To prepare imaging slides, coverslips of thickness No. 1.5H (0.170 mm ± 0.005 mm) were flamed and smeared with 0.1% Polyethylenimine (PEI) solution. Purified ookinetes or salivary gland sporozoites were fixed with 2%PFA in 1×PBS suspension and allowed to settle onto PEI-treated coverslips for 20 minutes and then probed with indicated antibodies (13.1 mouse monoclonal antibody for ookinetes and CSP antibody for sporozoites) by IFA, stained with DAPI, rinsed in water and mounted onto Vectashield on glass slides before sealing with nail varnish. Super-resolution images were acquired using a Deltavision OMX 3D-SIM System V3 BLAZE from Applied Precision (a GE Healthcare company) equipped with 3 sCMOS cameras, 405, 488, 592.5 nm diode laser illumination, an Olympus Plan Apo N 60 × 1.42NA oil objective, and standard excitation and emission filter sets. Imaging of each channel was done sequentially using three angles and five phase shifts of the illumination pattern as described previously49 (link). The refractive index of the immersion oil (Cargille) was adjusted to 1.516 to minimize spherical aberrations. Sections were acquired at 0.125 μm z steps. Raw OMX data was reconstructed and channel registered in SoftWoRx software version 6.1.3 (Applied Precision, a GE Healthcare company).
Deltavision omx 3d sim system v3 blaze
Manufactured by Cytiva
The Deltavision OMX 3D-SIM System V3 BLAZE is a high-resolution 3D structured illumination microscope (3D-SIM) designed for advanced cellular and molecular imaging. It utilizes structured illumination to achieve 2-3 times higher resolution than conventional widefield fluorescence microscopy.
Automatically generated - may contain errors
4 protocols using deltavision omx 3d sim system v3 blaze
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Super-resolution Imaging of Malaria Parasites
2
Immunofluorescence Imaging of Malaria Parasites
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To prepare imaging slides, coverslips of thickness No. 1.5H (0.170 ± 0.005 mm) were flamed and smeared with 0.1% polyethylenimine solution. Purified ookinetes or salivary gland sporozoites in suspension were fixed with 2% PFA in 1× PBS and allowed to settle onto polyethylenimine‐treated coverslips for 20 min and then probed with the indicated antibodies (13.1 mouse monoclonal antibody for ookinetes and CSP antibody for sporozoites) by immunofluorescence assay, stained with DAPI, rinsed in water, and mounted in Vectashield on glass slides before sealing with nail varnish. Super‐resolution images were acquired using a Deltavision OMX 3D‐SIM System V3 BLAZE (Applied Precision) equipped with three sCMOS cameras, 405‐, 488‐, 592.5‐nm diode laser illumination, an Olympus Plan Apo N 60 × 1.42 NA oil objective, and standard excitation and emission filter sets. Imaging of each channel was done sequentially using three angles and five phase shifts of the illumination pattern as described previously (Gustafsson et al., 2008 ). The refractive index of the immersion oil (Cargille) was adjusted to 1.516 to minimise spherical aberrations. Sections were acquired at 0.125 μm z steps. Raw OMX data were reconstructed and channel registered in SoftWoRx software version 6.1.3 (Applied Precision).
3
Super-resolution Imaging of DNA Repair Proteins
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Confocal imaging for γH2AX, RAD51, RPA, ssDNA (BrdU), BLM, BRCA2, FANCD2, Cyclin A and GFP (FAM35A and C20orf196) was performed as described in39 (link), for RAD51 and Cyclin A the pre-extraction step was omitted and cells were permeabilised for 15min in 0.2% Triton X-100 (Sigma) in PBS after fixation. Super-resolution images were acquired using a Deltavision OMX 3D-SIM System V3 BLAZE (Applied Precision, a GE Healthcare company) equipped with 3 sCMOS cameras, 405, 488, 592.5nm diode laser illumination, an Olympus Plan Apo N 60x 1.42NA oil objective, and standard excitation and emission filter sets. Imaging of each channel was done sequentially using three angles and five phase shifts of the illumination pattern as described58 (link). Sections were acquired at 0.125 μm z steps. Raw OMX data was reconstructed and channel registered in SoftWoRx software version 6.5.2 (Applied Precision, a GE Healthcare company). Voxelwise nearest-neighbour distances were measured for GFP-FAM35A signal relative to 53BP1 signal using a custom script (Butler R) for Fiji (https://github.com/gurdon-institute/OMX-Spatial-Analysis ). The script maps signal volumes using Kapur's maximum entropy thresholding method59 and measures distances using the exact signed 3D Euclidean distance transform with internal distances set to zero for display on the histogram. For all images, scale bars = 10μm.
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Super-resolution Imaging of DNA Repair Proteins
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