Imaging was performed under normal growth conditions using phenol-red free media. Cells were plated on Fluorodish poly-D-lysine coated plates (World Precision Instruments) and imaged using a 100X 1.4NA oil immersion objective on a Zeiss CSU spinning disk confocal microscope with a Yokogawa spinning disk scan head and an EM-CCD camera. 20–30 Z sections were obtained with a spacing of 0.5 μm and acquired at a rate of one stack every two seconds for 200 total time points or one stack every 40 seconds for 100 total time points. Laser intensity was 6% and exposure time was 30ms for 2 seconds intervals and 45ms for 40 seconds intervals. For fixed cell measurements, cells were fixed for 10 minutes in 4% paraformaldehyde in PBS and quenched 5 minutes in 0.1M Tris-HCl pH 7.4 and stained using a 1:2000 dilution of Alexa 488 conjugated rabbit monoclonal anti-GFP from Life Technologies (G10362). Axial elongation was corrected prior to calculating probe-to-probe distances (Figure S3 ).
Em ccd camera
Manufactured by Yokogawa
The EM-CCD camera is a specialized imaging device designed for low-light applications. It utilizes an Electron Multiplying Charge Coupled Device (EM-CCD) sensor to amplify weak signals, enabling high-sensitivity detection of faint light sources. The core function of the EM-CCD camera is to capture and digitize optical images with high quantum efficiency and low noise, making it suitable for applications that require accurate detection of subtle light signals.
Automatically generated - may contain errors
Lab products found in correlation
Spinning disk scan head, by Yokogawa (2 mentions)
Csu spinning disk confocal microscope, by Yokogawa (2 mentions)
Imagej 1.40g, by Adobe (2 mentions)
Plan apochromat objective, by Zeiss (2 mentions)
Eclipse ti, by Yokogawa (2 mentions)
Csu x1, by Yokogawa (2 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Csu 10, by Zeiss (1 mentions)
Inverted spinning disk confocal microscope, by Yokogawa (1 mentions)
Csu x1 spinning disk, by Yokogawa (1 mentions)
Csu x1 a1, by Yokogawa (1 mentions)
Ti e eclipse, by Yokogawa (1 mentions)
Chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Axio imager a1 microscope, by Zeiss (1 mentions)
Csu10, by Yokogawa (1 mentions)
8 protocols using em ccd camera
1
Quantitative Live-Cell Imaging of GFP Probes
2
Confocal Microscopy Imaging and Analysis
3
Live-cell Microscopy of Fluorescent Proteins
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Images and time-lapse series were collected with Nikon inverted spinning disk confocal microscope equipped with Yokogawa CSU-X1 Spinning Disk, EMCCD camera, laser launch including 445 nm (to image Cerulean and CFP), 488 nm (to image GFP), 514 nm (to image Venus and YFP), 561 nm (to image mCherry), and 647 nm (to image Cy5), focus drift correction through the Perfect Focus System, and 60× Plan Apo 1.40 NA oil/0.13 mm WD, and controlled by the NIS-Elements software. For live-cell imaging experiments, cells were seeded in # 1.5 coverslip bottom dishes and incubated in a complete DMEM cell culture medium (described above) at 37°C with 5% CO2 within a Tokai incubator on the microscope stage. Imaging experiments were performed at 60–80% confluency of stably transfected cells or 48–72 h after transient transfection, unless otherwise specified in the figure legend.
4
Multicolor Confocal Microscopy Protocol
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All samples were imaged on a Nikon Eclipse Ti microscope base equipped with a Yokogawa CSUX1 spinning disk confocal scanner unit, using 100x / 1.49 NA oil objective and Andor EM-CCD camera. Images were acquired using MetaMorph software. A single Z-slice in the center of cell nucleus was acquired per image, with an average of 100–200 images acquired per sample. The lasers used were: 405nm, 488nm, 561nm and 637nm.
5
Imaging and Tracking Cellular Structures
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Cell imaging was performed on a Zeiss Axiovert microscope equipped with a Cool Snap ES CCD camera (Ropper Scientific). Images were captured using 2×2 binning and 12 sequential z-planes were collected at 0.3-µm step intervals with an exposure time of 200 ms except for time-lapse video microscopy movies of Abp1–RFP and Sla1–RFP that were collected every second with five sequential z-planes (0.5 µm steps) and an exposure time of 100 ms.
