A prism-type total internal reflection fluorescence microscope was used to acquire fluorescence emission signals from Cy3 and Cy5 by a water immersion objective lens (UPlanApo 60×, Olympus). The laser scattering was rejected through a 550-nm long-pass filter. The fluorescence emission light was further separated into donor and acceptor signals with a 630- or 635-nm dichroic mirror (Chroma) and projected onto a back-illuminated electron-multiplying charge-coupled device (Andor) with a time resolution of 30 to 100 ms. Fluorescence signals of the donor and acceptor were amplified by a gain before camera readout. Thus, both recorded fluorescence intensities of Cy3 and Cy5 are in an arbitrary unit (a.u.), proportional to their photon counts. FRET efficiency is determined by the ratio of intensities, Intensityacceptor/(Intensitydonor + Intensityacceptor) after correcting for cross-talk between the donor and acceptor channels. All data were analyzed by MATLAB codes and plotted in Origin.
Uplanapo 60
Manufactured by Olympus
The UPlanApo 60x is a high-numerical aperture objective lens designed for use in Olympus microscopes. It provides a magnification of 60x and is optimized for a wide range of microscopy applications.
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Lab products found in correlation
Ixon du 897, by Oxford Instruments (1 mentions)
Ixon ultra 897 emccd camera, by Oxford Instruments (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Staphylococcal enterotoxin e, by Toxin Technology (1 mentions)
Nis elements c software, by Nikon (1 mentions)
Plan apo vc 60, by Nikon (1 mentions)
Uplanfl 100, by Olympus (1 mentions)
Mats u55r30, by Tokai Hit (1 mentions)
Inubg2h tizb, by Tokai Hit (1 mentions)
6 protocols using uplanapo 60
1
Single-Molecule FRET Microscopy Protocol
2
Single-molecule FRET using TIRF microscope
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A prism-type total internal reflection fluorescence (TIRF) microscope used in this single-molecule FRET experiment is same as before which previously described in the paper. Briefly, A 532-nm laser (Samba, Cobolt AB) was used to excite Cy3 and the emitted fluorescence was collected by objective (UPlanApo 60×, Olympus) and passed the emission filter (ZET405/488/532/642m, Chroma Technology Crop.) to eliminate the scattered light. The filtered signals were divided into donor and acceptor channels by a dichroic mirror (645dcxr, Chroma Technology Corp.) and recorded by an electron-multiplying charge-coupled device (iXon DU-897, Andor Technology). Data-acquisition time was set to 30 ms for recording real-time FRET traces and 500 ms for obtaining FRET histograms. All image data were processed using IDL and MATLAB codes.
3
Single-molecule TIRF Microscopy
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Single-molecule experiments were performed with a prism-type TIRF (total internal reflection fluorescence) microscope. Briefly, the fluorescence emission light from the donor (Cy3) and acceptor (Cy5) fluorophores was transmitted by a water-immersion objective lens (UPlanApo 60×, Olympus) and then collected through a 550-nm longpass filter to filter out scattered light from a 532 laser. The fluorescence emission spectrum was further divided into donor (green) and acceptor (red) signals with a dichroic mirror with a 630-nm cutoff (Chroma) and recorded by a back-illuminated electron-multiplying charge-coupled device (897 EMCCD, Andor) with the time resolution of 100-ms (for degradation assays) and 500-ms (for binding assays).
4
Single-Molecule FRET Dynamics Tracking
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FRET donor (Cy3) on the DNA was excited by a green laser (532 nm, 100 mW, Coherent Compass Laser). The fluorescence emission light from Cy3 and Cy5 was collected by a water immersion objective lens (UPlanApo 60×, Olympus) and then cleaned by a 550 nm long-pass fluorescence filter equipped in a total internal reflection fluorescence (TIRF) microscopy. The emission light was divided into donor and acceptor signals with a 635 nm dichroic mirror (Chroma) and was recorded by iXon Ultra 897 EMCCD camera (Andor). Both recorded fluorescence intensities of Cy3 and Cy5 were in an arbitrary unit (a.u.) since they were amplified by a gain factor. The data were saved in a video file format by a software written in Visual C++. Fluorescence intensities of single molecules were extracted by IDL software and FRET efficiency was calculated as the ratio of intensities, Accepter Intensity/(Donor Intensity + Acceptor Intensity) after amending cross-talk between the donor and acceptor channels. All data were analyzed with MATLAB codes and plotted in Origin software.
5
Visualization of TMEM16F Localization
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Raji cells were incubated for 2 h with 1 µg/ml of staphylococcal enterotoxin E (SEE; Toxin Technology) at 37°C. After extensive washing, 105 Raji cells were mixed with 105 Jurkat cells and plated onto poly-l -lysine-coated coverslips in 24-well plates, incubated for 30 min at 37°C, and then fixed with 2% paraformaldehyde (PFA). To determine the localization of TMEM16F, reporter (TMEM16F-RFP) or TCR-β were used to distinguish Jurkat from Raji cells. For immunofluorescence assays, samples were permeabilized with 0.1% Triton X-100, blocked with 10% FBS plus 2% goat serum in PBS, and stained for TCR-Vβ8 (cat. no. 555604; BD), followed by Alexa Fluor 555–conjugated goat anti–mouse IgG2b secondary antibody (A-21147; Life Technologies). All samples were mounted with ProLong Gold Antifade Mountant with DAPI (Life Technologies). Images were taken using a FV1000 confocal microscope with a water objective lens (UPlanAPO 60×; NA 1.20; Olympus). For 3D and z-axis image reconstruction, 20 confocal sections, 0.4 µm apart, were assembled using ImageJ/Fiji software (NIH).
6
Time-lapse Imaging of Cytoskeletal Dynamics
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Time-lapse images were captured with an inverted microscope (IX71; Olympus) equipped with a single-chip color CCD camera (DP70; Olympus) and an objective lens (LCPlanFl 20/0.40 NA; UPlanApo 60/0.90 NA; UPlanFl 100/1.30 NA Oil; Olympus).
During observation, cells were warmed on a thermoplate set to 37°C (MATS-U55R30; Tokai Hit). Images were captured every 5 min and analyzed using Lumina Vision version 2.4.2 software (Mitani Corporation). Images of cells expressing mCherry-NMHC-IIA and EGFP-NMHC-IIB were captured using an inverted microscope (Ti-E; Nikon) equipped with an oil-immersion objective lens (Plan Apo-VC 60/1.40 NA; Nikon). During observation, cells were warmed in an incubation chamber heated to 37°C (INUBG2H-TIZB; Tokai Hit). Images were captured and analyzed using NIS-Elements C software (Nikon).
During observation, cells were warmed on a thermoplate set to 37°C (MATS-U55R30; Tokai Hit). Images were captured every 5 min and analyzed using Lumina Vision version 2.4.2 software (Mitani Corporation). Images of cells expressing mCherry-NMHC-IIA and EGFP-NMHC-IIB were captured using an inverted microscope (Ti-E; Nikon) equipped with an oil-immersion objective lens (Plan Apo-VC 60/1.40 NA; Nikon). During observation, cells were warmed in an incubation chamber heated to 37°C (INUBG2H-TIZB; Tokai Hit). Images were captured and analyzed using NIS-Elements C software (Nikon).
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