Spinning disk confocal microscopy was used for analyzing biofilms. The 3D stacks were acquired on an Andor Dragonfly spinning disk confocal microscope equipped with an iXon 888 Ultra EMCCD camera and a Nikon Eclipse Ti inverted microscope. For this particular imaging, we used the Nikon PlanApo 60X 1.4 NA Oil immersion objective. Typically, a 3D stack would consist of 350 frames in two colors sampled according to the Nyquist criterion for optimal 3D reconstruction. 3D biofilm images were taken for each chosen sample with three biological replicates.
888 ultra emccd camera
Manufactured by Nikon
Sourced in Japan
The Nikon 888 Ultra EMCCD camera is a scientific imaging device designed for low-light applications. It features an electron-multiplying charge-coupled device (EMCCD) sensor that provides high sensitivity and signal-to-noise ratio. The camera is capable of capturing high-quality images and video in a wide range of scientific and research settings.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti inverted microscope, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Csu w1 spinning disk confocal unit, by Oxford Instruments (1 mentions)
Alc600 laser beam combiner, by Oxford Instruments (1 mentions)
Ti e microscope, by Yokogawa (1 mentions)
1.45 na oil immersion objective, by Nikon (1 mentions)
Scmos camera, by Oxford Instruments (1 mentions)
Eclipse ti epifluorescence microscope, by Nikon (1 mentions)
Nis elements software, by Nikon (1 mentions)
4 protocols using 888 ultra emccd camera
1
3D Biofilm Imaging with Spinning Disk Confocal
2
Quantifying Nuclear Protein Localization
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For immunofluorescence, images were collected on a Nikon TiE microscope equipped with a Yokogawa CSU-W1 spinning disk confocal unit (50-μm pinhole size), an Andor Borealis illumination unit, Andor ALC600 laser beam combiner (405 nm/488 nm), Andor IXON 888 Ultra EMCCD camera, and a Nikon 100×/1.45 NA oil immersion objective. The microscope was controlled by software from Nikon (NIS Elements, ver. 5.02.00). DAPI or Alexa 488 were excited with 405-nm and 488-nm lasers. Within each experiment, cells were imaged using the same settings on the microscope to compare signal intensities between cell lines. Fiji software was used to analyze images. The region of interest of the entire nucleus was manually selected or thresholded using the DAPI signal. Fluorescence intensities of these regions of interest were measured in the 488-nm channel. The mean fluorescence intensities were extracted for each cell line.
3
Quantifying Microscopic Droplet Distributions
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Samples caught on glass microscope slides were imaged with a CFI Plan Fluor 20 × objective mounted on a Nikon Eclipse Ti inverted microscope, equipped with an iXon Ultra 888 EMCCD camera, resulting in a resolution upon magnification of 0.65 µm pixel−1. For each sample, 210 fields of view, covering an area of 0.85 cm2 in total, were acquired in the FITC channel (gain 70, exposure time 100 ms) using NIS-Elements. Samples collected in tests involving human subjects were imaged with an Andor Zyla sCMOS camera and the same objective and microscope as above, resulting in a resolution upon magnification of 0.33 µm pixel−1. We acquired 153 fields of view, corresponding to 0.62 cm2. All images were downscaled by a factor of five prior to their analysis in scikit-image, Python. Droplets in each sample were counted as the markers of the watershed algorithm, which allows segmentation of touching objects in an image. A Gaussian smoothing filter was applied (standard deviation of five) to reduce pixel noise before a thresholding procedure using a manually selected value. Following morphological opening with a Boolean kernel of size seven, droplet centres were identified as the local maxima of the distance transform of the image, with the stipulation that centres should be more than 15 pixels from each other.
4
Imaging Kidney Tissue with smFISH
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Immunostained and smFISH-treated kidney sections were imaged on a Nikon Ti-Eclipse epifluorescence microscope equipped with an Andor iXON Ultra 888 EMCCD camera, using a 100× /1.45 Plan Apo Lambda oil objective (Nikon, Tokyo, Japan) and dedicated, custom-made fluorescence filter sets (Nikon). To cover large areas of the sectioned kidney, images of multiple adjacent areas were taken and combined using the tiling feature of the NIS Elements software (Nikon). For imaging of smFISH signals, z-stacks were collected with distances of 0.3–0.5 μm between planes in four fluorescence channels (GFP, Quasar 570, CAL FLuor Red 610, Quasar 670).
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