A custom-build microscope was used to reconstruct the light field behind the lenses. For this experiment, the drops were illuminated with a quasi-monochromatic plane wave. This was achieved by imaging the output of an optical fibre with 50 μm core in the back focal plane of an NPL × 20 objective (Leitz Wetzlar, NA=0.45) used as a condenser. A 540 nm bandpass filter with an 80 nm bandwidth was used to create quasi-monochromatic light. The light field behind the droplets was captured by scanning the focal plane in 5 μm steps using a Madcity Labs micro-stage, a × 20 Olympus objective (NA=0.75), a 200 mm focal length achromatic doublet tube lens and an Andor Zyla sCMOS 5.5 Camera. The light field data was analysed using MATLAB and ImageJ. The location and size of the droplets were determined from the images using ImageJ's measurement tools. The data from the focal scans were then entered into MATLAB to reconstruct the 3D light field behind individual droplet lenses, similar to the approach previously used to measure the light field behind retinal cell nuclei56 . After the light field was measured, the droplet lenses were placed in a microscope with horizontal optical axis and imaged from the side. This side view was used to determine the curvature and volume ratio of the drops.
5.5 scmos camera
Manufactured by Oxford Instruments
Sourced in Japan
The 5.5 sCMOS camera is a high-performance scientific camera that utilizes sCMOS (scientific Complementary Metal-Oxide-Semiconductor) sensor technology. It is designed to deliver exceptional image quality and sensitivity for a wide range of scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Ti e inverted microscope, by Nikon (3 mentions)
Ti microscope, by Oxford Instruments (2 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (2 mentions)
3xha egfp omp25, by Addgene (2 mentions)
Plan apo 20x n a 0.75 dic lens, by Oxford Instruments (2 mentions)
Spectra x, by Lumencor (2 mentions)
Motorized tirf illuminator, by Nikon (2 mentions)
Tie inverted microscope stand, by Nikon (2 mentions)
Na plan apo objective, by Nikon (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Eclipse ti e microscope, by Oxford Instruments (1 mentions)
Eclipse ti u microscope, by Oxford Instruments (1 mentions)
Anti cb1 antibody, by Cayman Chemical (1 mentions)
Ti e inverted microscope, by Oxford Instruments (1 mentions)
Rapamycin, by Thermo Fisher Scientific (1 mentions)
Gsk a1, by Merck Group (1 mentions)
Eclipse ti2 e microscope, by Oxford Instruments (1 mentions)
Dsri2 colour camera, by Nikon (1 mentions)
Eclipse te300, by Nikon (1 mentions)
Xa 21q22, by MetaSystems (1 mentions)
32 protocols using 5.5 scmos camera
1
Reconstructing Light Field Behind Droplet Lenses
2
Microfluidic Maintenance of C. elegans for Long-Term Imaging
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When animals were ∼72 h posthatching, they were loaded into the microfluidic devices along with a solution of 100 mg of E. coli ml−1 of liquid NGM (NGM without the agar). For animals continuing to receive the pharmacological treatment after development, the compound was introduced into the E. coli solution at the appropriate concentration before the concentrated bacteria solution was added to the device (Fig. 7 B). On each day for the remainder of the experiment, the devices were washed using liquid NGM to remove progeny and debris, and a fresh solution of bacteria was added to the device (Fig. 7 C,D). The arena of pillars and barriers in the outlet ports allow for the retention of adult animals and the filtering out of unwanted progeny, as has been previously demonstrated for C. elegans maintenance in microfluidic devices (Hulme et al., 2010 (link); Wen et al., 2012 (link); Xian et al., 2013 (link); Wen et al., 2014 (link)).
After clearing the devices of progeny and debris, and before adding fresh E. coli, animals were imaged in the microfluidic chambers (Fig. 7 C,D) for 45-s episodes at a rate of five frames per second. A Nikon Eclipse TI-E microscope with Andor Zyla sCMOS 5.5 camera was used. Any animals that remained stationary during the first image sequence, although few in number, were re-imaged until a movie including sufficient worm locomotion was obtained.
After clearing the devices of progeny and debris, and before adding fresh E. coli, animals were imaged in the microfluidic chambers (
3
Fluorescent Imaging of Microdroplet Arrays
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Fluorescent images of microdroplet arrays were acquired using a custom-built fluorescence microscope. Four laser lines were used for dye excitation: Vortran Stradus 532 nm (5.4 mW), Cobalt Mambo 594 nm (7.6 mW), Vortran Stradus 640 nm (8.6 mW), and Vortran Stradus 701 nm (7.2 mW). Optical powers stated were measured after the objective. The laser light was directed into a vibrating, 150 × 150 μm square core optical fibre, the end of which was imaged onto the sample via a 10×, 0.5 NA objective (S Fluor, Nikon) producing a flat, wide-field excitation profile over a 650 μm × 650 μm area. A filter cube changer stage was used to position the required excitation, dichroic, and emission filters in the optical path. Images were recorded with an Andor Zyla 5.5 sCMOS camera. The exposure time per laser for each field of view was 25.4 s (50.8 s for the 640 nm laser).
