Confocal microscopy was used to capture images of dendrites from the CA1 region of the hippocampus, based on previously described methods (75 (link), 78 (link)). A blinded experimenter performed all imaging. Images were captured with a Nikon (Tokyo, Japan) Ti2 C2 confocal microscope. The experimenter identified secondary dendrites from dye-impregnated neurons and captured three-dimensional z-stacks of those meeting the following criteria: (1 (link)) within 80 μm working distance of microscope; (2 (link)) relatively parallel with the surface of the coronal section; (3 (link)) no overlap with other branches; (4 (link)) minimum of 50 μm from the soma; (5 (link)) maximum of 110 μm from the soma. For each dendrite, z-stacks were captured with a 60X oil-immersion objective (Nikon Plan Apo, N.A. 1.40) using the following parameters: z-step: 0.1 μm; image size: 1024 × 512 px (0.04 μm x 0.04 μm x 0.1 μm); zoom: 4.8x; line averaging: 4; acquisition rate: 1 frame/sec. Captured images were deconvolved using Huygens Deconvolution System (16.05, Scientific Volume Imaging, the Netherlands) and the following settings: GMLE; maximum iterations: 10; signal to noise ratio: 15; quality: 0.003. Deconvolved images were saved in .tif format.
Ti2 c2 confocal microscope
Manufactured by Nikon
Sourced in Japan
The Ti2-C2 confocal microscope is a high-resolution imaging system designed for advanced scientific research. It features a confocal scanning mechanism that allows for the capture of clear, high-contrast images by eliminating out-of-focus light. The microscope is capable of producing detailed, three-dimensional images of various samples, making it a versatile tool for a wide range of applications.
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5 protocols using ti2 c2 confocal microscope
1
Confocal Imaging of Hippocampal Dendrites
2
Confocal Imaging and Image Processing
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Confocal images were acquired using either a Leica TCS-SP5 laser scanning confocal microscope or a Nikon Ti2-C2 confocal microscope. Images were saved using Leica LASF software or Nikon NIS-Elements software. Images were then exported and processed using Adobe Photoshop and Adobe Illustrator.
3
Confocal Imaging and Digital Processing
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Confocal images were acquired using either a Leica TCS-SP5 laser scanning confocal microscope, or a Nikon Ti2-C2 confocal microscope. Images were exported and processed using Adobe Photoshop and Illustrator.
4
Confocal Microscopy of Microbial Cultures
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The microfluidic device was mounted on the stage of a Nikon Ti2-C2 confocal microscope equipped with 60× (Apo Lambda S Oil, Nikon) and 100× (Apo TIRF Oil, Nikon) objective lenses. GFP was excited by a 488 nm laser and detected at 525 nm; Calcofluor White (CFW) and 4-MU were excited by a 405 nm laser and detected at 438 nm. Crosstalk was minimized by the use of line sequential mode. The images were stored and manually analyzed using the Nikon NIS-Elements software, which is compatible with the microscope. Fluorescence intensity was calculated by averaging over a region of interest (ROI) of 80 pixels or more. In the measured wavelength range, the autofluorescence of the hypha and the culture medium was weak and negligible compared to the fluorochrome.
5
Quantifying Neurodegeneration Markers in Brain
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Patient brain sections processed for autofluorescence and Sudan Black B were imaged at 20 × on a Nikon upright microscope or an EVOS M5000 imaging system (ThermoFisher). Images were taken of cortical layer II/III from 2–3 sections from each patient and averaged to give final values. The number of autofluorescent or Sudan Black B-positive particles per field of view was determined using ImageJ. Due to variability in background fluorescence and staining, the Triangle autothreshold function [99 (link)] was used to detect autofluorescence and the Yen autothreshold function [98 (link)] was used to detect Sudan Black B. Preliminary trials with these functions produced very similar thresholds as investigators blinded to sample identity.
Images of p-TDP-43, autofluorescence, and SCMAS from mouse brain sections were taken at 20X on an EVOS M5000 imaging system (ThermoFisher). Staining was quantified with ImageJ by applying a uniform threshold to all images. Immunofluorescent images for colocalization were obtained at 20X on a Nikon Ti2-C2 confocal microscope.
Images of p-TDP-43, autofluorescence, and SCMAS from mouse brain sections were taken at 20X on an EVOS M5000 imaging system (ThermoFisher). Staining was quantified with ImageJ by applying a uniform threshold to all images. Immunofluorescent images for colocalization were obtained at 20X on a Nikon Ti2-C2 confocal microscope.
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