For live-cell imaging, HL-60 neutrophils were collected, washed and resuspended with mHBSS containing 0.2% BSA, and then seeded on Lab-Tek chambered cover glasses (NUNC) coated with 50 μg/ml human fibronectin (Sigma-aldrich). Time-lapse microscopy was performed on a Leica Spinning Disk Confocal microscope. Chemoattractant fMLP (Sigma) was given to cells either globally or delivered by a Femtotips micropipette (Eppendorf). Total Internal Reflection Fluorescence (TIRF) microscopy was performed using a Nikon Eclipse Ti-E TIRF microscope. Fluorescence recovery after photobleaching (FRAP) was performed on Zeiss Axiovert 200 Laser scanning microscope with LSM510-Meta confocal module. For immuno uorescent staining, cells were xed, and then stained with Anti-PI 3-Kinase, p110γ (clone 17D7.2, Merck Millipore) in PBS containing 1% BSA as indicated in the Extended Experimental Procedures. Images were analyzed with ImageJ (NIH) as described in the Extended Experimental Procedures.
Eclipse ti e tirf microscope
Manufactured by Nikon
The Eclipse Ti-E TIRF microscope is a high-performance research-grade microscope system designed for advanced imaging techniques. It is equipped with a total internal reflection fluorescence (TIRF) illumination module, allowing for the selective excitation of fluorophores near the coverslip surface. This enables the visualization of dynamic processes and events occurring at the cell-substrate interface with high signal-to-noise ratio. The Eclipse Ti-E TIRF microscope provides researchers with a powerful tool for exploring a wide range of biological applications, including cell biology, membrane dynamics, and single-molecule studies.
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Lab products found in correlation
Lab tek chambered cover glasses, by Thermo Fisher Scientific (1 mentions)
Spinning disk confocal microscope, by Leica (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Lsm 510 meta confocal module, by Zeiss (1 mentions)
Axio imager m2 upright microscope, by Zeiss (1 mentions)
Lsm800 inverted confocal microscope, by Zeiss (1 mentions)
μ slide angiogenesis, by Ibidi (1 mentions)
4 protocols using eclipse ti e tirf microscope
1
Live-Cell Imaging of Neutrophil Chemotaxis
2
Planar Lipid Bilayer Imaging and Tracking
3
Imaging and Analysis of Phase-Separated Biomolecular Assemblies
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DIC images were captured using a Zeiss AxioImager M2 upright microscope. Raw, unprocessed DIC images are shown in the paper.
Epifluorescence images were captured with a Nikon Eclipse Ti-E TIRF microscope in epifluorescent mode. Raw, unprocessed images were analyzed using the Fiji (ImageJ) software. For illustration purposes, images were background corrected (SI Appendix, Supplementary Materials and Methods ). Methods of determination of size distribution and fluorescent partitioning are described in SI Appendix, Supplementary Materials and Methods .
FRAP experiments were performed at 23 °C using an inverted LSM800 confocal microscope (Zeiss).
For evaluation of droplet volumes, microscopic images were taken on a Zeiss Spinning Disk system at 24 °C. Image analysis methods are described inSI Appendix, Supplementary Materials and Methods .
Epifluorescence images were captured with a Nikon Eclipse Ti-E TIRF microscope in epifluorescent mode. Raw, unprocessed images were analyzed using the Fiji (ImageJ) software. For illustration purposes, images were background corrected (
FRAP experiments were performed at 23 °C using an inverted LSM800 confocal microscope (Zeiss).
For evaluation of droplet volumes, microscopic images were taken on a Zeiss Spinning Disk system at 24 °C. Image analysis methods are described in
4
Single-molecule TIRF microscopy
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A Nikon Eclipse Ti‐E TIRF microscope was used with apo TIRF 100× oil immersion objective (numerical aperture (NA) = 1.49) in epifluorescence mode. For fluorescence excitation, a 642‐nm laser (56RCS/S2799, Melles Griot), a 543‐nm laser (25‐LGP‐193–230, Melles Griot) and a Cyan 488‐nm laser (Coherent) were used according to the excitation spectra of the applied fluorescent probes. Fluorescence was deflected to a ZT405/488/561/640rpc BS dichroic mirror and recorded with a Zyla sCMOS (ANDOR) camera.
Samples (20‐μL) were visualized in μ‐Slide Angiogenesis (Ibidi) microscope slides at 25°C. Images were recorded using NIS‐Elements AR (Advanced Research) 4.50.00 software, using 2 × 2 pixel binning and 200‐ms laser exposure time during acquisition.
Samples (20‐μL) were visualized in μ‐Slide Angiogenesis (Ibidi) microscope slides at 25°C. Images were recorded using NIS‐Elements AR (Advanced Research) 4.50.00 software, using 2 × 2 pixel binning and 200‐ms laser exposure time during acquisition.
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