The 3 dpf embryonic brain, surrounding lymphatic vessels, and mrc1a+ cells inside the gfap+ labeled brain of Tg(mrc1a:egfp);Tg(gfap:nfsb-mCherry) animals were imaged using light sheet microscopy. Animals were anesthetized with Tricaine, enveloped in 1% low-melting point agarose, and mounted in a 100 μL glass capillary tube (Blaubrand, Germany). Light sheet microscopy was performed using a MuVi-SPIM system (Bruker) equipped with dual detection, 16x water-immersion objective lenses (N.A. 0.8, Nikon) and Orca Flash 4.0 V3 sCMOS cameras (Hamamatsu). Images were captured at 32X magnification using a 2x lens magnification changer. Image processing was performed using LuxControl software (version 3.4.0, Bruker). Further 3D surface reconstructions of the light sheet images of the gfap+ brain and mrc1a+ labeled vessels and cells inside gfap+ brain were created using IMARIS.
Orca flash 4.0 v3 scmos camera
Manufactured by Hamamatsu Photonics
Sourced in Japan
The ORCA-Flash 4.0 V3 is a scientific CMOS (sCMOS) camera developed by Hamamatsu Photonics. It features a 4.2-megapixel image sensor, a high dynamic range, and a fast readout speed. The camera is designed for a wide range of scientific applications that require high-performance imaging capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Sola se u nir light engine, by Lumencor (6 mentions)
Axio observer z1, by Zeiss (6 mentions)
Plan apo λ, by Nikon (5 mentions)
Ti2 e microscope, by Lumencor (3 mentions)
Perfect focus system, by Nikon (3 mentions)
Eclipse ti2 e microscope, by Yokogawa (2 mentions)
Mrd00205, by Nikon (2 mentions)
Ti e microscope, by Lumencor (2 mentions)
Ti e microscope, by Yokogawa (1 mentions)
Csu x, by Oxford Instruments (1 mentions)
Csu w1, by Hamamatsu Photonics (1 mentions)
Microamp optical adhesive film, by Thermo Fisher Scientific (1 mentions)
P96 1.5h n, by Cellvis (1 mentions)
Catalase, by Roche (1 mentions)
Ixon ultra emccd camera, by Oxford Instruments (1 mentions)
Ti2 microscope, by Nikon (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Bp 605 70, by Zeiss (1 mentions)
Ft 395, by Zeiss (1 mentions)
18 protocols using orca flash 4.0 v3 scmos camera
1
Visualizing Brain Lymphatics and Cells
2
Dual-Color Lattice Light Sheet Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were taken using spinning disk microscope with a 100x Apo objective, NA 1.4 (Nikon Ti-E microscope equipped with Yokogawa spinning disk unit CSU-X and an Andor iXon 897 camera).
For lattice light sheet microscopy, 1 M cells were seeded in 6 well plates containing 5 mm cover glasses (#1.5 thickness) and transfected with either 0.1 μg pCDNA3.1-eGFP-SpRng2(1-189) or with 0.5 μg pEGFP-IQGAP1 (# 30112, Addgene) and 0.5 μg pTK93 Lifeact-mCherry. Cells were imaged 16-22 hours post transfection. Cover glasses were mounted on the imaging chamber and DMEM medium was replaced by pre-warmed L-15 imaging medium (cat. no. 11415049, Gibco, Fisher scientific). Imaging was done at 37°C on a 3i second generation lattice-light-sheet microscope with a 0.71 NA LWD WI objective for excitation and a 1.1 NA WI objective for imaging and equipped with 2 Hamamatsu ORCA-Flash 4.0v3 sCMOS cameras for simultaneous dual color imaging providing a 62.5x magnification with 230x230x370 nm (x-y-z) resolution using 488 nm and 561 nm lasers for excitation. 3D volumes were recorded with 0.57 μm step size for 150 planes with 100 ms exposure.
For lattice light sheet microscopy, 1 M cells were seeded in 6 well plates containing 5 mm cover glasses (#1.5 thickness) and transfected with either 0.1 μg pCDNA3.1-eGFP-SpRng2(1-189) or with 0.5 μg pEGFP-IQGAP1 (# 30112, Addgene) and 0.5 μg pTK93 Lifeact-mCherry. Cells were imaged 16-22 hours post transfection. Cover glasses were mounted on the imaging chamber and DMEM medium was replaced by pre-warmed L-15 imaging medium (cat. no. 11415049, Gibco, Fisher scientific). Imaging was done at 37°C on a 3i second generation lattice-light-sheet microscope with a 0.71 NA LWD WI objective for excitation and a 1.1 NA WI objective for imaging and equipped with 2 Hamamatsu ORCA-Flash 4.0v3 sCMOS cameras for simultaneous dual color imaging providing a 62.5x magnification with 230x230x370 nm (x-y-z) resolution using 488 nm and 561 nm lasers for excitation. 3D volumes were recorded with 0.57 μm step size for 150 planes with 100 ms exposure.
3
Imaging Cells with Confocal Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells fixed and stained with DAPI, FOXO1 (Alexa 488) and Phalloidin (Alexa 568) were imaged using a Nikon Eclipse Ti2-E microscope equipped with a Yokogawa confocal scanner unit (CSU-W1), solid state diode lasers (405, 488 and 568 nm) and a Hamamatsu ORCA-Flash4.0 V3 sCMOS camera. Cells were imaged for mean intensity analysis using a 40x objective (CFI Super FLUOR; 0.9 NA) and the higher resolution images were captured with the 60x objective (Plan Apo; 1.40 NA oil). DIC images were collected for a single plane, while fluorescence images were collected every micron for 10 microns (the bottom slices including those below the nucleus and several out of focus planes were excluded from downstream mean intensity analysis; 7 slices were used for the analysis).
