Super-resolution 3D-SIM images were acquired on a DeltaVision OMX V4 (GE Healthcare) equipped with a 60x/1.42 NA PlanApo oil immersion objective (Olympus), 405, 488, 568 and 642 nm solid state lasers (100 mW) and sCMOS cameras (pco.edge). Image stacks of 1 µm with 0.125 µm thick z-sections and 15 images per optical slice (3 angles and 5 phases) with were acquired using immersion oil with a refractive index 1.524. Images were reconstructed using Wiener filter settings of 0.005 and optical transfer functions (OTFs) measured specifically for each channel with SoftWoRx 6.1.3 (GE Healthcare) to obtain super-resolution images with a twofold increase in resolution both axially and laterally. Images from different color channels were registered using parameters generated from a gold grid registration slide (GE Healthcare) and SoftWoRx 6.1.3 (GE Healthcare).
Softworx 6
Manufactured by GE Healthcare
Sourced in United States
SoftWoRx 6.5.2 is a software platform for image analysis and visualization. It provides a comprehensive set of tools for processing, analyzing, and displaying microscopy data.
Automatically generated - may contain errors
Lab products found in correlation
Gold grid registration slide, by GE Healthcare (4 mentions)
Deltavision omx v4, by GE Healthcare (4 mentions)
Deltavision omx sr, by GE Healthcare (3 mentions)
Uplansapo objective, by Olympus (3 mentions)
Deltavision omx v3, by GE Healthcare (3 mentions)
Uplansapo, by Olympus (3 mentions)
Deltavision omx system, by GE Healthcare (2 mentions)
Deltavision elite, by GE Healthcare (2 mentions)
Tetraspeck bead, by Thermo Fisher Scientific (2 mentions)
Deltavision omx sr microscope, by GE Healthcare (2 mentions)
Deltavision omx blaze, by GE Healthcare (2 mentions)
Scmos camera, by Olympus (1 mentions)
Planapo oil immersion lens, by Olympus (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Plan apochromat objective lens, by Olympus (1 mentions)
Mouse anti acetylated tubulin, by Merck Group (1 mentions)
Axioskop 2 plus, by Zeiss (1 mentions)
Mrm camera, by Zeiss (1 mentions)
Deltavision omx v4 blaze system, by GE Healthcare (1 mentions)
33 protocols using softworx 6
1
Super-resolution 3D-SIM Imaging Protocol
2
Super-resolution Imaging Acquisition and Analysis
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Super-resolution 3D-SIM images were acquired on a DeltaVision OMX V4 (GE Healthcare) equipped with a 60×/1.42 NA PlanApo oil immersion lens (Olympus), 405-, 488-, 568-, and 642-nm solid-state lasers and sCMOS cameras (pco.edge). Image stacks of 5–6 µm with 0.125-µm thick z-sections and 15 images per optical slice (3 angles and 5 phases) which were acquired using immersion oil with a refractive index 1.518. Images were reconstructed using Wiener filter settings of 0.003 and optical transfer functions measured specifically for each channel with SoftWoRx 6.1.3 (GE Healthcare) to obtain super-resolution images with a twofold increase in resolution both axially and laterally. Images from different color channels were registered using parameters generated from a gold grid registration slide (GE Healthcare) and SoftWoRx 6.1.3 (GE Healthcare).
3
Super-resolution Imaging of Cellular Structures
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Super-resolution 3D-SIM images were acquired on a DeltaVision OMX V4 (GE Healthcare) equipped with a 60×/1.42 NA PlanApo oil immersion lens (Olympus), 405, 488, 568 and 642 nm solid state lasers and sCMOS cameras (pco.edge). Image stacks of 10 μm with 0.125 μm thick z-sections and 15 images per optical slice (three angles and five phases) were acquired using immersion oil with a refractive index of 1.514. Images were reconstructed using Wiener filter settings of 0.008 for the 405 channel, 0.006 for the 528 channel, and 0.003 for the 508 and 683 channels. Optical transfer functions (OTFs) were measured specifically for each channel with SoftWoRx 6.1.3 (GE Healthcare) to obtain super-resolution images with a 2-fold increase in resolution both axially and laterally. Images from different color channels were registered using parameters generated from a gold grid registration slide (GE Healthcare) and SoftWoRx 6.5.2 (GE Healthcare). Volume renderings were generated in ImageJ v1.50g.
4
Super-resolution Structured Illumination Microscopy
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Superresolution 3D structured illumination microscopy images were acquired on a DeltaVision OMX SR (GE Healthcare) equipped with a 60×/1.42 NA PlanApo oil immersion lens (Olympus), 405-, 488-, 568-, and 640-nm solid-state lasers, and sCMOS cameras (pco.edge). Image stacks of 5–6 µm with 0.125-µm-thick Z sections and 15 images per optical slice (three angles and five phases) were acquired using immersion oil with a refractive index 1.518. Images were reconstructed using Wiener filter settings of 0.003 and optical transfer functions measured specifically for each channel with SoftWoRx 6.5.1 (GE Healthcare) to obtain superresolution images with a twofold increase in resolution both axially and laterally. Images from different color channels were registered using parameters generated from a gold grid registration slide (GE Healthcare) with the routines available in SoftWoRx 6.5.1.
