For dose-response experiments, static images were acquired by imaging cells on a 1% agarose pad made using the medium cells had been grown in using an upright fluorescence microscope (Zeiss, Thornwood, NY, USA) equipped with a Plan-Apo 1.4 100x phase objective, conventional epifluorescence filter set, and 1024 × 1024 back illuminated EM-CCD camera (Andor, South Windsor, CT, USA), producing an effective pixel size of 66 nm. Dynamic experiments were imaged using a Nikon Eclipse TiE enclosed in a temperature controlled chamber (Haison Technology, Taipei, Taiwan) set to 30°C in the case of C. crescentus and 37°C in the case of E. coli using a Plan-Apo 1.45 100x phase objective and 1024 × 1024 back illuminated EM-CCD camera (Andor, South Windsor, CT, USA), producing an effective pixel size of 86 nm. E. coli nutritional upshift experiments were performed using an ONIX Microfluidic Perfusion Platform (CellASIC, Hayward, CA, USA). C. crescentus perfusion experiments were performed using a low profile RC-31 flow chamber (Warner, Hamden, CT, USA). See Supplemental Experimental Procedures for further details.
Emccd camera
Manufactured by Oxford Instruments
Sourced in United Kingdom, Ireland, United States, Germany, Japan
The EMCCD camera is a specialized imaging device designed for low-light applications. It utilizes an electron-multiplying charge-coupled device (EMCCD) sensor to amplify the signal from incoming photons, allowing for the detection of extremely faint signals. The EMCCD camera is capable of delivering high quantum efficiency and low noise performance, making it suitable for a variety of scientific and research applications that require sensitive image capture.
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Lab products found in correlation
Matlab, by MathWorks (7 mentions)
Eclipse ti, by Nikon (6 mentions)
Nis elements software, by Nikon (5 mentions)
Eclipse ti inverted microscope, by Yokogawa (5 mentions)
Spinning disk confocal microscope, by Yokogawa (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Eclipse ti inverted microscope, by Nikon (4 mentions)
Fluo 4 am, by Thermo Fisher Scientific (4 mentions)
Metamorph software, by Molecular Devices (3 mentions)
Cysteamine, by Merck Group (3 mentions)
Elyra ps 1 microscope system, by Zeiss (3 mentions)
Csu10, by Yokogawa (3 mentions)
Ix71 inverted microscope, by Olympus (3 mentions)
Flash 4.0 camera, by Hamamatsu Photonics (3 mentions)
Celltirf 4line system, by Oxford Instruments (3 mentions)
Autoquant deconvolution software, by Media Cybernetics (3 mentions)
Eclipse ti e, by Nikon (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Spinning disk, by Oxford Instruments (2 mentions)
Coolsnap hq, by Teledyne (2 mentions)
203 protocols using emccd camera
1
Quantitative microscopy of bacterial cells
2
Single-Molecule TIRFM Imaging Protocol
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Total internal reflection
microscopy (TIRFM) was performed using a custom prism-based setup.
Videos were recorded using EMCCD camera (Andor) and Visual C++ smFRET
data acquisition software. Imaging was done at room temperature. Movies
were acquired using the red laser excitation at 640 nm for the first
and last 10 frames and using the green laser excitation at 532 nm
for the remaining frames at 20 frames per second. Short movies were
50 frames long, and long movies were stopped when 80% of molecules
have photobleached (∼2000 frames)
microscopy (TIRFM) was performed using a custom prism-based setup.
Videos were recorded using EMCCD camera (Andor) and Visual C++ smFRET
data acquisition software. Imaging was done at room temperature. Movies
were acquired using the red laser excitation at 640 nm for the first
and last 10 frames and using the green laser excitation at 532 nm
for the remaining frames at 20 frames per second. Short movies were
50 frames long, and long movies were stopped when 80% of molecules
have photobleached (∼2000 frames)
3
TIRF Microscopy of Rab37 Vesicle Trafficking
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The protocol was modified from Kuo’s report [16 (link)]. In brief, 293T (5 × 105) cells were seeded in 3.5 cm glass bottom dish. After 48 h, cells were transfected with RFP-Rab37 or GFP-PD-1 for 18 h. TIRF microscopy system was built on an inverted microscopy Olympus IX81 (Olympus, Tokyo, Japan) equipped with a high sensitivity EMCCD Camera (iXOn3897, Andor technology, New York, United States) and a UPON 100X oil objective lens (NA = 1.49, Olympus) to capture 100–200 nm images below the plasma membrane interface. We defined each green fluorescence spot as a cargo-containing vesicle and trafficking by Rab37 in cells, and then tracked each vesicle trafficking distance with trackIT software (Olympus). The cutoff length of moving vesicle was 3 μm. The vesicle trafficking event was measured as the ratio of moving vesicles to the total vesicles in cells. A total of at least 20 moving vesicles per cell were tracked and 6 cells were scored.
