Arabidopsis leaf protoplasts were prepared as described61 (link). For co-transfections, 10 μg of each vector were used to transfect 200 μl protoplasts (2×105 ml−1). After transfection, protoplasts were incubated for 10–12 hours before examined with a spinning-disk confocal system equipped with a CSU-W1 spinning-disk head (Yokogawa) and a iXon Ultra 888 EMCCD (Andor) on a DMi8 microscope body (Leica). Specifically, protoplasts were imaged with a HCX PL Apo 1.44 N.A. 100× oil immersion objective. GFP and YFP were excited at 488 nm. mCherry and mScarlet signals were excited at 561 nm. A TR-F525/50 or a TR-F593/46 bandpass emission filter (Semrock Brightline) was used for capturing GFP/YFP or mCherry/mScarlet signals. Confocal images were processed with the Fiji-ImageJ software62 (link). To quantify BiFC assays, p35S:mScarlet-ARA7 were co-transfected with BiFC vectors as a transfection control. Interactions of YN and YC fusion proteins were quantified as the percentage of cells with YFP signals of the total transfected cells (cells with mScarlet-ARA7 signals). Three independent sets of experiments were performed and quantified for each pair of YN and YC vectors. Note that BiFC quantification data summarized in Supplementary Fig. 5f was also shown in Figs. 4c , 4g-4i , and 5b as mean ± s.d.
Ixon ultra 888 emccd
Manufactured by Oxford Instruments
Sourced in United Kingdom
The IXon Ultra 888 EMCCD is a high-performance scientific camera designed for low-light imaging applications. It features an electron-multiplying charge-coupled device (EMCCD) sensor, allowing for enhanced signal detection and reduced noise levels. The camera is capable of capturing images with high sensitivity and fast frame rates, making it suitable for various scientific research and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Dmi8 microscope body, by Leica (3 mentions)
Csu w1 spinning disk head, by Yokogawa (3 mentions)
Tr f525 50, by IDEX Corporation (2 mentions)
Tr f593 46 bandpass emission filter, by IDEX Corporation (2 mentions)
Visiview software, by Visitron (2 mentions)
Ibeam smart, by Toptica (2 mentions)
Csu w1, by Yokogawa (2 mentions)
Tma dph, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mounting solution, by Thermo Fisher Scientific (1 mentions)
Zyla scmos camera, by Oxford Instruments (1 mentions)
P35g 1.5 10 c, by MatTek (1 mentions)
Fusion software, by Oxford Instruments (1 mentions)
D35c4 20 1.5 n, by Cellvis (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Plan fluor, by Nikon (1 mentions)
Plan apo, by Nikon (1 mentions)
Orca r2 ccd camera, by Hamamatsu Photonics (1 mentions)
Scanning electron microscope, by Hitachi (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
12 protocols using ixon ultra 888 emccd
1
Arabidopsis Leaf Protoplast Co-Transfection
2
E. coli Cell Membrane Fluidity Analysis
3
Multiorgan Tissue Immunofluorescence Imaging
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The kidneys, livers, lungs, and spleen were embedded in Tissue‐Tek (OCT compound) and kept frozen at −80 °C. Tissues were sectioned (5 µm thickness), fixed in acetone at −20 °C for 10 min and washed with PBS. The sections were blocked with 5% BSA for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. Sections were washed with PBS and incubated with secondary antibodies for 1 h at room temperature. Sections were next washed by PBS and mounted using Prolong Diamond antifade mounting solution containing DAPI (Invitrogen). Primary and secondary antibody dilution for different organ tissues are listed in Table S2 (Supporting Information). All sections were imaged using Axio Observer 7 epifluorescent microscope (Zeiss) equipped with iXon Ultra 888 EMCCD (Andor Technology) with or without SRRF with the same imaging parameters. Images (n = 70 per organ) were subjected to Fiji (ImageJ, NIH) for EP quantitative analysis with the same threshold. 5×5 tiled images were selected from 6 × 6 tiled images to remove uneven edges following tiling by Fiji (NIH).
4
Arabidopsis Leaf Protoplast Co-Transfection
5
Live-cell Confocal Microscopy of Aliphatic Alcohol Effects
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Microscopy was performed using a spinning disk confocal microscope system (Dragonfly, Andor Technology, Belfast, UK) with 63×/1.40 (magnification/numerical aperture) or 100×/1.40 HC PL APO objectives (Leica, Wetzlar, Germany), coupled with 1×, 1.5× or 2× motorized magnification changer. Image acquisition was controlled by Fusion software (Andor Technology) and images were captured using an iXon Ultra 888 EMCCD or Zyla sCMOS camera (Andor Technology). Sometimes, deconvolution of the images was also performed using the Fusion software (Andor Technology).
