Stressed and control cells were fixed for at least 30 min in 3.7% formaldehyde. Glass slides were coated with concanavalin A for cell attachment and cells were immersed in Vectashield mounting medium prior imaging. SIM was carried out with a DeltaVision OMX v4 BLAZE (Applied Precision) microscope on an inverted stand, a 4× sCMOS camera (Andor) and a 100×1.4 oil immersion UPlanSApochromat objective (Olympus). API softworx was used for driving the microscope and OMX SI reconstruction was carried out using Centos 4, the OMX SI reconstruction Linux box. A 488 nm DPSS laser and a 561 nm DPSS laser were used to image GFP and mCherry signal, respectively. At least five different Z-stacks (maximum Z-resolution) were recorded for each condition. Cell boundaries if shown (indicated by the white outline in fluorescence microscopy images) were drawn manually.
Deltavision omx v4 blaze
Manufactured by Cytiva
The DeltaVision OMX v4 BLAZE is a high-performance super-resolution microscope system designed for advanced live-cell imaging and high-resolution 3D analysis. The system utilizes structured illumination microscopy (SIM) technology to achieve resolutions beyond the diffraction limit of traditional light microscopes.
Automatically generated - may contain errors
4 protocols using deltavision omx v4 blaze
1
Stress-Induced Cellular Imaging by SIM
2
High-resolution Localization of HA-TbSAS-6
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Cells on the coverslips were coimmunostained with FITC-conjugated anti-HA monoclonal antibody and anti-TbSAS-6 polyclonal antibody. After thorough washing with PBS, cells were incubated with Alexa Fluor594-conjugated anti-rabbit IgG. Slides were examined under a DeltaVision OMX v 4 Blaze microscope (Applied Precision, GE Healthcare) to view the high-resolution localization patterns according to published procedures (34 (link)). The imaging data were then subjected to SI reconstruction and Image Registration using DeltaVision softWoRx software.
3
Super-Resolution Microscopy of Fluorescent Proteins
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Given that both McSA1 and pab27576 gave robust fluorescent signals, we were able to image them by SIM a super-resolution microscopy technique permitting a resolution of ~100 nm in x, y and of ~300 nm in z[44 (link)]. The IHC was done following the same protocol as for the confocal experiments, except that the McSA1 and pab27576 were used at a concentration of 1:1000. Images were taken on a DeltaVision OMX V4 Blaze super-resolution microscope (Applied Precision, GE Healthcare). Reconstructed images were visualized using the Volocity 3D Image Analysis Software (PerkinElmer, USA). Following image reconstruction and registration, serial z planes were assembled to form a 3D model using the Volocity Software. A video showing different rotations and magnifications of the 3D model was made using the video tools from the software (Additional file
1 : Video).
4
Structured Illumination Imaging of Yeast Cells
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For structured illumination, yeast cells were grown to exponential phase and concentrated to an OD of 5. 200 mL cell suspensions expressing Pub1-super folder GFP (sfGFP) and sfGFP alone under an ADH promoter were then subjected to 10-min heat stress using water baths set to 25 C, 37 C, and 42 C, respectively. Immediately after the stress treatment, cells were centrifuged for 1 min at 5,500 rpm, the supernatant was removed, and the cells were resuspended in 100 mL 3.7% formaldehyde in H 2 O. Samples were incubated at room temperature (RT) for 10 min. The fixed cells were washed with H 2 O and mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories) onto concanavalin-A-coated glass coverslips. Cells were imaged with a Deltavision OMX v4 BLAZE (Applied Precision), using a PlanApo N 603, NA 1.42, oil immersion objective (Olympus). sfGFP excitation was with a 488-nm laser. Structured illumination sections of 0.125 mm and 512 3 512 pixels were taken at 95 MHz, 10% 488-nm laser intensity, and 10-ms exposure time.
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