For performing single-molecule experiments, TIRF microscopy was performed using an Olympus excellence cell TIRF microscope equipped with 561-nm (200-mW) laser (Olympus, Tokyo, Japan) and a back-illuminated electron-multiplied charge-coupled device camera (C9100-13; Hamamatsu, Hamamatsu City, Japan). Fast single-molecule tracking used a digital complementary metal–oxide–semiconductor camera (ORCA-Flash4.0 V2 C11440-22CU; Hamamatsu). A 150× magnification objective with NA 1.45 (UAPON 150×/1.45; Olympus) was used for TIR illumination. The emitted light from the sample was filtered using a quad-band bandpass filter (FF01 446/523/600/677; Semrock, Rochester, NY). The microscope was enclosed in an incubation chamber maintained at 37°C and 5% CO2 (Olympus-PeCon). Localization and trajectory reconstruction were carried out as previously described (Janning et al., 2014 (link)). The theoretical localization precision of Halo-tau was 16.7 ± 0.2 nm. For further analysis, a mask was placed over the cell processes, and trajectories outside of this mask were removed.
Ff01 446 523 600 677
Manufactured by IDEX Corporation
The FF01-446/523/600/677 is a series of laser bandpass filters produced by IDEX Corporation. These filters are designed to isolate specific wavelength ranges from a broadband light source. The core function is to selectively transmit a desired wavelength while blocking unwanted wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
Di01 r405 488 561 635, by IDEX Corporation (2 mentions)
C9100 13, by Hamamatsu Photonics (1 mentions)
Uapon 150 1, by Olympus (1 mentions)
Orca flash4.0 v2 c11440 22cu, by Hamamatsu Photonics (1 mentions)
Ilas2, by Teledyne (1 mentions)
G 0543, by Merck Group (1 mentions)
C40 100mg, by Merck Group (1 mentions)
M6500, by Merck Group (1 mentions)
Uapon, by Olympus (1 mentions)
4 protocols using ff01 446 523 600 677
1
Single-Molecule TIRF Microscopy of Halo-Tau
2
Lattice Light Sheet Microscopy of Muller Organotypic Slices
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Muller organotypic slices were imaged using a custom-built lattice light sheet microscope (LLSM), which was a replica of the setup described previously24 (link). We used a dithered square lattice with an inner and outer NA of 0.44 and 0.55, respectively, giving a light sheet of constant thickness of approximately 0.6 μm over a length of 15 μm. Laser power incident on the illumination objective was controlled via an AOTF, and set to ~60 μW for 10 Hz and ~400 μW for 80 Hz acquisitions. The detection objective had an NA of 1.1 and a working distance of 2 mm. 3D volumes were acquired by a horizontal piezoelectric translator (step size 200 nm). A multiband emission filter (Semrock FF01-446/523/600/677) was used to block the light from the excitation lasers.
3
Multimodal Microscopy Imaging Protocol
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Epi-fluorescent images were acquired using a Nikon inverted Ti-S microscope equipped with a 100× oil immersion objective (CFI Plan Fluor ADH, NA 1.30; Nikon). Samples were illuminated using a light engine (Lumencor), and images were acquired using a charge-coupled device (CCD) camera (Coolsnap HQ; Photometrics). TIRF time-lapse images were acquired using a Nikon inverted Ti-S microscope equipped with a 100× oil immersion objective (CFI Apochromat TIRF, NA 1.49; Nikon) and an electron-multiplying CCD camera (Evolve 512; Photometrics) or sCOMS camera (Prime95B; Photometrics). Excitation laser (iLas2; Roper Scientific) was supplied at wavelengths of 488 nm (100 mW), 561 nm (100 mW), or 642 nm (100 mW) reflected from a multi-band dichroic mirror (Di01-R405/488/561/635; Semrock). The emitted light was filtered through multi-band emission filter (FF01-446/523/600/677; Semrock).
4
Single-Molecule TIRF Imaging Protocol
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The imaging buffer used was 10 mM tris containing 100 mM cysteamine hydrochloride (Sigma-Aldrich, M6500), glucose oxidase (4.0 mg/ml; Sigma-Aldrich, G0543), catalase (0.57 mg/ml; Sigma-Aldrich, C40-100MG), and 10% glucose (Sigma-Aldrich, 49158-1KG-F). pH was adjusted with 0.5 M NaOH to pH 8.3 to 8.5. Chamber wells were completely filled with buffer and sealed bubble free (air free) by a regular coverslip. Images were recorded using an inverted total internal reflection fluorescence (TIRF) microscope (Olympus IX-71) equipped with an oil-immersion objective lens (Olympus, UApoN, ×100 magnification, NA 1.49, TIRF) and a 647-nm laser (PhoxX 647, 140 mW, Omicron Laserage, Germany). A quad-edge dichroic beam splitter (Di01-R405/488/561/635, Semrock) and a quad-band excitation filter (FF01-446/523/600/ 677, Semrock) were used to remove the laser light before imaging the fluorescence on an EMCCD camera cooled to −50°C (DU-885-CS0-#VP, Andor) with an effective pixel size of 80 nm. Raw movies typically contained 3000 to 4000 images recorded at 30 to 90 Hz. Images were analyzed using rapidSTORM and custom-written routines in Matlab.
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