Experiments examining single-vesicle docking and fusion events were performed on a Zeiss Axiovert 35 fluorescence microscope (Carl Zeiss, Thornwood, NY), equipped with a ×63 water immersion objective (Zeiss; N.A. = 0.95) and a prism-based TIRF illumination. The light source was an OBIS 532 LS laser from Coherent Inc. (Santa Clara CA). Fluorescence was observed through a 610 nm band-pass filter (D610/60; Chroma, Battleboro, VT) by an electron multiplying CCD (DU-860E; Andor Technologies, Belfast UK). The prism-quartz interface was lubricated with glycerol to allow easy translocation of the sample cell on the microscope stage. The beam was totally internally reflected at an angle of 72° from the surface normal, resulting in an evanescent wave that decays exponentially with a characteristic penetration depth of ~100 nm. An elliptical area of 250 × 65 µm was illuminated. The laser intensity, shutter, and camera were controlled by a homemade program written in LabVIEW (National Instruments, Austin, TX).
Obis 532 ls laser
Manufactured by Coherent Inc
The OBIS 532 LS laser is a compact, low-noise, and highly stable laser that emits light at a wavelength of 532 nanometers. It is designed to provide a consistent and reliable light source for various laboratory applications.
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3 protocols using obis 532 ls laser
1
Single-Vesicle Docking and Fusion Visualization
2
Single Vesicle Docking and Fusion Imaging
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Experiments examining single-vesicle docking and fusion events were performed on a Zeiss Axiovert 35 fluorescence microscope (Carl Zeiss, Thornwood, NY), equipped with a 63x water immersion objective (Zeiss; N.A. = 0.95) and a prism-based TIRF illumination. The light source was an OBIS 532 LS laser from Coherent Inc. (Santa Clara CA). Fluorescence was observed through a 610 nm band pass filter (D610/60; Chroma, Battleboro, VT) by an electron multiplying CCD (DU-860E; Andor Technologies). The prism-quartz interface was lubricated with glycerol to allow easy translocation of the sample cell on the microscope stage. The beam was totally internally reflected at an angle of 72 o from the surface normal, resulting in an evanescent wave that decays exponentially with a characteristic penetration depth of ~100 nm. An elliptical area of 250 x 65 µm was illuminated. The laser intensity, shutter, and camera were controlled by a homemade program written in LabVIEW (National Instruments, Austin, TX).
3
Single-vesicle TIRF Microscopy Protocol
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Experiments examining single-vesicle docking and fusion events were performed on a Zeiss Axiovert 35 fluorescence microscope (Carl Zeiss), with a 63 × water immersion objective (Zeiss, numerical aperture, 0.95) and prism-based TIRF illumination. The light source was an OBIS 532 LS laser or an OBIS 488 LS laser from Coherent Inc. Fluorescence was observed through a 610 nm band pass filter (D610/60, Chroma) by an electron multiplying charge coupled device (CCD) (DU-860E, Andor Technology). The electron multiplying CCD (EMCCD) was cooled to −70°C, and the gain was set at 200. The prism-quartz interface was lubricated with glycerol to allow easy translocation of the sample cell on the microscope stage. The beam was totally internally reflected at an angle of 72° from the surface normal, resulting in an evanescent wave that decays exponentially with a characteristic penetration depth of ∼100 nm. An elliptical area of 250 μm × 65 μm was illuminated. The laser intensity, shutter, and camera were controlled by a homemade program written in LabVIEW (National Instruments).
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