to allow for extra space for the C&B Metabond to bind to the skull outside of the stacked cranial window. Fabricated stacked windows were stored in 70% ethanol prior to surgery.
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