All imaging was completed with a Carl Zeiss Axio Observer. Z1 inverted light/epifluorescence microscope equipped with ApoTome.2 optical sectioning and a Hamamatsu ORCA-Flash 4.0 V2 digital camera. Cells were viewed with a Plan-Apochromat 63×/1.40 Oil DIC M27 microscope objective. Throughout the experiments, cells were kept in a humidified incubator with 5% CO2 at 37 °C. Green fluorescence was detected using a fluorescence channel possessing excitation and emission wavelength filter sets of 450–490 nm and 500–550 nm, respectively. Both the intensity of fluorescence illumination and camera exposure time were held constant throughout all experiments. For time-lapse experiments, cells were imaged before treatment, with the location of each cell saved using the automated stage top. Cells were then either treated with 20 ng/mL of IL-6 or 100 nM of insulin and automatically imaged every 5 min for 30 min following treatment.
Orca flash 4.0 v2 digital camera
Manufactured by Hamamatsu Photonics
Sourced in Japan
The ORCA-Flash 4.0 (V2) is a digital camera developed by Hamamatsu Photonics. It features a 4 megapixel scientific CMOS image sensor with a pixel size of 6.5 μm. The camera supports a frame rate of up to 100 frames per second at full resolution.
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4 protocols using orca flash 4.0 v2 digital camera
1
Time-Lapse Imaging of Cytokine Response
2
Ca2+ Imaging of Neuronal Activity
3
Calcium Imaging and Network Analysis
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Two-minute videos of Ca2+ fluorescence were acquired with an ORCA-Flash 4.0 (V2) digital camera (Hamamatsu) via a 40x objective at 25 frames/second with a 512×512 pixel binning. Following imaging of spontaneous Ca2+ transients, fluorescent signals in response to extracellular stimulation were recorded in each field of view. Extracellular stimuli were delivered by 100 μs current pulses generated by a Master-9 stimulator (A.M.P.I) via a bipolar tungsten electrode positioned just outside the field of view in the stratum radiatum. The amplitude of the current pulses was controlled by a stimulus isolation unit (ISO-Flex, A.M.P.I.). Minimal stimulation was defined as minimal current intensity to produce visible fluorescence in any of the imaged cells. Network analysis of Ca2+ transients, was performed on the basis of undirected adjacency matrices thresholded to preserve 25% of the strongest correlation coefficients as described in our previous publication [32 (link)].
4
Microscopic Imaging of Transformed Tobacco Pollen Tubes
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Epifluorescence microscopy of transformed tobacco pollen tubes was done with an Olympus BX51 by using an UPlanSApo 910/ 0.40 objective (Olympus, Tokio, Japan). For mVenus-tagged constructs, the filter cube U-MWIB was used. Images were taken with an ORCA flash 4.0 V2 Digital Camera using the software HOKAWO 2.10 (Hamamatsu Photonics, Hamamatsu, Japan).
Confocal imaging of pectin stained by propidium iodide was performed with a Zeiss LSM510 confocal microscope using a Plan-Neofluar 940/1.30 oil immersion objective (Carl Zeiss, Oberkochen, Germany). Fluorescence of propidium iodide was observed after excitation with 561 nm and detected at 689-721 nm using the HFT 405/488/561 major beam splitter.
Fluorescence recovery after photobleaching analyses were carried out with a Zeiss LSM880 equipped with C-Apochromat 940/1.2 water immersion objective (Carl Zeiss). For mVenus imaging, optimal singletrack acquisition parameters were used (mVenus fluorescence was excited by a 514 nm laser, and the emission at 520-590 nm was recorded using a GaAsP detector).
Spinning disk confocal microscopy was carried out on the Nikon Ti-E platform with Yokogawa CSU-X1 spinning disk and sCMOS camera Andor Zyla, using a Plan Apo VC 960/1.20 water immersion objective (Nikon, Tokio, Japan)
Confocal imaging of pectin stained by propidium iodide was performed with a Zeiss LSM510 confocal microscope using a Plan-Neofluar 940/1.30 oil immersion objective (Carl Zeiss, Oberkochen, Germany). Fluorescence of propidium iodide was observed after excitation with 561 nm and detected at 689-721 nm using the HFT 405/488/561 major beam splitter.
Fluorescence recovery after photobleaching analyses were carried out with a Zeiss LSM880 equipped with C-Apochromat 940/1.2 water immersion objective (Carl Zeiss). For mVenus imaging, optimal singletrack acquisition parameters were used (mVenus fluorescence was excited by a 514 nm laser, and the emission at 520-590 nm was recorded using a GaAsP detector).
Spinning disk confocal microscopy was carried out on the Nikon Ti-E platform with Yokogawa CSU-X1 spinning disk and sCMOS camera Andor Zyla, using a Plan Apo VC 960/1.20 water immersion objective (Nikon, Tokio, Japan)
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