For analysis of microtubules and vesicle motion, cell imaging was performed on a confocal spinning disk inverted microscope (Nikon TI-E Eclipse) equipped with a Yokogawa motorized confocal head CSUX1-A1 and an Evolve EMCCD camera. A dual color acquisition of six sequential z-planes (0.3-µm steps) was performed every second with an exposure time of 50 ms and 100 ms for GFP–Snc1 and Bik1–RFP, respectively. All image manipulations, montages, and fluorescence-intensity measurements were performed using ImageJ (Schneider et al., 2012 (link)). Tracking analysis and dot number quantifications were performed using Icy (de Chaumont et al., 2012 (link)).
For analysis of microtubules and vesicle motion, cell imaging was performed on a confocal spinning disk inverted microscope (Nikon TI-E Eclipse) equipped with a Yokogawa motorized confocal head CSUX1-A1 and an Evolve EMCCD camera. A dual color acquisition of six sequential z-planes (0.3-µm steps) was performed every second with an exposure time of 50 ms and 100 ms for GFP–Snc1 and Bik1–RFP, respectively. All image manipulations, montages, and fluorescence-intensity measurements were performed using ImageJ (Schneider et al., 2012 (link)). Tracking analysis and dot number quantifications were performed using Icy (de Chaumont et al., 2012 (link)).
6
Imaging Phytohormone Responses in Seedlings
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To observe the response of ethylene (EIN3-GFP), auxin (DR5v2-GFP), and cytokinin (TCSn-GFP) reporter lines to MBZ, 14-day-old seedlings grown on ½ MS media were transferred to a Chambered Coverglass (Thermo Scientific) that was filled with treatment medium. For chamber preparation, 6 mL of ½ MS medium (1% agar) supplemented with the different treatments (DMSO, 10 μM MBZ, 1 μM IAA, 50 μM ACC, and 10 μM tZ) was filled to each chamber. After 20 min of medium solidification, the seedlings were placed on the media surface and the lateral roots were adjusted to positions suitable for imaging.
For imaging, a Zeiss CSU Spinning Disk Confocal Microscope equipped with laser 488 mm and a Yokogawa spinning disk scan head with EM-CCD camera were used to set up the image acquisition every 20 min for a total of 4 h. Three z stack positions were automatically captured at each time point under 10X objective and Fiji was used to quantify the average intensity of GFP fluorescence. For proper comparison at different treatment conditions of each reporter lines, we normalized the average fluorescence data as the percentages (or relative fluorescence intensity as text label). In detail, the first time point of each treatment is set as 100% and the relative fluorescence intensity from the second time point is calculated as the fold-change in percentages comparing the first time point.
For imaging, a Zeiss CSU Spinning Disk Confocal Microscope equipped with laser 488 mm and a Yokogawa spinning disk scan head with EM-CCD camera were used to set up the image acquisition every 20 min for a total of 4 h. Three z stack positions were automatically captured at each time point under 10X objective and Fiji was used to quantify the average intensity of GFP fluorescence. For proper comparison at different treatment conditions of each reporter lines, we normalized the average fluorescence data as the percentages (or relative fluorescence intensity as text label). In detail, the first time point of each treatment is set as 100% and the relative fluorescence intensity from the second time point is calculated as the fold-change in percentages comparing the first time point.
7
Multicolor Confocal Imaging of Cells
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All samples were imaged on a Nikon Eclipse Ti microscope base equipped with a Yokogawa CSUX1 spinning disk confocal scanner unit, using 100×/1.49 NA oil objective and Andor EM-CCD camera. Images were acquired using MetaMorph software. A single Z-slice in the center of cell nucleus was acquired per image, with an average of 100–200 images acquired per sample. The lasers used were: 405 nm, 488 nm, 561 nm and 637 nm.
8
Quantifying Collagen Localization in C. elegans
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Images were acquired using a Hammamatsu EM-CCD camera, a spinning-disk confocal microscope (CSU-10; Yokogawa Corporation of America) mounted on an AxioImager base (Carl Zeiss) with 40× and 100× Plan Apochromat objectives (1.4 NA) and controlled by μManager [101 ]. Worms were photobleached on a Zeiss AxioImager A1 microscope with a 100× plan-apochromat objective by exposing a portion of the uterine/vulval tissue to 561 nm light for 2 minutes. Acquired images were processed to enhance brightness/contrast using ImageJ 1.40g and Photoshop (CS6 Extended Adobe Systems, InC., San Jose, CA). Colocalization analysis was performed on confocal z-stacks using the “Coloc” module in IMARIS 7.4 (Bitplane, Inc., Saint Paul, MN). Measurements of collagen::mCherry intensity at the BM were collected in ImageJ by measuring the mean grey value of a 3 pixel width line drawn along the BM. Measurements at the surface of the muscle were acquired using the same method, except a 2 pixel width line was used. An equivalent line was drawn in an adjacent region of the worm to measure the background signal within the worm, which was subtracted from the signal at the BM or muscle surface.
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