4
Differential Interference Contrast Microscopy of Nucleosomes
5
High-Resolution Imaging of SARS-CoV-2 RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We imaged HCR RNA FISH samples on an inverted Nikon Ti2-E microscope equipped with a SOLA SE U-nIR light engine (Lumencor), an ORCA-Flash 4.0 V3 sCMOS camera (Hamamatsu), ×20 Plan-Apo λ (Nikon MRD00205), ×60 Plan-Apo λ (MRD01605) and ×100 Plan-Apo λ (MRD01905) objectives and filter sets for DAPI, Alexa Fluor 488, Alexa Fluor 594 and Atto647N. Our exposure times ranged from 100 ms–200 ms for most of the dyes except for DAPI, for which we used ~50 ms exposures. For experiments in Figs. 2 –4 , we first acquired tiled images in a single z-plane (scan) at ×20 magnification, from which we identified positions containing cells positive for SARS-CoV-2 and returned to those positions to acquire a z-stack at ×60 or ×100 magnification. For large area scans, we used Nikon Perfect Focus to maintain focus across the imaging area.
6
Multiplexed RNA and Protein Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We imaged RNA FISH samples on an inverted Nikon TI-E microscope equipped with a SOLA SE U-nIR light engine (Lumencor), an ORCA-Flash 4.0 V3 sCMOS camera (Hamamatsu), 20X Plan-Apo λ (Nikon MRD00205), 40X Plan-Fluor (MRH00401) and 60X Plan-Apo λ (MRD01605) objectives, and filter sets for DAPI, Cy3, Alexa Fluor 594, and Atto647N. For barcode ClampFISH and barcode HCR, we first acquired tiled images in a single Z-plane (scan) at 20X or 40X magnification, then, after identifying positions containing cells positive for resistant barcodes, we returned to those positions to acquire a Z-stack at 60X magnification. For subsequent rounds of single-molecule RNA FISH and ERK immunofluorescence, we acquired Z-stacks at 60X magnification. For scans, we used a Nikon Perfect Focus system to maintain focus across the imaging area.
7
Multiplexed RNA and Protein Imaging
8
Widefield and dSTORM Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Widefield images were acquired using a Nikon Ti-2 microscope equipped with a 60 × 1.49NA objective and an ORCA-Flash 4.0 v3 sCMOS camera (Hamamatsu, Hamamatsu City, Japan). Images were deconvolved with Nikon Elements by the Richard-Lucy method for 20 iterations. dSTORM experiments were conducted on a Nikon Ti-2 N-STORM microscope equipped with a 100 × 1.49NA oil immersion objective, 488 and 647 nm lasers, and an iXon ultra EMCCD camera (Andor, Oxford Instruments). 60,000–80,000 frames were collected with subcritical inclined excitation and reconstructed in Nikon Elements. dSTORM imaging buffer included glucose oxidase (Sigma, St Louis, Missouri), glucose (Sigma), catalase (Roche, Penzberg, Germany), and β-mercaptoethanol (Sigma)52 , 53 (link). A detailed dSTORM protocol has been described previously.15 (link)
9
Multicolor RNA FISH Imaging Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We imaged HCR RNA FISH samples on an inverted Nikon Ti2-E microscope equipped with a SOLA SE U-nIR light engine (Lumencor), an ORCA-Flash 4.0 V3 sCMOS camera (Hamamatsu), ×20 Plan-Apo λ (Nikon MRD00205), ×60 Plan-Apo λ (MRD01605), and ×100 Plan-Apo λ (MRD01905) objectives and filter sets for DAPI, Alexa Fluor 488, Alexa Fluor 594, and Atto647N. Our exposure times ranged from 100 to 200 ms for most of the dyes except for DAPI, for which we used ∼50-ms exposures. For RNA FISH HCR cell culture experiments in Fig. 1 , we acquired z-stack images using 50- to 100-ms exposure times. For the experiments depicted in Fig. 2 and 4 , we first acquired tiled images in a single z-plane (scan) at ×20 magnification, from which we identified positions containing cells positive for SARS-CoV-2 and returned to those positions to acquire a z-stack at ×60 or ×100 magnification. For large area scans, we used Nikon Perfect Focus to maintain focus across the imaging area. For the single-molecule RNA FISH experiments in Fig. S1 , we acquired z-stack images with 300- to 500-ms exposure times using green fluorescent protein (GFP) and Cy3.
10
Intracellular Brightness and Photostability Measurements
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular brightness and photostability measurements were carried out under Nikon Ti2-E widefield microscope equipped with Spectra III Light Engine (LumenCore), the ORCA-Flash 4.0 V3 sCMOS camera (Hamamatsu), and 20x/0.75 objective lens controlled by NIS Elements software using BFP (excitation 390/22 nm, emission 457/50 nm), GFP (excitation 475/28 nm, emission 535/46 nm), RFP (excitation 555/28 nm, 594/40 nm) channel. Intracellular photostability measurements were performed under continuous wide-field illumination at light power of 4.25 mW/mm2 and 0.91 mW/mm2 in HEK cells and 3.72 mW/mm2 in neurons. For high-quality images of HeLa cells expressing BFPs fusions Olympus FV3000-IX83 inverted confocal microscope was used, equipped with solid-state diode lasers (OBIS, Coherent) at 405 nm, bandpass exciter filter 360–370 nm, dichroic beam splitter DM410 and BA420-460, multi-alkali PMT (2CH Spectral detector) and U Plan S Apo 20x/0.75 and 40x/0.6 objective lenses. For OSER assay, Nikon Ti2-E widefield fluorescence microscope was used with the same optical configuration as previously stated. Cells were evaluated and counted by three researchers independently and blindly.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!