All live-cell imaging was performed with cells seeded in 35-mm glass-bottom dishes (P35G-1.5-10-C, MatTek Corp., Ashland, MA, USA or D35C4-20-1.5-N, Cellvis, Mountain View, CA, USA) mounted in a humidified chamber supplied with 5% CO2 inside a wrap-around environmental incubator (Okolab, Pozzuoli, Italy) with the temperature set at 37°C.
For acute treatments of live cells with aliphatic alcohols, the aliphatic alcohol was prepared in 10% FBS DMEM/F-12 medium, prewarmed to 37°C and added onto cells mounted on the microscope stage. Time-lapse microscopy was performed before and immediately after the addition of aliphatic alcohol. For the control, cells were imaged under the same acquisition conditions except that the cells were treated with 10% FBS DMEM/F-12 medium alone.
All live-cell imaging was performed with cells seeded in 35-mm glass-bottom dishes (P35G-1.5-10-C, MatTek Corp., Ashland, MA, USA or D35C4-20-1.5-N, Cellvis, Mountain View, CA, USA) mounted in a humidified chamber supplied with 5% CO2 inside a wrap-around environmental incubator (Okolab, Pozzuoli, Italy) with the temperature set at 37°C.
For acute treatments of live cells with aliphatic alcohols, the aliphatic alcohol was prepared in 10% FBS DMEM/F-12 medium, prewarmed to 37°C and added onto cells mounted on the microscope stage. Time-lapse microscopy was performed before and immediately after the addition of aliphatic alcohol. For the control, cells were imaged under the same acquisition conditions except that the cells were treated with 10% FBS DMEM/F-12 medium alone.
6
Wide-field and Confocal Microscopy Imaging
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Wide-field imaging was performed with a 20X, 0.70 NA long-distance objective (Plan Fluor, Nikon) using an inverted Nikon-Ti wide-field microscope (Nikon, Japan) equipped with an Orca R-2 CCD camera (Hamamatsu Photonics, Japan).
Confocal images of cells were collected using a 40X, 1.3 NA, oil immersion objective (Plan-Apo, Nikon) with a Nikon-Ti Eclipse spinning disk confocal microscope (Nikon, Japan) equipped with an iXon Ultra 888 EMCCD (Andor, UK).
After sputtering of approximately 5 nm of Gold/Palladium on the surface, samples were imaged using a scanning electron microscope (Hitachi High Technologies Europe, Germany) with detection of signal from secondary electrons.
Confocal images of cells were collected using a 40X, 1.3 NA, oil immersion objective (Plan-Apo, Nikon) with a Nikon-Ti Eclipse spinning disk confocal microscope (Nikon, Japan) equipped with an iXon Ultra 888 EMCCD (Andor, UK).
After sputtering of approximately 5 nm of Gold/Palladium on the surface, samples were imaged using a scanning electron microscope (Hitachi High Technologies Europe, Germany) with detection of signal from secondary electrons.
7
Imaging Cell Adhesion on LN-511 Coated Slides
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Ibidi µ-Slide 8-well chambers were coated with 10 µg/mL Biolaminin LN-511 (BioLamina) diluted in PBS with 1 mM CaCl2 and 0.5 mM MgCl2 overnight at 4°C. Coated wells were rinsed with PBS and immediately filled with S-L-2i medium to which 4 × 104 to 6 × 104 cells were plated. Cells were imaged 24 h after seeding on a Nikon Ti2-E Eclipse inverted microscope equipped with a Yokogawa CSU W1 spinning disk confocal scanning unit, two back illuminated EMCCD iXon-Ultra-888 (Andor) cameras and CFI Plan Apochromat Lambda 100×/1.45 oil immersion objective (Nikon). Fluorescence was excited with 488-nm iBeam Smart (Toptica) and 561-nm Cobolt Jive (Cobolt) lasers, and images (pixel size 0.13 µm) were acquired using VisiView software (Visitron Systems GmbH) with the following settings: 100% laser intensity, 500-msec exposure time, EMCCD GAIN 100 for all mNeonGreen-tagged proteins and 25% laser intensity, 200-msec exposure time, EMCCD GAIN 100 for mCherry-U2AF2. The cells were kept at 37°C and 5% CO2 during all treatments and imaging.
8
Laminin-coated 8-well live cell imaging
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Ibidi µ-Slide 8-well chambers were coated with 10 µg/ml Biolaminin diluted in PBS with 1 mM CaCl2 and 0.5 mM MgCl2 at 4°C overnight. Coated wells were rinsed with PBS and immediately filled with S-L-2i medium to which 4 -6 x 10 4 cells were plated.
