Total internal reflection fluorescence microscopy (TIRF-M) and phase contrast microscopy were performed using a Nikon Eclipse Ti equipped with a Nikon Plan Apo λ 100X 1.45 objective and a Hamamatsu ORCA-Flash4.0 V2 (C11440-22CU) sCMOS camera. Except where specified, fluorescence time-lapse images were collected by continuous acquisition with 1,000 ms exposures. Microscopy was performed in a chamber heated to 37 °C.
Orca flash4.0 v2 c11440 22cu
Manufactured by Hamamatsu Photonics
The ORCA-Flash4.0 V2 C11440-22CU is a scientific-grade CMOS camera designed for a variety of imaging applications. It features a 4-megapixel, back-illuminated CMOS image sensor with low noise and high quantum efficiency. The camera provides fast readout speeds and high sensitivity, making it suitable for applications such as fluorescence microscopy, low-light imaging, and high-speed phenomena capture.
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Lab products found in correlation
6 protocols using orca flash4.0 v2 c11440 22cu
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TIRF and Phase Contrast Microscopy
2
Fluorescent Imaging of Intact Neurons
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Single-Molecule TIRF Microscopy of Halo-Tau
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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.
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TIRF and Phase Contrast Microscopy
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Total internal reflection fluorescence microscopy (TIRF-M) and phase contrast microscopy were performed using a Nikon Eclipse Ti equipped with a Nikon Plan Apo λ 100X 1.45 objective and a Hamamatsu ORCA-Flash4.0 V2 (C11440-22CU) sCMOS camera. Except where specified, fluorescence time-lapse images were collected by continuous acquisition with 1,000 ms exposures. Microscopy was performed in a chamber heated to 37 °C.
5
Structured Illumination Microscopy Imaging
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Microscopic observations were performed by structured illumination microscopy to produce wide-field stacks using an Optigrid module (Leica-microsystems MAAF DM 16000B). All stacks were acquired using an X63 oil N.A. 1.4 objective and a digital CMOS Camera (ORCA-Flash4.0 V2 C11440-22 CU – Hamamatsu) at an optimal resolution such that lateral and axial resolution were respectively XY = 0.103 µm and Z = 0.2 µm. For better resolution, some confocal images were acquired with a Zeiss LSM 800 with an X63 oil N.A. 1.4 objective and a voxel size XY = 0.60 µm and Z = 0.2 µm. Final image numbers are given for each dataset in Table 1 .
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Fluorescent Imaging of Intact Neurons
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Neurons were cultured and transfected as described above, but were left intact (unpatched). They were imaged with an ORCA-Flash4.0 V2 C11440-22CU (Hamamatsu) scientific CMOS camera as described above, except that we used a 480/20-nm excitation filter (Chroma HQ480/20x).
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