The surface available biotin content of each the electrospun meshes was quantified using different concentrations of fluorescein isothiocyanate labeled streptavidin (FITC-streptavidin) using a Leica DMI6000 B confocal microscope equipped with a Nipkow (CSU-X1) spinning disk (Yokogawa) and a Hamamatsu ImagEM EMCCD camera imaging through a 10× objective. The samples were excited using a Coherent Sapphire laser at 488 nm, and fluorescent images were captured using a Chroma ET bandpass 525/50 filter to capture the 529 nm wavelength emission from the FITC-streptavidin. An automated stage controlled via a μManager plugin for ImageJ (Version 1.45, NIH)33 (link) was used to capture a montage of images and a custom Matlab script was used to create a single image of the entire mesh composed of many 10× images stitched together. ImageJ was used to manually segment each mesh as well as an internal blank space within each image to establish the average background fluorescence for each image.
Csu x1 spinning disk
Manufactured by Hamamatsu Photonics
The CSU-X1 is a spinning disk confocal scanning unit designed for high-speed and high-resolution imaging. It utilizes a rotating Nipkow disk with multiple pinholes to rapidly scan a sample, providing optical sectioning capabilities. The CSU-X1 is compatible with a variety of microscope systems and can be integrated with cameras and other imaging equipment to enable efficient and flexible imaging solutions.
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Lab products found in correlation
Imagem em ccd, by Hamamatsu Photonics (1 mentions)
Ultraview vox, by PerkinElmer (1 mentions)
Volocity, by PerkinElmer (1 mentions)
Imagem x2 camera, by Hamamatsu Photonics (1 mentions)
Cell observer sd microscope, by Yokogawa (1 mentions)
Imagem emccd camera, by Hamamatsu Photonics (1 mentions)
Oil immersion objective, by Zeiss (1 mentions)
2 orca fusion camera, by Hamamatsu Photonics (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Imagem ccd camera, by Hamamatsu Photonics (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Rat anti cd31, by BD (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Fusion bt camera, by Hamamatsu Photonics (1 mentions)
Ti2e microscope, by Yokogawa (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Orca flash 4 camera, by Hamamatsu Photonics (1 mentions)
Em ccd camera, by Hamamatsu Photonics (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
9 protocols using csu x1 spinning disk
1
Quantifying Surface Biotin Content in Electrospun Meshes
2
Live Imaging of Dorsal Aorta Development
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Embryos were imaged on an inverted microscope (UltraVIEW VOX, Perkin Elmer) equipped with a Yokogawa CSU-X1 spinning-disk, an ImagEM-X2 camera (Hamamatsu), 488 nm and 561 nm laser lines for excitation, a Zeiss LD C-Apochromat 40x corrM27 water objective (NA 1.1, WD 0.62 mm at cover glass 0.17 μm) and the Volocity (Perkin Elmer, http://www.perkinelmer.com/ ) acquisition software. Optical Z planes were spaced by 0.6 μm, the power of the lasers (around 150 mW) and exposure time (between 50 ms and 200 ms) were adjusted depending on the fluorophore and the transgenic line used. In all cases, acquisitions of a Z stacks containing the whole dorsal aorta was reduced to 1 min or less and spaced every 2 min. For TL sequences longer than 1 hr, samples were maintained at 28.5°C using an Okolab cage incubator and the objective was immersed in Immersol W 2010 oil (Zeiss, Cat#: 444969-0000-000).
3
Visualizing embryo division dynamics
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Worms at the larval L4 stage were incubated on 6 cm RNAi feeding plates for 48 hr at 20°C. In the experiment using the div1(or148ts) mutant (Figure 2D–E ), all worms were shifted to the restrictive temperature of 25°C for 24 hr, prior to imaging. For each experimental condition, five embryos were dissected from different worms in M9 medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 5 g / L NaCl, 0.25 g / L MgSO4) and mounted together on a 2% agarose pad. Embryos were then recorded simultaneously at 24°C, taking images every 10 s with 75% laser power and 200 milliseconds exposure time. Time lapse images were acquired using an Zeiss Cell Observer SD microscope with a Yokogawa CSU-X1 spinning disk, HAMAMATSU C13440 camera, fitted with a PECON incubator, using the ZEN blue software, and then processed with ImageJ software (National Institutes of Health) as previously described (Sonneville et al., 2017 (link)).