Cells were imaged 24 hours after seeding on a Nikon Ti2-E Eclipse inverted microscope equipped with Yokogawa CSU W1 spinning disk confocal scanning unit, two back illuminated EMCCD iXon-Ultra-888 (Andor) cameras and CFI Plan Apochromat Lambda 100x/1.45 oil immersion objective (Nikon). Fluorescence was excited with 488 nm iBeam Smart (Toptica) and 561 nm Cobolt Jive (Cobolt) lasers and images (pixel size 0.13 µm) were acquired using VisiView software (Visitron Systems GmbH) with the following settings: 100% laser intensity, 500 ms exposure time, EMCCD GAIN 100 for all mNeonGreen-tagged proteins and 25% laser intensity, 200 ms exposure time, EMCCD GAIN 100 for mCherry-U2AF2. The cells were kept at 37°C and 5% CO2 during all treatments and imaging.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Cells were imaged 24 hours after seeding on a Nikon Ti2-E Eclipse inverted microscope equipped with Yokogawa CSU W1 spinning disk confocal scanning unit, two back illuminated EMCCD iXon-Ultra-888 (Andor) cameras and CFI Plan Apochromat Lambda 100x/1.45 oil immersion objective (Nikon). Fluorescence was excited with 488 nm iBeam Smart (Toptica) and 561 nm Cobolt Jive (Cobolt) lasers and images (pixel size 0.13 µm) were acquired using VisiView software (Visitron Systems GmbH) with the following settings: 100% laser intensity, 500 ms exposure time, EMCCD GAIN 100 for all mNeonGreen-tagged proteins and 25% laser intensity, 200 ms exposure time, EMCCD GAIN 100 for mCherry-U2AF2. The cells were kept at 37°C and 5% CO2 during all treatments and imaging.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
9
Liquid-Liquid Phase Separation Droplet Imaging
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We respectively mixed the solution
of (PR)12 and (PR)20 (final concentration was
100 μM) and poly-A RNA (final concentration was 0.5 mg/mL) in
the volume ratio of 1:1 in a phosphate buffer solution (final concentration
was 10 mM) at room temperature. After mixing the fresh solution, we
dripped 10 μL of solution on the cover glass surface and then
sandwiched it with the other cover glass to form a thin solution film.
As a result, the LLPS droplets were observed under an oil-immersion
lens (NA = 1.4) via an inverted fluorescence microscope (Olympus IX73)
with an electron-multiplying charge-coupled device (iXon-Ultra888
EMCCD, Oxford Instruments) in a bright field. The exposure time was
set as 2 s, and the light source was a white light-emitting diode
(LED) light positioned up the sample.
of (PR)12 and (PR)20 (final concentration was
100 μM) and poly-A RNA (final concentration was 0.5 mg/mL) in
the volume ratio of 1:1 in a phosphate buffer solution (final concentration
was 10 mM) at room temperature. After mixing the fresh solution, we
dripped 10 μL of solution on the cover glass surface and then
sandwiched it with the other cover glass to form a thin solution film.
As a result, the LLPS droplets were observed under an oil-immersion
lens (NA = 1.4) via an inverted fluorescence microscope (Olympus IX73)
with an electron-multiplying charge-coupled device (iXon-Ultra888
EMCCD, Oxford Instruments) in a bright field. The exposure time was
set as 2 s, and the light source was a white light-emitting diode
(LED) light positioned up the sample.
10
Wetting Dynamics of LLPS Droplets
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We used the same two
groups of mixture solution forming LLPS droplets. Then, we placed
a poly(dimehylsiloxane) (PDMS) film (3 mm in thickness) with a hole
(5 mm in diameter) on the substrate surface. The hole was filled with
20 μL of mixture solution. Meanwhile, we covered the other same
cover glass on the top of the PDMS hole to avoid solution evaporation
during measurement periods. To observe the wetting process of LLPS
droplets on the solid interface, we defined the time scale from 0,
50, 90, and 140 min. All observations were under an oil-immersion
lens (NA = 1.4) via an inverted fluorescence microscope (Olympus IX73)
with an electron-multiplying charge-coupled device (iXon-Ultra888
EMCCD, Oxford Instruments) in a bright field. The exposure time was
set as 2 s, and the light source was a white light-emitting diode
(LED) positioned up the sample.
groups of mixture solution forming LLPS droplets. Then, we placed
a poly(dimehylsiloxane) (PDMS) film (3 mm in thickness) with a hole
(5 mm in diameter) on the substrate surface. The hole was filled with
20 μL of mixture solution. Meanwhile, we covered the other same
cover glass on the top of the PDMS hole to avoid solution evaporation
during measurement periods. To observe the wetting process of LLPS
droplets on the solid interface, we defined the time scale from 0,
50, 90, and 140 min. All observations were under an oil-immersion
lens (NA = 1.4) via an inverted fluorescence microscope (Olympus IX73)
with an electron-multiplying charge-coupled device (iXon-Ultra888
EMCCD, Oxford Instruments) in a bright field. The exposure time was
set as 2 s, and the light source was a white light-emitting diode
(LED) positioned up the sample.
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