Orca flash 4.0 v3 scmos camera

Manufactured by Nikon

The ORCA-Flash 4.0 V3 sCMOS camera is a high-performance scientific imaging device produced by Nikon. It features a large sensor with a resolution of 4.2 megapixels and a high frame rate of up to 100 frames per second. The camera utilizes sCMOS (scientific Complementary Metal-Oxide Semiconductor) technology to provide low noise, high quantum efficiency, and a wide dynamic range.

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Lab products found in correlation

Sola se u nir light engine, by Lumencor (5 mentions) Perfect focus system, by Nikon (3 mentions) Ti e inverted fluorescence microscope, by Lumencor (3 mentions) Plan fluor 10x 0, by Nikon (1 mentions)

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ORCA Flash 4.0 (13 citations) SCMOS camera (6 citations) Flash 4.0 camera (2 citations)

5 protocols using orca flash 4.0 v3 scmos camera

Single-Molecule RNA FISH Imaging Protocol

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To image single-molecule RNA FISH, nuclei, and colorimetric dye signal, we used a Nikon TI-E inverted fluorescence microscope equipped with a SOLA SE U-nIR light engine (Lumencor), a Hamamatsu ORCA-Flash 4.0 V3 sCMOS camera, and 4X Plan-Fluor DL 4XF (Nikon #MRH20041/MRH20045), 10X Plan-Fluor 10X/0.30 (Nikon #MRH10101) and 60X Plan-Apo λ (#MRD01605) objectives. We used the following filter sets to acquire signal from different fluorescence channels: 31000v2 (Chroma) for DAPI, 41028 (Chroma) for Atto 488, SP102v1 (Chroma) for Cy3, 17 SP104v2 (Chroma) for Atto 647N, and a custom filter set for Alexa Fluor 594. We tuned the exposure times depending on the dyes used (Cy3, Atto 647N, Alexa Fluor 594, and DAPI). For large scans, we used a Nikon Perfect Focus system to maintain focus across the imaging area. For imaging RNA FISH signal at high magnification (≥60X), we acquired z-stacks of multiple Z-planes and used the maximum intensity projection to visualize the signal.

Jain N., Goyal Y., Dunagin M.C., Cote C.J., Mellis I.A., Emert B., Jiang C.L., Dardani I.P., Reffsin S, & Raj A. (2023). Retrospective identification of intrinsic factors that mark pluripotency potential in rare somatic cells. bioRxiv.

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Imaging RNA FISH and Nuclei with Advanced Microscopy

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To image RNA FISH and nuclei signal, we used a Nikon TI-E inverted fluorescence microscope equipped with a SOLA SE U-nIR light engine (Lumencor), a Hamamatsu ORCA-Flash 4.0 V3 sCMOS camera, and 4X Plan-Fluor DL 4XF (Nikon MRH20041/MRH20045), 10X Plan-Fluor 10X/0.30 (Nikon MRH10101) and 60X Plan-Apo λ (MRD01605) objectives. We used the following filter sets to acquire different fluorescence channels: 31000v2 (Chroma) for DAPI, 41028 (Chroma) for Atto 488, SP102v1 (Chroma) for Cy3, 17 SP104v2 (Chroma) for Atto 647N, and a custom filter set for Alexa 594. We tuned the exposure times depending on the dyes used (Cy3, Atto 647N, Alexa 594, and DAPI). For large scans, we used a Nikon Perfect Focus system to maintain focus across the imaging area

Reffsin S., Miller J., Ayyanathan K., Dunagin M.C., Jain N., Schultz D.C., Cherry S, & Raj A. (2023). Single cell susceptibility to SARS-CoV-2 infection is driven by variable cell states. bioRxiv.

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Tracking Drug Resistance Mechanisms in Melanoma

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Drug-naive WM989 A6-G3s were transduced with unique barcodes as described above. Cells were plated in 6-well plates at a density of 100,000 cells per well. One plate was fixed in formaldehyde after 24 h using a protocol described above and the second plate was treated with medium containing 1 μM PLX after 24 h. This drug treatment was continued for three weeks which was enough time for resistant colonies to form. This second plate containing resistant populations was fixed in formaldehyde. Wells were imaged on a Nikon TI-E inverted fluorescence microscope equipped with a SOLA SE U-nIR light engine (Lumencor), a Hamamatsu ORCA-Flash 4.0 V3 sCMOS camera, and 10× Plan-Fluor 10×/0.30 (Nikon MRH10101). Images were analysed using the custom Raj Lab image processing software NimbusImage via the CellPose tool^{67 (link)}. Average intensity for each of the cells was calculated also using NimbusImage.