4
Fluorescent Imaging of Span 80 Emulsions
5
Spinning Disk Confocal Microscopy
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Before imaging, samples were mounted on ProLong (P36962, Invitrogen). Imaging was performed on a microscope setup based on an inverted Axio Observer.Z1 microscope (Zeiss), a Yokogawa CSU-X1 spinning disk, and a 2 ORCA Fusion camera (Hamamatsu). ZEN 2 acquisition software was used for setup-control and data acquisition. Illumination was performed using different lasers (405 nm, 488 nm, 561 nm). Cells were inspected with a 63 × 1.4 oil immersion objective (Zeiss). Images were taken in z-stack focal-planes with distances of 500 nm for a maximal intensity projection.
6
Embryo Processing and Analysis Protocols
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Embryos were processed as previously described (Wythe et al., 2013 (link)). For lacZ embryos, beta galactosidase activity was detected using Salmon Gal (Sigma) prior to processing for in situ hybridization. Immunostaining of MADM samples for GFP and Myc was as previously described (Zong et al., 2005 (link)). Antibodies used on cryosections include: Rabbit anti-GFP (Invitrogen, 1:1000), Goat anti-Myc (Novus, 1:200), Rat anti-CD31 (BD Pharmingen, 1:100), Mouse anti-tropomyosin (DSHB clone CH-1, 1:50). Whole-mount lacZ and indirect immunofluorescent images were obtained using a Leica dissecting microscope and camera with the Leica LAS Montage extended focus function. Confocal images were obtained on a Nikon ECLIPSE Ti 2000 confocal microscope with a Yokogawa CSU-X1 spinning disk and Hamamatsu ImagEM CCD camera. Images were processed using Volocity software (Perkin Elmer). All images, including immunofluorescent, in situ hybridization, and LacZ images are representative images. At least 5 embryos (in situ hybridization and LacZ) or 3 independent sections (immunofluorescence) were examined for each experiment. Images shown represent average or representative expression levels.
7
Quantifying Cells in Spinal Cord Lesions
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For spinal cord lesions, slides were imaged with confocal microscopy (Zeiss LSM 800). Images were blinded with cage number and ear tag before quantification. Cells were counted with image J in the lesioned area defined by Hoechst staining, and then calculated as per mm2 or as percentages. The average of results from 2–6 spinal cord sections from each mouse was defined as n of one. For each group, n = 3–8 mice were used for statistics. For RAW 264.7 cells, microglia, and mixed glial cultures with AKA treatment were imaged with confocal microscopy (Zeiss LSM 800) and counted with image J. Results from five views of images in each condition (n = 3–4 repeats in total) were examined for statistics. For primary oligodendrocyte cultures with IL4I1 treatment, all views of images from 2 independent experiments were used for quantification. For primary oligodendrocyte cultures with AKA treatment, stained oligodendrocytes were imaged on a Nikon Ti2-E microscope with a Yokogawa CSU-X1 spinning disk and a Hamamatsu FusionBT camera. Cells were analyzed using NIS Elements software. Results from 3 independent experiments (3 rat pups) were used for statistics.
8
Immunofluorescence Imaging of Neural Stem Cells
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Immunofluorescence samples were imaged using a SP5 confocal microscope (Leica) with a 40× oil objective lens (Leica). For fixed NSCs, three random regions of each coverslip were imaged with a 1 µm z-step. For each brain section, the left and right DGs in every 12th 40 µm section along the rostrocaudal axis were imaged, with a 1 µm z-step through the whole 40 µm section. For the reactivation experiment in quiescent control and β-catdel ex3-6 NSCs and quiescent Catnblox(ex3)/wt NSCs, an Axio Imager.Z2 microscope with a 20× objective (Zeiss) with Hamamatsu Orca Flash 4 camera and Apotome 2 technology for optical sectioning was used. Images of the comparison between DG and SVZ for immunolabelling of β-catenin were acquired with an Axio Observer microscope (Zeiss) equipped with a Yokogawa CSU X1 spinning disk and a Hamamatsu EMCCD camera.
9
Multimodal Microscopy for Cellular Imaging
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Images were acquired on a Nikon Ti-E system fitted with a Yokogawa CSU-X1 spinning disk head, Hamamatsu Orca Flash 4.0 v2 digital CMOS camera, Perfect Focus system, and a Nikon LU-N4 solid state laser launch (15 mW 405, 488, 561, and 647 nm) using the following objectives: 100x 1.49 NA Apo TIRF oil immersion, 40x 1.3 NA Plan Fluor oil immersion, and 20x 0.75 NA Plan Apo. This microscope was powered through Nikon Elements AR software on a 64-bit HP Z440 workstation.
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