The cells were plated at 50% confluence glass cover slips overnight: plasmid transfection used GFP–iBox–Pak4cat and FLAG–iBox–Pak4cat constructs at a ratio of 4:1, to promote crystal nucleation. The cover slips were transferred to a Chamlide magnetic chamber (Live Cell Instruments, Seoul, Korea) with 5% CO2 at 37 °C for live imaging on an Zeiss Axiovert 200 M live-cell imaging with a × 10 objective. We imaged multiple chosen regions for 8 h at 6-min intervals. To measure crystal growth rate, we used instead a Nikon Eclipse Ti microscope equipped with spinning disk confocal attachment (Yokogawa CSU-22 module) to avoid photo-damage. The cells were imaged at × 60 1.4 numerical aperture (NA) objective at 2-min intervals. For SIM and confocal imaging, cells were fixed in non-hardening mounting media (Vectashield). The slides were imaged by Delta vision OMX SIM with a × 100 1.4 NA objective. Confocal imaging used an Olympus FV1000 upright system with a × 60 1.42NA objective. The three-dimensional stacks were analysed by IMARIS software.
Eclipse ti microscope
Manufactured by Yokogawa
Sourced in Japan
The Eclipse Ti microscope is a high-performance optical microscope designed for advanced research and imaging applications. It features a sturdy and ergonomic design, providing a stable platform for precise observation and analysis. The Eclipse Ti offers a range of advanced features, including a high-resolution optical system, versatile illumination options, and compatibility with a variety of imaging techniques and accessories. This microscope is suitable for a wide range of applications in fields such as cell biology, materials science, and biomedical research.
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Lab products found in correlation
Du897 emccd camera, by Oxford Instruments (7 mentions)
Csu x1 spinning disk, by Agilent Technologies (6 mentions)
Laser line module, by Oxford Instruments (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Laser line module, by Agilent Technologies (2 mentions)
Polygon400, by Mightex (2 mentions)
Prime 95b cameras, by Leica (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Apotome 2, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Zen imaging software, by Zeiss (1 mentions)
Csu w1, by Oxford Instruments (1 mentions)
4.2 plus camera, by Oxford Instruments (1 mentions)
Laser module, by Oxford Instruments (1 mentions)
Ti2 e microscope, by Hamamatsu Photonics (1 mentions)
Fusionbt scmos camera, by Hamamatsu Photonics (1 mentions)
Csu x1, by Hamamatsu Photonics (1 mentions)
Scsuw1 confocal scanner unit, by Yokogawa (1 mentions)
Zyla 4.2 plus camera, by Oxford Instruments (1 mentions)
Ix70 microscope, by Yokogawa (1 mentions)
21 protocols using eclipse ti microscope
1
Live-cell imaging of crystal nucleation
2
Quantifying BBB Permeability Using Thioflavin S
3
Fluorescence Microscopy for Cellular Imaging
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Standard fluorescent microscopy was conducted on a widefield Zeiss Axio Observer.Z1 microscope using a Plan-Apochromat 63X/1.40 Oil DIC M27 objective and an ORCA-Flash 4.0 CMOS Camera. The Zeiss Apotome 2.0 was used for structured illumination microscopy using three phase images. All image processing were done using the Zen imaging software from Zeiss. Confocal microscopy was done using a Nikon Eclipse Ti microscope equipped with a W1 Yokogawa Spinning disk with 50 um pinhole disk and an Andor Zyla 4.2 Plus sCMOS monochrome camera. A 60X/1.4 Plan Apo Oil objective or a 100X/1.45 Plan Apo Oil objective was used.
4
Optogenetic Microscopy Imaging Setup
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Cells were maintained at 37 °C with 5% CO2 for the duration of an imaging experiment. Confocal microscopy was performed on a Nikon Eclipse Ti microscope with a prior linear motorized stage, a Yokogawa CSU-X1 spinning disk, an Agilent laser line module containing 405, 488, 561, and 650 nm lasers, ×60 oil emersion objective, and an iXon DU897 EMCCD camera.
For optogenetic microscope experiments, blue light from the XLED1 system was delivered through a Polygon400 digital micromirror device (DMD; Mightex Systems) to control the temporal dynamics of light inputs. We applied specific temporal patterns to an image by drawing ROIs within the Nikon Elements software package and using custom macros to turn on and off the light. To attenuate 450 nm light, we dithered the DMD mirrors to apply light 50% of the time, and set our 450 nm LED to 50% of its maximum intensity.
