2D time-lapse imaging was performed on a Nikon Eclipse Ti inverted fluorescence microscope with a 100× (NA 1.40) oil-immersion objective (Nikon Instruments) and controlled with μ-Manager v1.4. Cells grown on agarose pads or in CellASIC microfluidic chambers were maintained at targeted temperature during imaging with an active-control environmental chamber (HaisonTech). An iXon3 888 EMCCD camera (Andor) was used for fluorescence time-lapse microscopy experiments and a Zyla 5.5 sCMOS camera (Andor) was used for millisecond phase imaging of cell separation. For fluorescence, a 49002-ET-EGFP filter set (Chroma, ET470/40, 495LP, ET525/50) was used to image NADA, 6-FlTre, fDHPE, and O-TMM-DBF; an 49008-ET-mCherry filter set (Chroma, ET560/40, 585LP, ET630/75) was used to image FM, TDL, 6-TMR-Tre, and rDHPE; and a 49006-ET-Cy5 filter set (Chroma, ET620/60, 660LP, ET700/75) was used to image O-TMM-647. Images were taken with a time interval of 3 or 5 min.
Ixon3 888 emccd camera
Manufactured by Oxford Instruments
The IXon3 888 EMCCD camera is a high-performance imaging device designed for low-light applications. It features an electron-multiplying CCD (EMCCD) sensor with a resolution of 1024 x 1024 pixels and a pixel size of 13 x 13 μm. The camera provides low readout noise and high quantum efficiency, making it suitable for a wide range of scientific and research applications that require high sensitivity and low-light detection.
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Lab products found in correlation
Zyla 5.5 scmos camera, by Oxford Instruments (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Immersol w 2010, by Zeiss (1 mentions)
Csu w1, by Yokogawa (1 mentions)
Chelex 100, by Bio-Rad (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Zyla 4.2 scmos camera, by Oxford Instruments (1 mentions)
Metamorph 7, by Molecular Devices (1 mentions)
4 protocols using ixon3 888 emccd camera
1
Fluorescence Microscopy for Cell Imaging
2
Drosophila Pupal Wing Imaging Protocol
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To prepare the Drosophila pupal wing samples for image collection, pupae at appropriate ages were fixed to double-sided tape and the pupal case above the left wing was removed. The pupae were then placed on a small drop of water or Immersol W 2010 (Zeiss 444969-0000-000) in a glass bottom dish with the left side facing downward6 (link),7 (link),46 (link),66 (link). The fixed time-point images other than Fig. 3a, b and time-lapse images shown in Fig. 6l, m were acquired using an inverted confocal microscope (A1R; Nikon) equipped with a ×60/NA1.2 Plan Apochromat water-immersion objective at 25 °C. Other images were acquired using an inverted confocal spinning disk microscope (Olympus IX83 combined with Yokogawa CSU-W1) equipped with an iXon3 888 EMCCD camera (Andor), an Olympus ×60/NA1.2 SplanApo water-immersion objective, and a temperature control chamber (TOKAI HIT), using IQ 2.9.1 (Andor)66 (link). After imaging, we confirmed that the pupae survived to at least the pharate stage.
3
Real-time monitoring of zinc release from cells
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CASK-targeted siRNA or non-silencing control siRNA transfected INS-1E cells were bathed in Chelex-100 (BioRad)-treated, zinc-free KRBH containing 4 μM FluoZin (ThermoFisher) inside an OKOlabs incubator that was maintained at 37 °C and mounted on a Nikon Ti-E microscope. Zn2+ released from the cells will react with FluoZin in the medium and results in the fluorescence enhancements. TIRFM was performed using a 60× NA 1.65 objective with a 488-nm laser introduced into the excitation light path through the LApps H-TIRF module (Nikon) angled to generate a thin evanescent wave of ¡-90 nm. Fluorescence was detected using an Andor iXon3 888 EMCCD camera running at 30 Hz (exposure time ~33 ms). Background fluorescence was removed by subtracting a 100-frame running average projection. Two separate methodologies were compared to detect release events and found to give similar results. These were an in house software package “TIRF explorer” (10.1109/ICCVW.2009.5457651) and a simple analysis pipeline developed in Fiji33 (link) and cell profiler (PMC3072555) using object detection. The speed of the release events precluded us measuring the event kinetics and so we opted for a simple metric of the event size or magnitude. This was defined as the event area multiplied by the event intensity identified from a maximum projection of the frames that contained the event.
4
Spinning Disk Confocal Microscopy of Egg Chambers
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Imaging was performed on a Nikon Eclipse Ti-E double-stage microscope equipped with a Yokogawa CSU-W Spinning Disk confocal scanner and a piezoelectric z-stage (737.2SL; Physik Instrumente), using 40× 1.15NA water immersion objective, and 488 and 561 nm laser lines for excitation of GFP and mCherry, respectively. An Andor iXon3 888 EMCCD camera with a 2× postmagnification lens was used for time-lapse acquisitions, while an Andor Zyla 4.2 sCMOS camera with no further postmagnification was used for snapshot images of whole egg chambers for analyses, as shown in Fig. 1 and Fig. S1 . Images were acquired at 73 focal planes with 0.5 μm z-spacing. The x–y pixel size was 162 nm. During time-lapse microscopy, images were acquired every 30 s (in experiments shown in Figs. 2 and 3 ; Fig. S2 ; and Fig. S3, A–C ) or every 60 s (in experiments shown in Fig. 4 and Fig. S3, D–F ) for 60–90 min. To assess Par protein dynamics at longer timescales (Figs. 5 and 6 ), images were acquired every 5 min for at least 150 min. Snapshots and time-lapse acquisitions during mechanical manipulation were acquired using Andor IQ3 software (Andor Technologies). All ablation experiments were performed using Metamorph 7.10 (Molecular Devices Inc.). Experiments were performed at 25°C room and microscope stage temperature.
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