Cells fixed and stained with DAPI, FOXO1 (Alexa 488) and Phalloidin (Alexa 568) were imaged using a Nikon Eclipse Ti2-E microscope equipped with a Yokogawa confocal scanner unit (CSU-W1), solid state diode lasers (405, 488 and 568 nm) and a Hamamatsu ORCA-Flash4.0 V3 sCMOS camera. Cells were imaged for mean intensity analysis using a 40x objective (CFI Super FLUOR; 0.9 NA) and the higher resolution images were captured with the 60x objective (Plan Apo; 1.40 NA oil). DIC images were collected for a single plane, while fluorescence images were collected every micron for 10 microns (the bottom slices including those below the nucleus and several out of focus planes were excluded from downstream mean intensity analysis; 7 slices were used for the analysis).
Csu w1
Manufactured by Hamamatsu Photonics
The CSU-W1 is a compact confocal scanning unit designed for fluorescence microscopy applications. It features a high-speed rotary Nipkow disk that enables rapid and efficient optical sectioning of samples. The unit is compatible with a range of microscope systems and provides a simple solution for integrating confocal capabilities into various imaging setups.
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Lab products found in correlation
Flash 4.0 camera, by Hamamatsu Photonics (2 mentions)
Lunv solid state laser launch, by Nikon (2 mentions)
Elements ar 4, by Nikon (2 mentions)
Stage top chamber, by Okolab (2 mentions)
Glass bottom dishes, by MatTek (2 mentions)
Ti2 inverted microscope, by Yokogawa (2 mentions)
Eclipse ti2 e microscope, by Yokogawa (1 mentions)
Orca flash 4.0 v3 scmos camera, by Hamamatsu Photonics (1 mentions)
Flash 4 scmos camera, by Hamamatsu Photonics (1 mentions)
Ti e microscope, by Yokogawa (1 mentions)
Ix83 microscope, by Yokogawa (1 mentions)
Orca fusion cmos camera, by Hamamatsu Photonics (1 mentions)
Eclipse ti, by Yokogawa (1 mentions)
Plan apo, by Nikon (1 mentions)
Flash 4, by Hamamatsu Photonics (1 mentions)
Spinning disk confocal microscope, by Yokogawa (1 mentions)
Orca flash 4 v3, by Hamamatsu Photonics (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
Orca flash 4.0 cmos camera, by Hamamatsu Photonics (1 mentions)
Orca fusion scmos camera, by Nikon (1 mentions)
14 protocols using csu w1
1
Imaging Cells with Confocal Microscopy
2
Quantifying Crypt Area in Organoid Images
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Images for crypt area quantification were acquired with Nikon Eclipse TI equipped with a Yokagawa CSU W1 spinning disk and a Hamamatsu Flash 4.0 camera. 3D DAPI images were acquired via a 4× 0.2 NA air objective with 405 nm excitation and a standard DAPI emission filter. Images were collected with 50-micron spacing in the z dimension. Crypt segmentation was performed in Fiji (Schindelin et al., 2012 (link)) by first maximum projecting whole well images and then Gaussian blurring with a standard deviation of 4 pixels. Next, we subtracted a 100-pixel radius rolling background. Next, we thresholded the image at 2.5% of the maximum DAPI intensity and filled the holes in the resulting objects. Finally, we filtered out objects with an area less than 750 pixels to avoid noise and contaminants. Some organoids were very close to each other and were not successfully separated by the above algorithm. Those organoid masks were separated by drawing a dark line between them. Finally, the areas of the organoids were measured as the number of pixels contained within each mask.
3
Imaging Tissue Sections with Spinning Disk Confocal
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Tissue sections on the slides were imaged using the Nikon Ti-E microscope coupled to a Yokogawa CSU-W1 spinning disk using the Hamamatsu Flash 4 sCMOS camera. The images were captured using 60× Plan Apochromat (NA 1.4) oil objective. The sections to image were manually identified using a 10× objective (NA 1.45). The images were obtained at 100% laser power for far red 633 nm, red 561 nm, green 488 nm and DAPI 405 nm lasers. For each channel the following filters were used: DAPI, ET455/50m; green, ET525/36m; red, ET605/70m; far red, ET700/75m. A Nikon Elements Job ‘Tiler’ was used to capture the images if tiles were taken, and the image was stitched later in Fiji (https://imagej.net/software/fiji ) using the grid/collection stitching plugin. The order of experiments was Lambda (z-series), so each color was imaged in z before moving to the next color. For obtaining a z-stack through the tissue section, each slice imaged was 1 µm apart and a range of 20 steps was taken. Images were obtained in the order of 633 (for the 647 probe), 561 (for the 555 probe), 488 (for the atto 488 probe) and 405 (for DAPI). On a slide, the whole tissue section was imaged, and for each slide a single row was imaged all the way across for each genotype of embryo. All images were stitched using Fiji before further processing.
