Mounted ovaries were visualized and imaged using a Nikon Ti-E-PFS inverted microscope equipped with a Yokogawa CSU-X1 spinning disk confocal unit. The 40x and 20× 1.4 NA Plan Apo Lambda objective lenses were used to capture images. The system is also equipped with a self-contained 4-line laser module (excitation at 405, 488, 561, and 640 nm), and an Andor iXon 897 EMCCD camera. EGFP and the Alexa 488-conjugated secondary antibody were excited using the 488 nm laser and detected with the 525 nm emission filter. The Rhodamine Red™-X-conjugated secondary antibody and the FM® 4-64 Dye were excited at 540 nm and detected with the 570 nm filter. DAPI was excited at 358 nm and collected with the 461 nm filter. For each image, the Nikon Ti-E internal focus motor was used to take 12 z-series optical sections with a step size of 2 microns. Four to five images were taken above and below the mid-section of the ovary and were subsequently compiled together. The gamma, brightness, and contrast were adjusted (identically for compared image sets) using NIS Elements Ar imaging software. For whole-ovary fluorescent images, 64 images were taken with the 20x objective lens to cover the whole ovary. These pictures were automatically stitched together into a single image using the NIS Elements Ar imaging software. Multiple stage positions were collected using a Prior ProScan motorized stage.
897 emccd camera
Manufactured by Oxford Instruments
The 897 EMCCD camera is a high-performance imaging device designed for low-light applications. It features an electron-multiplying CCD (EMCCD) sensor that enhances the camera's sensitivity, enabling the capture of high-quality images in challenging lighting conditions.
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Lab products found in correlation
Ti e pfs inverted microscope, by Yokogawa (1 mentions)
Csu x1 spinning disk confocal unit, by Yokogawa (1 mentions)
Eclipse ti e inverted microscope, by Nikon (1 mentions)
Coolled pe2, by Lumencor (1 mentions)
Axiovert 40 cfl, by Zeiss (1 mentions)
Poly l lysine solution, by Merck Group (1 mentions)
Ti e microscope, by Nikon (1 mentions)
N storm inverted microscope, by Nikon (1 mentions)
Nis elements, by Adobe (1 mentions)
Plan apo lambda, by Nikon (1 mentions)
Ti e pfs, by Nikon (1 mentions)
Csu x1 spinning disk unit, by Oxford Instruments (1 mentions)
Csu x1 spinning disk unit, by Nikon (1 mentions)
Ti e microscope, by Yokogawa (1 mentions)
Csu x1 spinning disk unit, by Yokogawa (1 mentions)
Ti e pfs inverted microscope, by Nikon (1 mentions)
Csu x spinning disk, by Oxford Instruments (1 mentions)
Csu x1, by Oxford Instruments (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
Dmi8 microscope, by Oxford Instruments (1 mentions)
12 protocols using 897 emccd camera
1
Imaging Mounted Ovaries Using Confocal Microscopy
2
Imaging Bacterial Dynamics in Microfluidics
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For image acquisition, a Nikon Eclipse Ti-E inverted microscope was used, with Nikon CFI Plan Apo Lambda DM 100X Oil objective, CoolLED pE-2 or Lumencor Aura illumination system (470 or 485 nm LED, respectively, for excitation of GFP) and Andor iXon 897 EM-CCD camera. The following filters were employed: excitation filter bandpass 470/40 nm, dichroic mirror 495 nm and emission filter 525/50 nm (AHF Analysentechnik F46-470). Focus was maintained by Nikon’s PFS3 system. Acquisition was started within 10 min (generally ~7 min) after the bacteria had been introduced in the microfluidic system and every 10 min phase contrast images and GFP signal (200 ms exposure time) were acquired at multiple positions. The microscope was controlled by NIS Elements v4.51 software.
3
Microscopic Imaging Techniques
4
Yeast Cell Imaging with Spinning Disc Confocal
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The glass-slide part of the “Glass bottom dishes” (Mattech, #s: P50G-1.5-14-) was covered with 0.1% w/v Poly-L-Lysine solution (SIGMA-ALDRICH, No. P 8920) in order to mount the yeast cells. To diminish the autofluorescence, the yeast cells were grown in minimal medium (1.7 g/l YNB, 0.04 g/l CSM-His (Bio101, Inc. and 2% (w/v) dextrose) with 20 μg/ml extra adenine. All procedures were carried out at 25°C and according to the protocol, described by Silva and co-workers
[62 (link)] (Cite). Observations were made by CFI Apo TIRF 100X Oil 1.49 NA objective mounted on Yokogawa CSU-X1 spinning disc confocal microscope (Andor Revolution XD system with Nikon TiE microscope stand and incubator for temperature and humidity control). Data were documented by iXon 897EMCCD camera with TiCAM. All time-lapse experiments were run using the following parameters: 11 Z-stacks, 0.5 μm apart, acquired with 18% laser power on 488 nm and 200 ms exposure. Acquisition was made on every 5 min for 12–16 h. Maximum intensity projections of the stacks were prepared using ImageJ. Images and movies were processed by ImageJ software.