4
Imaging Clec9a+ Dendritic Cells in CNS
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uDISCO clarified CNS, spinal cord or embryo from Clec9acreRosaLSLtdtomato mice were imaged on a Miltenyi-LaVision BioTec Ultramicroscope II light-sheet microscope. Tissues were mounted on a sample holder and imaged in BABB-D4 solution with an Olympus MVPLAPO 2x /0.5 NA with a protective dipping cap (WD > 5.7 mm). Tissues were excited with a bi-directional 561nm wavelength distributed across three Gaussian light-sheets with a NA of 0.09 and exposed for 200ms. The step size between each image was 5 μm. Images from both light-sheets were acquired with an Andor Zyla 5.5 sCMOS camera and merged using the blend function in Imspector Pro software. A zoom of 1.25x-1.6x was used for imaging of E11.5 embryos. A zoom of 0.63x was used for imaging of whole CNS and volumes were stitched using BigStitcher (Hörl et al., 2019 (link)) in Fiji/ImageJ software (Schindelin et al., 2012 (link)). DNGR-1 traced surfaces were generated using Imaris software (Bitplane). Given the lower tdTomato fluorescence of DNGR-1 traced cDCs (Figures S1 C and S5 C) a low laser power was used to preferentially illuminate the bright tdTomato ependymal cell compartment in injured spinal cords (Figure 7 A).
5
Fluorescence Microscopy Image Acquisition and Analysis
6
Multi-channel Fluorescence Imaging of Cellular Delivery
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Fluorescence images were acquired on a Nikon Eclipse TE 2000 microscope equipped with an Andor Zyla 5.5 sCMOS camera. Image acquisition was controlled using Micro-Manager software. A custom Python script interfacing with Micro-Manager was used to acquire multichannel images from the 24-well plate–format LEPDs. The images of cells after intracellular delivery were acquired with an exposure time of 400 ms under ×4/×10/×20/×40 magnification. For the time-lapse imaging of the calcein depletion experiments, an image was acquired every second with an exposure time of 200 ms using the multidimensional acquisition module of Micro-Manager.
7
Immunocytochemical Detection of CB1 Receptors
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Transfected COS7 cells were grown on 24-well glass bottom plate and fixed with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer for 10 min. Cells were then treated with 10% normal goat serum for 30 min, followed by an incubation with anti-CB1 antibody (1:2000, Cayman Chemicals, Ann Arbor, MI, USA, Cat. No: 10006590) for 2 h. After washing, goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488 (1:1000, Invitrogen) was applied for 1 h, then cells were covered with Vectashield-DAPI. Antibodies were diluted in PBS (pH 7.4) containing 1% normal goat serum. All incubation steps were carried out at room temperature.
Imaging of immunostained COS7 cells was carried out with an Olympus IX-81 inverse microscope attached to a DSD2 an Andor Zyla 5.5 sCMOS camera. Images were acquired using a 60× PlanApo N oil-immersion objective (NA: 1.40) and selecting the “high signal” disk of DSD2, and processed with Adobe Photoshop CS6.
The specificity of anti-CB1 antibody has extensively been characterized earlier in our laboratory (Hegyi et al., 2009 (link)). To test the specificity of the immunostaining protocol, transfected COS7 were incubated according to the immunostaining protocol described above with primary antibodies omitted or replaced with 1% normal goat serum. No immunostaining was observed under these conditions.
Imaging of immunostained COS7 cells was carried out with an Olympus IX-81 inverse microscope attached to a DSD2 an Andor Zyla 5.5 sCMOS camera. Images were acquired using a 60× PlanApo N oil-immersion objective (NA: 1.40) and selecting the “high signal” disk of DSD2, and processed with Adobe Photoshop CS6.
The specificity of anti-CB1 antibody has extensively been characterized earlier in our laboratory (Hegyi et al., 2009 (link)). To test the specificity of the immunostaining protocol, transfected COS7 were incubated according to the immunostaining protocol described above with primary antibodies omitted or replaced with 1% normal goat serum. No immunostaining was observed under these conditions.
8
Mitochondrial Function Under Hypoxia
9
Live-Cell Microscopy Imaging Protocol
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For live-cell microscopy experiments, cells were cultured and seeded as described in previous sections. Time-lapse wide-field microscopy was performed as detailed in Tan and Huang, 2017 (link), with cells maintained in 95% air and 5% CO2 at 37°C in an environmental chamber. Images were collected with a Nikon (Tokyo, Japan) 20x/0.75 NA Plan Apo objective on a Nikon Eclipse Ti inverted microscope, equipped with a Lumencor SOLA or Lumencor SPECTRA X light engine. Fluorescence filters used in the experiment are DAPI (custom ET395/25x – ET460/50m – T425lpxr, Chroma), CFP (49001, Chroma), YFP (49003, Chroma), Cherry (41043, Chroma), and Cy5 (49006, Chroma). Images were acquired using Andor Zyla 5.5 sCMOS camera every 6–7 min at 2 × 2 binning. Exposure times for each channel were 25–50 ms for DAPI; 150–250 ms for CFP; 150–250 ms for YFP; 300–500 ms for Cherry; and 300–500 ms for Cy5.
10
Immunofluorescence Microscopy Imaging Protocol
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