4
High-Resolution Fluorescence Microscopy Protocol
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All imaging was conducted on an Olympus IX83 microscope fitted with a Yokogawa CSU-W1 Dual Disk SoRa, dual Hamamatsu Orca Flash 4.0 V3 sCMOS cameras, and Plan S-Apo 40x & 100x Si Oil Objectives (NA 1.25, 1.35) operated by cellSens software.
5
Spinning Disk Confocal Microscopy for Super-Resolution Imaging
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Sixteen-bit fluorescence images were acquired using an Olympus IXplore spinning disk confocal microscope (equipped with the Yokogawa CSU-W1 with 50 µm pinhole disk and a Hamamatsu ORCA Fusion CMOS camera). A 60× oil immersion objective (NA 1.42) in combination with a 3.2× magnification lens (equalling 192× total magnification) was used for super-resolution imaging of fixed cells and z-stacks with a 0.24 µm slice interval were acquired. These z-stacks were then processed using the Olympus 3D deconvolution software (cellSens Dimension 3.1) (constrained iterative deconvolution, using automatic background removal and noise reduction, filter using advanced maximum likelihood algorithm and five iterations). Finally, ‘maximum-z’ projection images of the deconvoluted z-stacks were generated. Olympus 3D deconvolution software (cellSens Dimension 3.1) was used for analysis. Nuclear foci were counted manually and at least 50 cells per condition were imaged in each experiment. Quantification of the foci was performed manually based on maximum intensity projections.
6
Imaging of Neomycin-Induced Hair Cell Damage
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Images were acquired using a Nikon Ti Eclipse with Yokogawa CSU-W1 spinning disk head equipped with a Hamamatsu Flash 4.0 sCMOS. Objective lenses used were a Nikon Plan Apo 40 × 1.15 NA LWD (water) and a Nikon Plan Apo 20 × 0.75 NA.
For live imaging experiments, larvae were immobilized with tricaine (MS-222) up to 150 mg/L and mounted in glass bottom dishes (MatTek) with 0.8% low melting point agarose dissolved in 0.5x E2 with tricaine (100 mg/L). Time lapse recordings were started 10-min after addition of neomycin (300μM) on top of the agarose. Temperature was kept constant at 28.5 °C using a Stage Top Chamber (OkoLab).
A Nikon LUNV solid state laser launch was used for lasers 405, 445, 488, 561, and 647 nm. Emission filters used on the Nikon were 480/30, 535/30, 605/70.
All image acquisition was performed using Nikon Elements AR 4.6 (Nikon) software.
For live imaging experiments, larvae were immobilized with tricaine (MS-222) up to 150 mg/L and mounted in glass bottom dishes (MatTek) with 0.8% low melting point agarose dissolved in 0.5x E2 with tricaine (100 mg/L). Time lapse recordings were started 10-min after addition of neomycin (300μM) on top of the agarose. Temperature was kept constant at 28.5 °C using a Stage Top Chamber (OkoLab).
A Nikon LUNV solid state laser launch was used for lasers 405, 445, 488, 561, and 647 nm. Emission filters used on the Nikon were 480/30, 535/30, 605/70.
All image acquisition was performed using Nikon Elements AR 4.6 (Nikon) software.
7
Quantifying Crypt Area in Organoid Images
8
Live Cell Imaging of Paclitaxel Effect on Mitosis
9
Imaging Intact Vertebrate Skulls
10
Imaging and Quantifying Lipid Droplets
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All cells were imaged using either a Nikon Ti2 inverted microscope equipped with a 100 × (1.4 NA) Plan-Apo oil immersion objective and a Yokogawa CSU-W1 spinning disk confocal attached to a Hamamatsu ORCA-FLASH4.0 CMOS camera. Cells in either DMEM FluoroBrite (Thermo Fisher Scientific) medium supplemented with 5% FBS or EBSS lacking phenol red (MilliporeSigma) were imaged at 37 °C and 5% CO2. Images stacks were captured at 16-bit 2048 × 2044 resolution with an axial spacing of 0.2 μm using the Nikon Elements Software package. All images were captured blindly and randomly, which involved imaging the nearest cell to a random set of x-y coordinates that contained actin fluorescence (depending on the experiment). Captured images were blinded again, and image analysis was performed using the software Fiji (https://imagej.net/Fiji ). Specifically, LD number and diameters were scored manually, and 2D/3D colocalization analysis was performed using the Fiji Coloc2 analysis tool. Specifically, LD diameter was measured in the plane where a given LD’s diameter was the largest and LD density was determined by dividing the total number of LDs by the area of the cell’s basement membrane.
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