Spinning disc confocal fluorescence microscopy was done using either a Leica HC PL APO 63×/1.40 oil immersion objective with 2× zoom on a Leica Yokogawa CSU-W1 spinning disc confocal scanner unit with iXon Life 888 EMCCD camera and VisiView software for spinning disc confocal microscopy (for images and data generated for Figures 1B , 1D , 4B and 4C ) or a Nikon PLAN APO λD 60X/1.42 oil immersion objective with 1X zoom on a Nikon Ti2 microscope with a Yokogawa CSU-W1 spinning disc confocal scanner unit, Gataca Systems Live-SR super resolution module and Kinetix 22 Scientific CMOS (sCMOS) camera for super resolution spinning disc confocal microscopy with NIS AR software (for images and data generated for Figures 1G , 1H , 3B , 4A , 4D , and S4A –D ). Laser scanning confocal microscopy was done using Leica HC PL APO CS2 63×/1.40 oil immersion objective with 2X zoom on a Leica TCS SP8 laser confocal microscope with photomultiplier tubes (PMT) detectors and Leica Application Suite X software (for images generated for Figure 1 I). TIRF microscopy was done using a Nikon 60X Apochromat TIRF oil objective with 1X zoom on a Nikon Ti2 microscope with Gataca Systems Ilas2 TIRF platform and iXon Ultra 897 EMCCD camera and NIS AR software (for images generated for Figure S2 ). For all microscopy, Dictyostelium cells were in development buffer (DB).
Life 888 emccd camera
Manufactured by Oxford Instruments
The Life 888 EMCCD camera is a high-performance scientific imaging device designed for low-light applications. It features a back-illuminated EMCCD sensor and provides high quantum efficiency, low noise, and fast frame rates. The camera is suitable for a range of scientific and research applications requiring sensitive image capture.
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Lab products found in correlation
Eclipse ti e, by Nikon (2 mentions)
W1 confocal spinning disk, by Yokogawa (2 mentions)
W1 spinning disk confocal, by Nikon (2 mentions)
A1r confocal, by Yokogawa (2 mentions)
A1r point scanning confocal system, by Nikon (2 mentions)
Wide field epifluorescence microscope, by Nikon (2 mentions)
Ultra 897 emccd camera, by Oxford Instruments (1 mentions)
Application suite x software, by Leica (1 mentions)
Csu w1, by Yokogawa (1 mentions)
Uplanfln, by Olympus (1 mentions)
Ix73 fluorescence microscope, by Olympus (1 mentions)
Uplsapo na 1, by Olympus (1 mentions)
Dp26 camera, by Olympus (1 mentions)
D9542, by Merck Group (1 mentions)
A21070, by Thermo Fisher Scientific (1 mentions)
Ab13970, by Abcam (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Fd35 100, by World Precision Instruments (1 mentions)
6 protocols using life 888 emccd camera
1
Multimodal Microscopy Techniques for Dictyostelium
2
Bioluminescent Imaging of Brain Tumors
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100 μL ETZ (6.8 mM) in the in vivo injection buffers was delivered to 8-week-old, anesthetized BALB/cJ mice via tail vein. BLI was subsequently performed with a UVP BioSpectrum dark box, a Computar Motorized ZOOM lens (M6Z1212MP3), and an Andor iXon Life 888 EMCCD camera. Mice were placed 21 cm away from the front of the lens with no emission filter. Other imaging conditions and data analysis procedure were identical to the descriptions in the above section. The same procedure was used to examine mice with untransfected HEK 293 T cells intracranially injected into the hippocampus.
3
Live Cell Microscopy Imaging Protocol
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Pictures of living cells were acquired using an Olympus IX73 fluorescence microscope fitted with a 40 X objective (0.75NA UPlanFLN, Olympus) or a 20 × 0.40 NA objective (LCAChN 0.4NA, Olympus), a X-Cite Series 120Q lamp (Lumen dynamics), a DP26 camera (Olympus) and using the adequate filters set. For time series, cells were seeded in glass-bottom dishes (from MatTek or ibidi µSlide) in phenol red-free Leibovitz’s L-15 medium containing 10% FBS and pen./strep. For each time point, z-stacks were acquired using a Andor/Olympus Yokogawa CSU-X1 confocal spinning disk fitted with 60 X (UPLSAPO NA 1.35, Olympus) or 100 X (UPLSAPO NA 1.4, Olympus) objectives, a Andor iXon Life 888 EM-CCD camera and with temperature and humidity control. The white scale bars on representative pictures represent 10 µm.
4
Immunostaining of Paxillin in ZMEL Cells
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1 d before immunostaining, ZMEL-GFP cells and ZMEL-mCherry cells that express WT-Paxillin were plated on either glass bottom dishes (FD35-100; World Precision Instruments) or collagen-coated dishes (0.5 kPa, SV3520-COL-0.5; 50 kPa, SV3520-COL-50; Matrigen). Cells were fixed with 4% PFA and permeabilized in 0.1% Triton X-100/TBS for 5 min and then blocked with 1% BSA + 1% FBS for 1 h. Cells were incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature. Primary antibodies used were chicken anti-GFP (1:500, ab13970; Abcam) and rabbit anti-pY118 Paxillin (1:500, NBP2-24459; Novus Biologicals). Secondary antibodies used were Alexa Fluor 488 goat anti-chicken IgY(H + L) secondary antibody (1:500, 103-545-155; Jackson Immunoresearch), Alexa Fluor 633 goat anti-rabbit IgG(H + L) secondary antibody (1:500, A-21070; Thermo Fisher Scientific), and DAPI (D9542; Sigma-Aldrich). Imaging was performed using either a Zeiss LSM 880 (Carl Zeiss) with a 63×/1.40 oil immersion objective with three photomultiplier tubes and two GaSP detectors or a Leica Yokogawa CSU-W1 spinning disc confocal microscope with a Leica PL APO 63×/1.40 oil immersion objective and an iXon Life 888 EMCCD camera at room temperature.
5
Multi-Modal Microscopy Imaging Protocol
6
Multi-Modal Microscopy Imaging Protocol
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Images were acquired using a Nikon wide-field epifluorescence microscope, Nikon A1R confocal, and Yokogawa W1 spinning disk confocal. All microscopes were run using Nikon Elements software. The widefield microscope was an inverted Nikon Eclipse TiE system with a 120BOOST LED-based illumination system and equipped with a Photometrics HQ2 CoolSnap camera and motorized XY stage. The Nikon A1R point scanning confocal system was run on an inverted Nikon Eclipse TiE base with 405-, 488-, 568- and 647nm excitation laser lines and four detectors: two GaAsP and two Alkali PMTs with a motorized XY stage. The Yokogawa W1 spinning disk confocal has an inverted Nikon Eclipse TiE base and 100mW 405-, 490-, 561-, and 640nm lasers, equipped with an Andor iXon 888 Life EMCCD camera linked with a 10-position filter wheel and a motorized XY stage. The spinning disk system was enclosed in an environmental chamber with temperature and local [CO2] control.
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