For optogenetic microscope experiments, blue light from the XLED1 system was delivered through a Polygon400 digital micromirror device (DMD; Mightex Systems) to control the temporal dynamics of light inputs. We applied specific temporal patterns to an image by drawing ROIs within the Nikon Elements software package and using custom macros to turn on and off the light. To attenuate 450 nm light, we dithered the DMD mirrors to apply light 50% of the time, and set our 450 nm LED to 50% of its maximum intensity.
5
Confocal Imaging of Fluorescently Labeled Samples
6
Timelapse Imaging of Cell Lines
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Timelapse imaging of NIH3T3 cells, SYF cells, and HEK293T cells was performed on a Nikon Eclipse Ti microscope with a Yokogawa CSU-X1 spinning disk, an Agilent laser module containing 405, 488, 561, and 650 nm lasers, and an iXon DU897 EMCCD camera, using ×40 or ×60 oil objectives. Timelapse imaging of MCF10A cells on polyacrylamide substrata was performed on a Nikon Ti2-E microscope with a CSU-W1 SoRa spinning disk, a Hamamatsu FusionBT sCMOS camera, using a ×20 air objective with ×2.8 magnification optics.
7
Quantifying Axon Initial Segment Morphometry
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Antibody-labeled neuron cultures were observed on an inverted Nikon Eclipse Ti microscope equipped with a Yokogawa CSU-X1 spinning disk confocal head, a Hamamatsu Flash 4.0 scientific C-MOS camera, a Nikon 40X oil 1.4 NA Plan Apo objective, and 405 nm, 488 nm, 561 nm, and 640 nm lasers. From each coverslip, 20 unique fields of cells with at least 20 cells per treatment group were imaged. All analyses were performed by 4 examiners (MNB, YL, NNF, and NK) blinded to the treatments using the FIJI version of ImageJ. More specifically, we analyzed: 1) the shortest distance between the nucleus and the AIS (nucleus-AIS gap) measured using the “straight line tool”; 2) mean TRIM46 immunofluorescence intensity within the AIS measured using an “AIS Analysis” plugin (https://github.com/ksiller/aisanalyzer ) created by KHS; and 3) AIS length (region-of-interest; ROI outlining the AIS thresholded at 50% fluorescence intensity of a skeletonized line measuring the length of the AIS) measured using the “AIS Analysis” plugin. The plugin processes pairs of fluorescent images and corresponding FIJI/ImageJ ROI files. The output includes automated nucleus and AIS ROIs that aid in the quantification of AIS morphometric parameters (Supplementary Figure 1 ).
8
Perfusion-Fixed Mouse Brain Imaging
9
Fluorescence Imaging of Protein Aggregation
10
Imaging of Light-Inducible Transcription Factor
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For imaging, 35 mm glass bottom dishes with 20 mm well (Cellvis, #D35-20-1.5-N) were used. Glass was first treated with 10 μg/mL of fibronectin in PBS for 30 min in 37oC. 293 T UAS-dGFP cell line was plated on dish and allowed to adhere onto the plate. For mCherry tagged Gal4LOVSK22 tracking experiment, 2500 ng of pLZA144 encoding Gal4LOVSK22-VP64-mCherry was transfected to 293 T UAS-dGFP cells with the FuGENE HD Transfection Reagent following the protocol in manual and kept in dark first. 4 h after transfection, cells were either switched to blue light illumination or kept in dark for another 20 h. Then cell media was aspirated and cells was fixed in 4% PFA in PBS for 15 min in room temperature, followed by PBS washing for three times. Cells were then stained with 2 μg/mL DAPI for 15 min in room temperature, followed by PBS wash for three times and kept in 4oC before imaging. Imaging was done using Nikon Eclipse Ti microscope with a Prior linear motorized stage, a Yokogawa CSU-X1 spinning disk, an Agilent laser line module containing 405, 488, 561 and 650 nm lasers, an iXon DU897 EMCCD camera, and a 40X oil immersion objective lens. Several images of fixed cells in the 405, 488 and 561 channels were collected.
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