Cells were grown in respective media, centrifuged (20817 g for 2 min). and were mounted on agarose (2% w/v) pads for microscopy. Images were taken in z-sections of 0.2 µm step size using a Delta Vision microscope (GE Healthcare) fitted with 100×1.4 NA objective and Cool-SNAP HQ2 camera. Images were acquired using FITC and TRITC filters. Image processing and quantification were performed with SoftWorx (GE Healthcare) and Fiji (NIH) software. For colocalization analysis, images were de-convolved and background subtracted, and colocalized entities were either quantified manually by using the cell counter plugin or automatically by using the colocalization highlighter plugin in Fiji for all z-sections. Representative colocalizations events quantified manually were also confirmed by line profile and colocalization measurement options in SoftWorx (GE Healthcare). All the supplementary movies are of a single z-plane.
Softworx
Manufactured by GE Healthcare
Sourced in United States
SoftWoRx is a software suite developed by GE Healthcare for the analysis and processing of microscopy data. It provides a range of tools for image acquisition, visualization, and quantification, enabling researchers to gain insights from their microscopy experiments.
Automatically generated - may contain errors
Lab products found in correlation
Deltavision elite, by GE Healthcare (9 mentions)
Leibovitz s l 15, by Thermo Fisher Scientific (9 mentions)
Deltavision microscope, by GE Healthcare (5 mentions)
Softworx software, by GE Healthcare (4 mentions)
Deltavision omx v4 blaze microscope, by GE Healthcare (3 mentions)
Prism 9, by GraphPad (3 mentions)
Deltavision elite system, by GE Healthcare (3 mentions)
Oil planapo n uis2 objective, by Olympus (2 mentions)
Deltavision omx v4 blaze, by GE Healthcare (2 mentions)
1.4 na objective, by Olympus (2 mentions)
Deltavision imaging system, by GE Healthcare (2 mentions)
35 mm glass bottom plate, by MatTek (2 mentions)
Concanavalin a (cona), by Merck Group (2 mentions)
1.4 na oil immersion objective, by Olympus (2 mentions)
Deltavision omx v4 microscope, by Olympus (2 mentions)
Plan super apochromat oil immersion objective, by Olympus (2 mentions)
X83 inversed microscope, by Olympus (2 mentions)
No 1.5 glass bottomed dish, by MatTek (2 mentions)
Eight well dishes, by Ibidi (2 mentions)
Deltavision elite microscope, by GE Healthcare (2 mentions)
82 protocols using softworx
1
Microscopy Imaging of Subcellular Colocalization
2
Quantitative Analysis of Nascent Transcription
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Visualization of nascent transcription by 5-ethynyl-uridine (EU) labeling post-UV was performed as described [42 (link)]. Briefly, siRNA-transfected cells grown on coverslips were mock-treated or irradiated with UV (6 J/m2). Cells were allowed to recover for either 3 or 24 h and pulse-labelled with 400 μM of EU for 1 h prior to harvesting. In all cases, 24 h prior to the RSR assay, cells were grown in complete media containing 1% FBS to favor incorporation of EU. Labeled cells were processed as described in the above Flow cytometry section except that the concentration of Alexa647-azide was increased to 10 μM in the Click-iT reaction, and DAPI staining was performed in an analysis buffer that does not contain RNase. Microscopy was performed using a DeltaVision fluorescence microscope equipped with SoftWorx (GE Healthcare). Images were analyzed using a custom Python 3.6 script. Nuclei were segmented with DAPI staining channel images using Otsu’s thresholding, followed by extraction of the average fluorescence intensity per cell in the other channel.
3
High-Resolution Microscopy Imaging Protocol
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Tissue sections were imaged using x40 objective on either a Deltavision restoration microscope with a Photometrics CoolSnap QE camera (GE Healthcare, IL) (images in Fig. 2 ), or the InCell 2200 (GE Healthcare, IL) (images in Fig. 5 ). Images were then deconvolved and stitched using either SoftWoRx (version 6.1.3) for Deltavision images, or InCell analyser software for InCell images (GE Healthcare, IL). All subsequent image analysis was performed using Fiji (Madison version).
In some cases multiple Z-planes were imaged using a x60 objective on the Deltavision System with 0.1 um z-spacing (Transmitted/Founder HIV images in Fig.6e and Supplementary Fig. 3 ). Huygens Professional 18.10 (Scientific Volume Imaging, The Netherlands, http://svi.nl ) CMLE algorithm, with SNR:20 and 40 iterations, was used for deconvolution of Z-stacks. XY, XZ, YZ projections were generated using the Huygens Orthoslicer feature.
In some cases multiple Z-planes were imaged using a x60 objective on the Deltavision System with 0.1 um z-spacing (Transmitted/Founder HIV images in Fig.