4
Imaging Blood Flow in Zebrafish
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Blood flow videos were obtained by imaging control and ablated Tg(gata1:DsRed; vmhc:mCherry-NTR) fish at six dpf (24 hpt). Fish were imaged with a Leica Sp5 (Wetzlar, Germany) resonance scanner at 50–100 frames per second, at 512 × 128 pixels of resolution. 10 z-stacks were obtained, each separated by 10 μm for a total of 2 min. Bright field blood flow videos for two dpf wild-type control and trpv4 -/- mutant were recorded by an Andor iXon EMCCD camera at a frame rate of 40 ms/ frame.
5
Tracking Microtubule Dynamics in HeLa Cells
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Time-lapse movies of HeLa cells expressing tubulin–RFP were acquired on an Olympus IX71 epifluorescent microscope equipped with an EMCCD camera (Ixon, Andor, Belfast) using a 60×1.42 N.A. oil objective. To track single MTs at the cell periphery, acquired movies were subjected to a bandpass filter (20:2 pixels) in ImageJ, background subtracted using a rolling ball radius of 15 pixels, and a 3D Gaussian blur filter was applied. Resulting movies were overlaid with the originals to avoid image-processing-derived artefacts and single MT length was measured over time from a defined starting point proximal to the cell periphery. Frequency of catastrophe, growth rate and time spent in growth phase were quantified.
6
Microscopic Intestinal Lumen Imaging
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Still images of the intestinal lumen were taken with an Olympus IX81 microscope, csu-xi Yokogawa spinning disk, and Andor iXon EM CCD camera. They were analyzed using Metamorph software (version 7.8.0.0, Molecular Devices, Sunnyvale, CA). Images of a micrometer taken at the same magnification were used to calibrate the measuring tool. This tool was then used to determine the width of the intestinal lumen.
7
Single-molecule FRET analysis of DNA dynamics
8
In vivo Calcium Imaging with YC3.60
9
Immunostaining of Drosophila Larval Neurons
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Third-instar larvae were dissected, fixed in Bouin’s fixative or 4% PFA in PBS, and immunostained as previously described (Eaton et al., 2002 (link); Harris et al., 2015 (link)). Dissected third instar larvae were fixed with PFA (4%) and incubated overnight at 4 C with primary antibodies (mouse anti Flag 1:50; rabbit anti-RFP 1:100; rabbit anti-Dlg, 1:1000; anti-Syt1 1:1000, anti-Brp 1:100, Life Technologies). Alexa-conjugated secondary antibodies and goat anti-HRP were used (Jackson Laboratories 1:500). Images were acquired with either a Zeiss LSM700 confocal microscope equipped with Zen software using a 63X 1.6 NA oil immersion objective or an upright epifluorescence deconvolution confocal microscope (Axiovert 200, Zeiss) equipped with a 100X objective (N.A. 1.4), cooled CCD camera (CoolSnap HQ, Roper Scientific). Slidebook 5.0 (3I, Intelligent Imaging) was used for capturing, deconvolving and analyzing images. Structured illumination microscopy (Nikon LSM 710 equipped with 63X objective and Andor Ixon EMCCD camera) was used to perform Brp-GFP and MCTP-Flag colocalization experiments. Bouton numbers and Brp numbers and densities were quantified as described previously (Harris et al., 2015 (link)).
10
Super-Resolution Imaging of Single Molecules
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dSTORM imaging was performed using an Elyra PS.1 microscope (Carl Zeiss Microscopy) equipped with a Plan-Apochromat 100×/1.46 oil objective and a liquid cooled EMCCD camera (Andor Technology). Imaging was carried out in MEA imaging buffer as previously described (36 (link)). In short, fresh stock solutions (1 M cysteamine in 360 mM HCl, 10% glucose in PBS, 70 mg/ml glucose oxidase in PBS, and 20 mg/ml catalase in PBS) were prepared the day before imaging and stored at 4°C and mixed directly before imaging to final concentrations of 0.124 M cysteamine (Sigma), 44.8 mM HCl, 8.6% glucose, 1.08 mg/ml glucose oxidase from Aspergillus niger (Sigma), and 0.0773 mg/ml catalase from bovine liver (Sigma) in PBS. Imaging was performed in 12.8 × 12.8-μm areas in an inclined total internal reflection fluorescence microscope mode (37 (link)). Single molecule fluorescence detection on the EMCCD camera was acquired with 100 × 100-nm pixel size, 20-ms Exposure time, and 100 Gain. 20,000 image frames were acquired for each channel. Both channels were imaged sequentially in 500 frame sequences and the appropriate filters and lasers for each dye were used (642 nm for Alexa Fluor 647 and 488 nm for Atto 488). The images were analyzed with the ImageJ plugin SMLocalizer (38 (link)).
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