For live-cell oblique illumination, neurons were visualized at 50-Hz (10,000–20,000 frames by image streaming) and 20-ms exposure, at 37°C on a microscope equipped with an ILas2 double-laser illuminator (Roper Technologies), a CFI Apo TIRF 100× 1.49 NA objective (Nikon), and an Evolve512 delta EMCCD camera (Photometrics). Image acquisition was performed using Metamorph software (MetaMorph Microscopy Automation and Image Analysis Software, version 7.7.8; Molecular Devices). A quadruple beam splitter (LF 405/488/561/635-A-000-ZHE; Semrock) and a QUAD band emitter (FF01-446/510/581/703-25; Semrock) were used.
Cfi apo tirf 100 1.49 na objective
Manufactured by Nikon
The CFI Apo TIRF 100× 1.49 NA objective is a high-numerical aperture objective designed for total internal reflection fluorescence (TIRF) microscopy. It features a magnification of 100× and a numerical aperture of 1.49, which enables the efficient collection of fluorescence signals near the sample surface.
Automatically generated - may contain errors
Lab products found in correlation
Ilas2 double laser illuminator, by Roper Technologies (3 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Lf 405 488 561 635 a 000 zhe, by IDEX Corporation (1 mentions)
Ff01 446 510 581 703 25, by IDEX Corporation (1 mentions)
Tirf microscope, by Roper Technologies (1 mentions)
Infinity 2 5 m camera, by Teledyne (1 mentions)
Ixon 897 ultra em ccd camera, by Oxford Instruments (1 mentions)
4 protocols using cfi apo tirf 100 1.49 na objective
1
Live-cell Oblique Illumination Microscopy
2
Live-cell TIRF Microscopy Protocol
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For live-cell TIRF microscopy, transfected cells were visualized on a TIRF microscope (Roper Technologies) equipped with an ILas2 double laser illuminator (Roper Technologies), a CFI Apo TIRF 100× (1.49-NA) objective (Nikon), and an Evolve512 delta EMCCD camera (Photometrics). Image acquisition was performed using Metamorph software (version 7.7.8; Molecular Devices). Cells were bathed in buffer A.
3
Quantitative Live-Cell Imaging with TIRF Microscopy
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An inverted microscope (Nikon Ti-E) in combination with a CFI Apo TIRF 100 × 1.49 NA objective (Nikon) was used. Bright-field and fluorescence images were acquired using an iXon 897 Ultra EMCCD camera (Andor) coupled to an additional 2.0 × lens (Diagnostic Instruments DD20NLT). Phase-contrast imaging was performed with an Infinity 2–5 M camera (Lumenera). To track JF549 bound to HaloTag, a 553 nm laser (SLIM-553, Oxxius, 150 mW) with a power density of 3 kW/cm2 on the sample plane was used in stroboscopic illumination mode with 3 ms laser pulses per 30 or 60 ms camera exposures. The microscope was controlled using the μManager software package. Data acquisition from multiple positions was performed using custom μManager plugins.
4
Single-Particle Tracking of Caveolins in Cells
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Single-particle tracking photoactivated localization microscopy of MCF and MDCK cells transfected with Cav1-mEos2 was performed on the Roper Scientific total internal reflection fluorescence (TIRF) microscope equipped with an iLas2 double-laser illuminator (Roper Technologies), a CFI Apo TIRF 100× (1.49 NA) objective (Nikon), and an Evolve512 delta EMCCD camera (Photometrics). Images were acquired using Metamorph software (version 7.78; Molecular Devices) at 50 Hz, and 16,000 frames were acquired per cell. A 405-nm laser was used to photoconvert mEos2, with simultaneous 561-nm exposure to excite the photoconverted mEos2. For stochastic photoconversion of mEos2 molecules, a low amount (3–5%) of 405-nm laser and 75–80% of 561-nm laser was used. Data analysis was performed as previously described (Bademosi et al., 2017 (link); Kasula et al., 2016 (link)) using PALM-Tracer, a plugin in Metamorph software (Molecular Devices).
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