Imaging was done with a widefield imaging system based on an Olympus IX83 inverted microscope body equipped with a home-made image splitter coupled to a sCMOS camera (ORCA Flash V2, Hamamatsu) as sketched in Fig. 1 . Excitation was done in epifluorescence mode by a supercontinuum white light laser (Fianium) coupled to a high power AOTF (Fianium), which was controlled through an FPGA-RT unit (National Instruments) coded with Labview. This unit synchronized the alternated laser excitation with the camera acquisition. Images were acquired at 37 °C with Micromanager and a 40x objective. The donor fluorophore was excited at 442nm (power 200 muW), the acceptor at 515nm (power 240 muW). The fluorescence emission was first separated from the excitation via a triple line beamsplitter (Brightline R442/514/561 Semrock) in the microscope body. The fluorescence emission was further splitted with a beamsplitter at 510nm (Chroma) and filtered with a 475/50 filter (BrightLine HC, Semrock) for the donor channel and a 519/LP longpass filter (BrightLine HC, Semrock) for the acceptor channel. Hence, in two camera snapshots, four images were obtained with all combinations of donor/acceptor excitation and donor/acceptor emission.
Orca flash v2
Manufactured by Hamamatsu Photonics
Sourced in Germany
The ORCA Flash V2 is a scientific camera designed and manufactured by Hamamatsu Photonics. It features a high-resolution, high-sensitivity CMOS image sensor that is capable of capturing images and video with exceptional quality. The camera is suitable for a wide range of scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager z2 microscope, by Zeiss (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Brightline hc, by IDEX Corporation (1 mentions)
Deltavision deconvolution microscope, by Cytiva (1 mentions)
Ti e microscope, by Hamamatsu Photonics (1 mentions)
Softworx software, by GE Healthcare (1 mentions)
Eclipse fn1, by Nikon (1 mentions)
M205 fa stereomicroscope, by Leica (1 mentions)
Dmi8 inverted microscope, by Leica (1 mentions)
Dfc9000 gt camera, by Leica (1 mentions)
8 protocols using orca flash v2
1
Widefield imaging system for FRET analysis
2
Imaging and Analysis of EGFP-H4 Localization
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Cells transfected or not with EGFP-H4 were stained with Hoechst 33342 (10 μM, 30 min at 37°C) and imaged on a spinning disk CSU-W1 (Yogogawa, Andor) microscope (DMI8 Leica, Germany) equipped with lasers at 405 nm (100 mW) and 488 nm (150 mW) operating at 10% power and sCMOS camera (Orca-Flash V2+, Hamamatsu). Z stacks of EGFP-H4 (emission filter: 500–550 nm) and Hoechst (emission filter: 425–475 nm) images were acquired (300 ms per slice) through a 63× NA = 1.4 oil objective. Pixel-by pixel analysis on the equatorial plane was performed with MATLAB routines on nuclei segmented as above (Supplementary Figure S1C ). To assess for possible biases, this segmentation approach was compared to line-scan analyses across intensity images of nuclei, where peripheral pixels were defined as those between the last border pixel from both line ends below about the background intensity plus its standard deviation and the next local minimum of intensity, and central pixels elsewhere in between.
3
Microscopy Techniques for Cellular Dynamics
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All microscopy, excluding the experiments for Figure 5 , was performed at 30°C on a Delta Vision Deconvolution Microscope (Applied Precision), using InsightSSITM Solid State Illumination of 488 and 594 nm, an Olympus UPLS Apo 60x or 100x oil objective with 1.4NA and softWoRx software (GE lifesciences). Detection was done with a CoolSNAP HQ2 camera. Microscopy to study Msn2 dynamics was performed on a Nikon Ti-E microscope equipped with a Hamamatsu Orca Flash V2 using a 40X oil immersion objective (1.3NA). Fluorescence excitation was performed using an LED illumination system (Excelitas 110-LED) that is triggered by the camera.
