Imagem x2 em ccd camera

Spinning disk images were captured using a Leica DMI6000 microscope, equipped with a Yokogawa CSU-X1 spinning disk confocal scanner unit and a Hamamatsu ImagEMX2 EM-CCD camera. For high resolution images, a Leica 100× 1.49 NA oil immersion objective was utilized (as shown in Fig. 1B, 1C, 2B, 2C. 3B–3D, 4C, 4D, Appendix Fig. S4A). Broader field views were obtained using a Leica 20× 0.4 NA air objective (as shown in Fig. 3E and 3F).
Confocal images were acquired on a Leica SP8 microscope equipped with a 63 × 1.4 NA oil immersion objective. For detection, the system employed either hybrid (HyD) detectors (SI Appendix Fig. S2 and S3) or PMT detectors (Fig. 4E).
Total Internal Reflection Fluorescence (TIRF) images were captured using a TIRF/iLAS2 TIRF/FRAP module (Biovision) mounted on a Leica DMI6000 microscope base equipped with Leica 100× 1.49 NA oil immersion objective (Fig. 4B) or a DeltaVision OMX SR system, equipped with an Olympus 60 × 1.49 NA oil immersion objective for TIRF and ring-TIRF capabilities (Fig. 2D). Highly inclined and laminated optical sheet (HILO) microscopy for single molecule imaging was captured using DeltaVision OMX SR system, equipped with an Olympus 60 × 1.49 NA oil immersion objective for TIRF (Fig. 5B–5F).

TIRF images were captured using a TIRF/iLAS2 TIRF/FRAP module (Biovision) mounted on a Leica DMI6000 microscope base equipped with a plan apo 100 × 1.49 NA TIRF objective and a 405/488/561/647 nm Laser Quad Band Set filter cube for TIRF applications (Chroma). Illumination was provided by an integrated laser engine equipped with multiple laser lines (405 nm-100mw/445 nm-75mw/488 nm-150mw/514 nm-40mw/561 nm-150mw/637 nm-140mw/730 nm-40mw, Spectral). Confocal images were captured using a Yokogawa spinning disk and a 405/488/561/647 nm Laser Quad Band Set filter cube (Chroma) with a plan apo 63 × 1.40 NA objective. Images were acquired using a Hamamatsu ImagEMX2 EM-CCD camera.
In Figure 7, TIRF images were captured using a Leica TIRF-module mounted on a Leica DMi8 equipped with a plan apo 100 × 1.47 NA TIRF objective and a Quad Band set filter cube for TIRF applications. Illumination was provided by an integrated laser system equipped with multiple laser lines (405 nm-50mw/488 nm-150mw/561 nm-120mw/638 nm-150mW). Images were acquired using LASX software a Hamamatsu Flash 4.0 V3 CMOS camera.

TIRF images were captured using a TIRF/iLAS2 TIRF/FRAP module (Biovision) mounted on a Leica DMI6000 microscope base equipped with a Hamamatsu ImagEMX2 EM-CCD camera with a 100 × 1.49 NA objective. Images were acquired using MetaMorph software. Experiments using TIRF microscopy are described in detail in the following methods sections: Steady State Reconstitution Assays, Actomyosin Contraction Assays, Actin Binding Assays on SLBs, and Activation of Jurkat T Cells.

Ca²⁺-imaging was performed using HT Functional Drug Screening System FDSS/µCELL (Hamamatsu Photonics) at nominal 37 °C. The FDSS/µCell system enables the indirect recording of changes of intracellular Ca²⁺ [Ca²⁺]_i via a Ca²⁺-sensitive fluorescent dye. The fluorescence signal of a complete 384-well plate is acquired at once with a high-speed and high-sensitivity digital ImagEM X2 EM-CCD camera (Electron Multiplying Charge-Coupled Device, Hamamatsu Photonics), but with limited spatial resolution. Therefore, the software only determines the mean fluorescence signal of each well. The signals of individual cells could not be captured. For compound application, the integrated dispenser head with 384 pipette tips was used, which can add the test compound to all wells simultaneously. The cells were preincubated with Cal-520 AM (AAT Bioquest) at a concentration of 1 µM for 1 h at 37 °C. For recording, the medium was exchanged by a buffer solution containing [mM]: 135 NaCl, 5 KCl, 0.2 MgCl₂, 2.5 CaCl₂, 10 HEPES and 10 D-glucose, pH 7.4. Test compound application was executed after obtaining a 1.5 min baseline recording. Where applicable, a second application was executed 4.5 min after the first application. The total recording never exceeded 8 min.

Cells were seeded onto 35-mm cover-glass bottom cell culture dishes (Cat. #: FD35-100, World Precision Instruments) and transfected with 1 μg of pmCherry paxillin (Cat. #: 50526, Addgene). Media were changed 18–24 h after transfection, and cells were allowed to recover for 24 h. Images were acquired every 20 s for 20 min on a Total Internal Reflection Fluorescence (TIRF)-Spinning Disk Spectral Diskovery System (Spectral Applied Research, Richmond Hill, ON) based on a Leica DMI 6000 microscope stand (Quorum Technologies, Puslinch, ON) equipped with a Leica Plan-Apochromat 63x/1.47NA oil DIC objective, ImagEM X2 EM-CCD camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan), and Chamlide CU-501 top-stage incubator system (Live Cell Instrument, Seoul, South Korea). Each cell was illuminated with a 561-nm diode laser set to 22.7% (or ~ 74 μW power). An ET600/50m emission filter (Chroma, Bellows Falls, VT) was used to capture mCherry fluorescence. The camera exposure time was set to 500 ms with an EM gain of 255 and read speed of 22 MHz. A total internal reflection fluorescence (TIRF) prism was used to limit fluorescence excitation to a depth of 80 nm.

Microscopic images were taken by an EMCCD camera (ImagEM X2 EM-CCD camera, Hamamatsu Photonics K.K.). For fluorescence imaging of C. reinhardtii cells, a fluorescence lamp (X-Cite 120PC Q, Excelitas Technologies Corp.), a 488/10 nm single bandpass excitation filter (Semrock, Rochester, NY), and a 440/521/607/700 quad-bandpass emission filter (Semrock, Rochester, NY) were used. During the 7-day experiment period, fluorescence images were taken every 4 h with 50 ms exposure using the cellSens imaging software (Olympus Life Science).