[62 (link)] (Cite). Observations were made by CFI Apo TIRF 100X Oil 1.49 NA objective mounted on Yokogawa CSU-X1 spinning disc confocal microscope (Andor Revolution XD system with Nikon TiE microscope stand and incubator for temperature and humidity control). Data were documented by iXon 897EMCCD camera with TiCAM. All time-lapse experiments were run using the following parameters: 11 Z-stacks, 0.5 μm apart, acquired with 18% laser power on 488 nm and 200 ms exposure. Acquisition was made on every 5 min for 12–16 h. Maximum intensity projections of the stacks were prepared using ImageJ. Images and movies were processed by ImageJ software.
5
Multicolor Super-resolution Imaging of Diatoms
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Diatoms were imaged on a Nikon N-STORM inverted microscope with a 100x Oil Immersion TIRF objective with a numerical aperture of 1.49. Four fiber-coupled lasers are available for illumination at 405, 488, 561 and 647 nm, respectively. Corresponding filter sets from Semrock, Chroma and AHF were used to block out all unwanted fluorescence and scattered laser light (see SI Mat/Met for a detailed description). The image was projected onto an Andor Ixon 897 EMCCD camera. To visualize the chloroplasts, the red 647 nm laser was used at low power (1.5 mW end of fiber) for 100 ms. For screening of the FPs, the appropriate excitation wavelength (SI Table S1) was chosen at a power of 40 mW and an exposure time of 100 ms. The photo-conversion test was performed by illuminating the sample with 405 nm light for 1 second at 20 mW. Super-resolution images were recorded at 20 fps with an excitation power of 80 mW. Image stacks of at least 1000 images were used (1000 for Fig. 3A,D and SI Fig. S5, 1200 for Fig. 3E , 2500 for Fig. 3F ) to ensure a sufficient localization count. For mEOS3.2 and Dendra2 green excitation is sufficient to localize pre-converted FPs. Dronpa is dark in its ground state, a continuous 405 nm activation of 8 mW in addition to the green excitation was required.
6
Live Sample Imaging with Confocal Microscopy
7
Imaging HeLa Cells with Spinning Disk Microscopy
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HeLa cells were imaged with a Nikon Ti-E microscope equipped with a Yokogawa CSU X-1 spinning disk unit, a 60× objective (Plan Apo VC, oil, DIC, NA 1.4), Perfect Focus System and the Nikon NIS elements software. Images were acquired with a Andor iXon 897 EMCCD camera. Photo-activation was achieved with a single pulse of the 440 nm laser light, intensity set to 20%, for 1 s. During photo-activation CFPs were imaged using a 440 nm laser line, a triple dichroic mirror (440, 514, 561 nm), and a 460–500 nm emission filter. RFPs were imaged using a 561 nm laser line, a triple dichroic mirror (405, 488, 561 nm), and a 600–660 nm emission filter.
8
Live Cell Imaging at 37°C
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Live cell imaging was performed under 5% CO2 humidified conditions, at 37°C on a laser spinning-disc confocal attached to a Nikon Ti equipped with an Andor iXon 897 EMCCD camera.
9
Live Cell Imaging Protocol
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Fluorescent images of live or fixed samples were captured either using spinning-disk confocal microscopy as described previously in Golden et al. (2009) (link) or using a Nikon Ti-E-PFS inverted microscope equipped with a 60× 1.4NA Plan Apo Lamda objective. The Ti-E-PFS system is outfitted with a Yokogawa CSU-X1 spinning disk unit, a self-contained four-line laser module (excitation at 405, 488, 561, and 640 nm), and Andor iXon 897 EMCCD camera. Confocal images were acquired using Openlab 4.0 or NIS-Elements and processed using ImageJ 1.38X and Adobe Photoshop CS5 software. All images shown are single focal planes unless noted.
10
Live-Cell FRAP Imaging of GFP Dynamics
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Live imaging of GFP Fluorescent Recovery After Photobleaching (FRAP) in HEK-293T cells was performed with a Nikon-Ti microscope (X40 oil objective) paired with the Andor Frappa device, Yokogawa CSU-X spinning disk, and an iXon897 EMCCD camera. FRAP on complete cells was done using a 488nm laser at 15 mW power. Dual color images of GFP and Cy3 miRNAs were acquired prior to FRAP, and for 20 consecutive times afterward at 30-s intervals. Live imaging was performed in a controlled CO2 and 37 °C chamber. Image analysis was performed with FIJI.
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