4
Cytological Analysis of Ovule Primordia
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For cytological examination of cleared ovule primordia, flower buds from wild-type and mutant plants were harvested and fixed in formalin-acetic acid-alcohol solution (40% formaldehyde, glacial acetic acid, 50% ethanol; in a 5:5:90 vol ratio) for at least 24 hr at room temperature. After fixation, samples were washed two times with 100% ethanol and stored in 70% ethanol. Gynoecia of 0.2–0.6 mm in length were removed from the flowers with fine needles (1 mm insulin syringes), cleared in Herr’s solution (phenol: chloral hydrate: 85% lactic acid: xylene: clove oil in 1:1:1:0,5:1 proportions), and observed by differential interference contrast microscopy using a Zeiss Axioimager Z2 microscope and 40X or 63X oil immersion lenses. Picture acquisition was done with a sCMOS camera (Hamamatsu ORCA Flash V2). Nuclei area measurements were carried out with ImageJ software, using the manual contour tool ‘Oval’.
5
Calcium Signaling Dynamics in Cells
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Cells were incubated with the labelled calcium indicator Fluo-4AM (ThermoFisher Scientific, F14201) in culture medium for 45 min (10 µM, 37 °C). Cell were incubated for 5 min (37 °C) to allow for de-esterification of AM esters. For baseline measurements of calcium signalling, artificial CSF (aCSF) was perfused over the cells for 5 min (see Table 2 ). 5HT (10 µM in dH2O, 14927) was perfused for 5-minutes suspended in aCSF before a 5-min aCSF washout period. A minimum time-course of 3-min has been used in previous research to investigate 5HT induced calcium dynamics at similar compound concentrations [12 (link), 13 (link)]. Imaging was performed using a fluorescence microscope (Nikon Eclipse FN1) with a 20× objective. Fluo-4AM fluorescence was excited at 488 nm and captured every 2 s to create a time-lapse (Hamamatsu Orca Flash V2). The fluorescence was calculated from five regions of interest containing approximately 3–10 cells using Fiji software (ImageJ, NIH).
aCSF constituent used for perfusion over cells for baseline calcium measurements and as a vehicle for serotonin.
| Component | Final concentration (mM) |
|---|---|
| NaCl | 126 |
| KCL | 2.5 |
| NaHCO3 | 26 |
| KH2PO4 | 1.25 |
| MgSO4 | 1 |
| CaCl2 | 2 |
| Glucose | 10 |
pH 7.3–Continuous bubbling with CO2 throughout the experiment.
6
Callose Detection in Apomictic Ovules
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Ovaries and anthers collected at pre-anthesis stages (white or pale-yellow anthers) from independent T2 plants of apomictic events T314 15.1 and T314 37.7 (and of replicated samples collected on T3 plants of event T314 15.1) were fixed in Carnoy’s or FAA fixatives and rinsed in 70% ethanol. Dissected ovaries were mounted in Hoyer’s clearing medium and gently squashed by pressure on the coverslip to expose ovules. Callose detection using aniline blue staining was performed as described60 (link). Observation and imaging were done on a Zeiss Axioimager Z2 microscope equipped with DIC, CFP emission filter; ×40 or ×63 (NA1,4) oil immersion lenses, and a sCMOS camera (Hamamatsu ORCA Flash V2). Images were processed using ImageJ software.
7
Intravital Confocal Microscopy Setup
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Intravital imaging was performed using a Leica DM6 FS upright microscope equipped with a motorized stage. The microscope is fitted with HCX PL Fluotar 5x/0.15, HC Fluotar ×25/0.95 and HC PL APO ×40/1.10 objectives lens (Leica), mounted on an optical table to minimize vibration and totally enclosed in a biosafety cabinet (Noroit). The microscope is coupled to a Yokogawa CSU-W1 confocal head modified with Borealis technology (Andor). Four laser excitation wavelengths (488, 561, 642, and 730 nm) were used in fast succession and visualized with the appropriate long-pass filters. Fluorescence signals were detected using a sCMOS 2048 × 2048 pixel camera (Orca Flash v2 + , Hamamatsu). Metamorph acquisition software (Molecular devices) was used to drive the confocal microscope.
8
Motility Assays of Exponentially Growing Cells
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Motility assays were performed as described previously (37 (link)). Briefly, exponentially growing cells were harvested (3 min, 6,000 × g, RT) and resuspended in 1% CTT to a density of 7 × 109 cells mL−1. Five-microliter volumes of cell suspensions were placed on 0.5% and 1.5% agar (Invitrogen) supplemented with 0.5% CTT and incubated at 32°C for 24 h. Cells were imaged using a M205FA stereomicroscope (Leica) and a DMi8 inverted microscope (Leica) equipped with a Hamamatsu ORCA-Flash V2 digital CMOS camera (Hamamatsu Photonics) and DFC9000 GT camera (Leica), respectively.
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