5
Superresolution Imaging Using 3D-SIM
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Superresolution 3D structured illumination microscopy (SIM) images were acquired on a DeltaVision OMX SR (GE Healthcare) equipped with a 60× 1.42 NA PlanApo oil immersion lens (Olympus); 405-, 488-, 568-, and 640-nm solid state lasers; and sCMOS cameras (pco.edge). Image stacks with 0.125-µm-thick z-sections, and 15 images per optical slice (three angles and five phases) were acquired using immersion oil with a refractive index 1.516. Images were reconstructed using Wiener filter settings of 0.003 and optical transfer functions (OTFs) measured specifically for each channel with SoftWoRx 6.5.2 (GE Healthcare) to obtain superresolution images with a twofold increase in resolution both axially and laterally. Images from different color channels were registered using parameters generated from a gold grid registration slide (GE Healthcare) and SoftWoRx 6.5.2 (GE Healthcare).
6
Structured Illumination Microscopy of Axonemes
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Immunofluorescence microscopy was performed as described previously (Zhao et al., 2019 (link)). Briefly, the slides with attached intact axonemes were treated overnight at 4°C with blocking buffer containing the diluted primary antibody (mouse anti-acetylated tubulin, Sigma-Aldrich, clone 6-11B-1, 1:1,000). On the next day, the slides were washed four times over 1 h with blocking buffer, and then treated for 1 h with blocking buffer containing the secondary antibody (F(ab′)2-goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568, Invitrogen, A11019, 1:1,000) and Streptavidin (Alexa Fluor 488, Invitrogen, A32354, 1:200). The samples were mounted and examined as before (Zhao et al., 2019 (link)) using structured illumination microscopy performed on a DeltaVision OMX system (GE Healthcare) with a 1.42 NA 60× Plan-Apochromat objective lens (Olympus) and immersion oil with a refraction index of 1.512. Structured illumination microscopy images were reconstructed with softWoRx 6.1.3 (GE Healthcare). Capture times and adjustments were the same for images with the same antibody. Image brightness and contrast were adjusted using ImageJ (National Institutes of Health).
7
Flagella Imaging and Measurement Techniques
8
Super-Resolution Imaging of HCMV in LifeAct-GFP-NLS HFFs
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LifeAct-GFP-NLS-expressing HFFs were infected with WT HCMV (MOI of 3) and fixed at RT in 3.7% formaldehyde-DPBS at 24 hpi. 3D-SIM data were collected at the Cell Biology Microscopy Facility at Harvard Medical School using a DeltaVision OMX V4 Blaze system (GE Healthcare) equipped with a 60×/1.42-numerical-aperture (NA) Plan Apo oil immersion objective lens (Olympus), 488 solid state laser, and a pco.edge 5.5 scientific complementary metal oxide semiconductor (sCMOS) camera. z-stacks were acquired with a z-step of 125 nm and with 15 raw images per plane (five phases, three angles). Spherical aberration was minimized using immersion oil matching. Super-resolution images were computationally reconstructed from the raw data sets with a channel-specific measured optical transfer function (OTF) and a Wiener filter constant of 0.001 to 0.002 using softWoRx 6.1.3 (GE Healthcare).
9
Immunofluorescence Staining of Prometaphase Cells
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Cells were plated on glass ø10 mm coverslips (#1.5H) > 24 hr. before fixation. Cells were pre-extracted with 37 C PHEM-TX100 (PIPES, HEPES, EGTA, 2 mM MgCl2, 0.2% Triton X-100) for 1 min. at room temperature after which cells were fixed in freshly prepared 4% paraformaldehyde/PBS (pH7.2) for 5 mins. at room temperature. Cells were washed with PBS and blocked in 3% BSA/ PBS (blocking buffer) for 30 min. at room temperature. Primary antibody dilutions were made in blocking buffer and coverslips were incubated at 4 C overnight inverted. Next, cells were washed in blocking buffer + 0.1% Triton X-100 followed by 1 hr. incubation with secondary antibodies diluted in blocking buffer. Coverslips were washed in blocking buffer and a final wash step in ddH2O before mounting onto glass slides using Prolong Gold antifade (Thermo Fisher Scientific). Images were acquired on a DeltaVision Elite (GE Healthcare) with a 100x 1.40NA UPlanSApo objective (Olympus) and SoftWorx 6.0 software (GE Healthcare). Images were acquired with 0.15mm Z-intervals and then deconvolved and maximum intensity projections were made using SoftWorx. Early prometaphase cells were selected based on the shape of DAPI signal.
10
Structured Illumination Microscopy of E. coli
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E. coli cells were pelleted and washed with phosphate-buffered saline and resuspended to an appropriate density for microscopy. Cell suspensions were incubated on an agarose pad for 30 min and mounted in VECTASHIELD Antifade Medium (Vector Laboratories). Samples were imaged on a DeltaVision OMX V4 microscope with the Blaze 3D structured illumination module equipped with a 60× oil objective [numerical aperture (NA), 1.42]. Illumination was performed with a 642-nm laser with the emitted light collected through a 683/40 band-pass filter. For each SIM image, nine axial planes were captured with a spacing of 125 nm, and the data were reconstructed with the SoftWoRx 6.0 software package (GE Healthcare).
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