Chao D.C., Scheinhorn D.J, & Stearn-Hassenpflug M. (1998). Impact of renal dysfunction on weaning from prolonged mechanical ventilation. Critical Care, 1(3), 101-104.

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Multi-Modal Imaging of RNA FISH and Nuclei

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To image RNA FISH and nuclei signal, we used a Nikon TI-E inverted fluorescence microscope equipped with a SOLA SE U-nIR light engine (Lumencor), a Hamamatsu ORCA-Flash 4.0 V3 sCMOS camera, and 4× Plan-Fluor DL 4XF (Nikon MRH20041/MRH20045), 10× Plan-Fluor 10×/0.30 (Nikon MRH10101) and 60× Plan-Apo λ (MRD01605) objectives. We used the following filter sets to acquire different fluorescence channels: 31000v2 (Chroma) for DAPI, 41028 (Chroma) for Atto 488, SP102v1 (Chroma) for Cy3, 17 SP104v2 (Chroma) for Atto 647N, and a custom filter set for Alexa 594. We tuned the exposure times depending on the dyes used (Cy3, Atto 647N, Alexa 594, and DAPI). For large tiled scans, we used a Nikon Perfect Focus system to maintain focus across the imaging area. For imaging RNA FISH signals in tissue sections, we acquired

z

-stacks (three positions) at 60× magnification, and used maximum intensity projection to visualize the signal. For bright-field imaging of resistant colonies, we used a Nikon Eclipse TS2-FL with an Imagingsource DFK 33UX252 camera and 4× Plan-Fluor 4×/0.13 (Nikon MRH20041) objective. For time-lapse imaging of the emergence of drug-resistant colonies, we used an IncuCyte S3 Live Cell Imaging Analysis System (Sartorius) with a 4× objective on WM989 A6-G3 tagged with an mCherry nuclear reporter (H2B–mCherry).

Chao D.C., Scheinhorn D.J, & Stearn-Hassenpflug M. (1998). Impact of renal dysfunction on weaning from prolonged mechanical ventilation. Critical Care, 1(3), 101-104.

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Multicolor RNA FISH Imaging of Cardiac Cells

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We imaged RNA FISH samples on a Nikon TI-E inverted fluorescence microscope equipped with a SOLA SE U-nIR light engine (Lumencor), a Hamamatsu ORCA-Flash 4.0 V3 sCMOS camera, and × 4 Plan-Fluor DL 4XF (Nikon MRH20041/MRH20045), × 20 Plan-Apo λ (Nikon MRD00205), and × 60 Plan-Apo λ (MRD01605) objectives. As multiple layers of cells are generated through cardiac directed differentiation, we acquired z-stacks (0.5 μm spacing between slices) encompassing multiple complete cells at × 60 magnification. To acquire different fluorescence channels, we used the following filter sets: 31000v2 (Chroma) for DAPI, 41028 (Chroma) for Atto 488, SP102v1 (Chroma) for Cy3, and 17 SP104v2 (Chroma) for Atto 647N, and a custom filter set for Alexa 594. We tuned the exposure times depending on the dyes used: 500 ms for probes in Cy3, Atto 647N, and Alexa 594 and 10 ms for DAPI probes. We also acquired images in the Atto 488 channel with a 200 ms exposure as a marker of autofluorescence.
We used the same microscope setup to take × 4 magnification brightfield short videos demonstrating the prevalence of contractile activity prior to fixation of cells for RNA FISH.

Jiang C.L., Goyal Y., Jain N., Wang Q., Truitt R.E., Coté A.J., Emert B., Mellis I.A., Kiani K., Yang W., Jain R, & Raj A. (2022). Cell type determination for cardiac differentiation occurs soon after seeding of human-induced pluripotent stem cells. Genome Biology, 23, 90.

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