4
Quantifying Ser2P Accumulation at Transgenes
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Immunostaining used a 1:1,000 dilution of rabbit anti-RNA polII COOH-terminal domain repeat YSPTSPS (phospho S2) antibody (Abcam, ab193468) followed by a 1:500 dilution of secondary anti-rabbit Alexa Fluor 647 (Invitrogen, A-21244). To measure Ser2P accumulation adjacent and over the transgene, projected images of the Z-stacks showing the transgene were generated from conservative, iterative deconvolved (Softworx, GE Healthcare) images. Using ImageJ (National Institutes of Health), integrated intensity measurements were made within a manually defined region of interest surrounding the Ser2P foci near the transgene. Background integrated intensity from a nearby region of the same area was subtracted from these measurements.
5
Z-stack imaging via Deltavision Elite
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Image acquisition: For each timepoint, Z-stacks were acquired on a Deltavision Elite (GE Life Sciences). Samples were illuminated through a 60x/1.42NA objective lens with excitation light passing through a bandpass filter of 475/28. Fluorescence emission was collected through a bandpass filter of 525/48 and sampled on an Edge 5.5 sCMOS camera (PCO) at 108nm and 200nm in the lateral and axial axes respectively. To minimize spherical aberration, the immersion oil refractive index was matched until point spread functions from within the sample itself were approximately symmetrical as described by Hiraoka et al. Biophys, 1990. Z-stacks were deconvolved using the constrained iterative algorithm with an appropriately matched optical transfer function in SoftWorx (GE Healthcare).
6
Live-cell Analysis of Mitotic Regulators
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Live-cell analysis was performed on a Deltavision Elite system using a × 40 oil objective (GE Healthcare). Cells were seeded in eight-well Ibidi dishes (Ibidi) in advance and before filming, the media was changed to Leibovitz's L-15 (Life Technologies). Appropriate channels were recorded for 18 h and data analysed using Softworx (GE Healthcare). MEFs were transfected with Bub1-Venus constructs by electroporation using the Neon Transfection System (Invitrogen) according to manufacturer's instruction. A total 1 μM 4-OHT and 200 nM taxol were added 24 and 2 h before filming, respectively. Statistical analysis was done using Prism software.
7
Quantifying Fluorescent Protein Intensity at Subcellular Structures
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Acquired microscopy images were deconvolved using the SoftWorx package (GE Healthcare Life Sciences). Projected images are used for image display. Display of single optical sections is specified in figure legend (Fig 3 ). We used the SoftWorx measuring tools to determine the fluorescence intensity of line scans. Pixel intensities were plotted along the line as shown in Figs 1D, 1E and 3B . To determine the Tub4-RFP intensity at the SPB (Figs 7D and S4B ), we defined a 6 x 6 pixel area as the position of SPB. The mean background intensity was subtracted from the region of interest to yield the net intensity of Tub4-RFP.
8
3D-SIM Imaging Data Acquisition and Reconstruction
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Acquire 3D-SIM dataSingle molecule according to manufacturer’s guidelines and good imaging practices, balancing signal–noise and bleaching while correcting for spherical aberration [22 (link), 23 (link)]. Check raw data with the open source ImageJ plugin SIMcheck (Fig. Representative output from SIMcheck. For detailed explanation of these plots and statistics see [18 (link)]
Perform image reconstruction using commercial software that accompanies the instrument (in our case, SoftWORX by GE for the OMX V3) or an open-source alternative, such as fairSIM [24 (link)]. For multichannel imaging you will need to register channels using Tetraspec beads data and an appropriate Image registration software.
Check the quality of reconstruction using SIMcheck (Fig.
9
3D-SIM Imaging of mCherry-MamY in Cells
10
Super-Resolution Imaging of Synaptic Proteins
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Targets were selected based on the SynCAM 1 signal. Three-dimensional SIM images were acquired with the OMX V4 Blaze system (GE Healthcare, UK), using the 405 nm, 488 nm, 568 nm, and 647 nm laser lines, a 60× 1.42 N.A. oil objective (Olympus), an immersion oil with a refractive index of 1.518 and standard emission filters at 125 nm z-sectioning. Multicolour registration with an error below 40 nm was done using 100 nm fluorescent beads (TetraSpeck, T7284, Thermo Fisher Scientific). Images were acquired with the DeltaVision OMX acquisition software (GE Healthcare), and images were reconstructed with softWoRx (GE Healthcare). Parameters for the reconstruction can be found in the Supporting information (S1 Files ). The quality of 3D SIM reconstructions was tested with SIMcheck [54 (link)]. The superresolution channels 642, 568, and 488 show a good signal-to-noise ratio and no signs of hexagonal artefacts. Fast Fourier transformed images uncover a high amount of information below the diffraction limit. Due to the limited brightness and stability of the Alexa Fluor 405 dye, the signal-to-noise ratio and resolution in the 405 nm channel were limited. We thus decided to use this channel only for vGlut1 as a reference for the segmentation of mature synapses and not as a structural superresolution readout. An image acquisition parameter log file is included in S1 Files .
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