Freshly prepared samples were placed on the microscope setup, and movies of typically 300 frames with a frame rate of 10 Hz were recorded. We used a home build setup arranged around an inverted microscope (IX70, Olympus) with a 1.45 NA oil immersion objective (PLAPON 60×0TIRFM, Olympus). Samples were illuminated using a 633 nm HeNe laser (circularly polarized light, 1.4 kW/cm2) in oblique illumination mode to excite the samples slightly above the glass-cell interface, minimizing in this way any potential artifacts associated with the proximity of the cell membrane to the glass substrate. The fluorescence emission of the ATTO-647N was separated from the excitation light using a dichroic mirror (Semrock, FF500/646-Di01). A 660 nm long pass filter (Semrock, BLP01-635R-25) then selectively allowed the fluorescent emitted light to be detected by an EMCCD (Hamamatsu) camera. Temperature was maintained at 37°C with 5% CO2 by a custom made incubator built around the microscope stage. In experiments where cells were stimulated with CCL21 and/or ICAM-1, movies of 30 s were recorded before, and at one-minute intervals after stimulation up to 10 minutes.
Blp01 635r 25
Manufactured by IDEX Corporation
The BLP01-635R-25 is a laser module that generates a 635 nanometer red laser beam with a power output of 25 milliwatts. It is a compact, self-contained unit that can be used in a variety of laboratory and industrial applications.
Automatically generated - may contain errors
4 protocols using blp01 635r 25
1
Single-Molecule Imaging of Cell Signaling
2
Single-molecule QD655 tracking setup
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Single QD655 tracking was performed using a custom setup built around an inverted microscope (IX70, Olympus) equipped with a 1.49 numerical aperture oil immersion objective (Apon ×60 total internal reflection fluorescence, Olympus). Samples were excited using a 488-nm laser (Sapphire 488–150CW CDRH) in oblique illumination configuration. A dichroic mirror (Semrock, FF500/646-Di01) was used to direct the laser light onto the sample while allowing transmission of the QD fluorescence emission, which was directed onto a CMOS camera (Hamamatsu, ORCA-Flash 4.0) after further long-pass filtering (Semrock, BLP01–635R-25). A custom-made incubator built around the microscope allowed the samples to be maintained at 37 °C with 5% CO2 during the measurements. During a typical experiment, movies of 1000 frames were recorded every 60 s at a frame rate of 62 Hz.
3
Single-Molecule Wnt3A Imaging Protocol
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White-light transmission and single-molecule fluorescence images were acquired with a modified fluorescent microscope based on an Olympus IX71 inverted microscope, equipped with an infinity-corrected oil immersion objective (Olympus UPlanApo, ×100, 1.4 NA), operating in highly inclined and laminated optical sheet imaging mode (23 (link)) to reduce the excitation volume, and detected on a 512 × 512 pixel EMCCD (Andor I-Xon2, 897) at a rate of 20–30 ms per frame for Wnt3A imaging. The general epifluorescence setup has been described previously (24 (link)); here, the filters used were a dichroic mirror (Di01-R635-25x36; Semrock) and a 635 nm long pass filter (BLP01-635R-25; Semrock). Laser excitation was provided by a 635 nm solid-state laser (Blue Sky Research, FTEC2-635-V50PFM, 638 nm FiberTECII laser) at a power density of ∼250 W/cm2.
4
Single-Molecule TIRF Imaging
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Before imaging, the cells were washed in PBS and then added to the rCD2 constructs and QDot-containing bilayer (preparation described above). Imaging was performed using a custom-built total internal reflection (TIRF) setup using a 100× Apo TIRF, numerical aperture 1.49 objective (Nikon, Tokyo, Japan), creating a TIRF illumination at the glass water interface. Fluorescence was recorded through a beam splitting system (Dual-View; BioVision Technologies, Exton, PA) using a dichroic mirror and filters (for 488 or 633 emission, FF605-Di02 (dichroic; Photometrics, Tucson, AZ), FF03-525/50-25 (filter, 488 emission), and BLP01-635R-25 (filter, 633 emission), all Semrock, Rochester, NY). 1000 frames per experiment were acquired with an exposure time of 30 ms, yielding an overall recording time of 1 min. The camera (Cascade II; Photometrics) and shutter (SH05; Thorlabs, Newton, NJ) were operated using Micromanager (Vale Lab, University of California-San Francisco, San Francisco, CA). The two Dual-View channels were aligned using TetraSpec Microspheres (0.1 μm, fluorescent blue, green, orange, and dark red; Thermo Fisher Scientific) routinely to a precision of ∼120 nm.
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