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 .
1.42 n a oil objective
Manufactured by Olympus
Sourced in United Kingdom
The 60× 1.42 N.A. oil objective is a high-magnification microscope objective designed for use with oil immersion techniques. It has a numerical aperture of 1.42, which allows for the capture of a large amount of light and enables high-resolution imaging. The objective is suitable for a variety of microscopy applications that require detailed, high-magnification observations.
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Lab products found in correlation
Softworx, by GE Healthcare (1 mentions)
Tetraspeck, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Softworx suite, by GE Healthcare (1 mentions)
Deltavision elite system, by GE Healthcare (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Eclipse ti2 microscope, by Nikon (1 mentions)
Softworx software, by Cytiva (1 mentions)
Fv1000, by Olympus (1 mentions)
Tetraspeck microsphere, by Thermo Fisher Scientific (1 mentions)
Prism 6.0 for mac os x, by GraphPad (1 mentions)
6 protocols using 1.42 n a oil objective
1
Super-Resolution Imaging of Synaptic Proteins
2
Yeast Heat Shock RNA Visualization
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Yeast strains carrying integrated GFP reporters were grown in selective media overnight at room temperature into log phase and shifted directly to 37°C for 3 h. After temperature shift, cells were fixed for 15 min using 4% paraformaldehyde, and poly(A)-RNA was detected using 2 ng of TYE 563–labeled LNA oligo-dT probe (Exiqon) and 4 ng of Quasar 670–labeled ITS1 probe (Biosearch Technologies) by FISH (Cole et al., 2002 (link)). After the final wash, slides were dipped in 100% ethanol for 10 s and air dried, mounting medium with DAPI was then applied to each sample, and a coverslip was affixed. Imaging was performed on a DeltaVision Elite microscope system equipped with a front-illuminated sCMOS camera driven by Softworx 6 using an Olympus 60×/1.42 NA oil objective. Image analysis was performed in FIJI (Schindelin et al., 2012 (link)).
3
Immunofluorescence Imaging of FOXJ1
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Cells were grown on 12 mm glass coverslips as described above and fixed for 10 min in either 4% paraformaldehyde (PFA) (for FOXJ1) at RT or 100% ice-cold methanol at −20°C. The samples were blocked in PBS with 0.2% Triton X-100 and 10% FBS before incubation with the primary and secondary antibodies. The cells were counterstained with DAPI (10 μg ml−1; Sigma-Aldrich) and mounted in ProLong Gold Antifade (Invitrogen). Immunofluorescence images were collected using a Deltavision Elite system (GE Healthcare) controlling a Scientific CMOS camera (pco.edge 5.5). Acquisition parameters were controlled by SoftWoRx suite (GE Healthcare). Images were collected at RT using an Olympus 60× 1.42 NA oil objective at 0.2 mM z-sections and subsequently deconvolved in SoftWoRx suite.
4
Live-cell Imaging Protocols and Microscopy
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All live-cell imaging was performed at 37°C in mHBS supplemented with 2% FCS. TIRF-SIM and 3D-SIM imaging were performed on a GE DeltaVision OMX SR microscope (Cytiva) equipped with a 60 × 1.42 NA oil objective (Olympus). For 3D-SIM, z-stacks were acquired at 0.125 µm increments. Raw data were reconstructed using Softworx software (Cytiva) with a Wiener filter constant of 0.002–0.003. Airyscan imaging was performed using an LSM 880 Zeiss confocal microscope equipped with Airyscan and using a Plan-Apochromat 63 × 1.4 NA oil objective. Airyscan image reconstruction was performed using Zeiss ZEN imaging software. TFM was imaged using a Nikon Eclipse Ti2 microscope equipped with a 60 × 1.2 NA water objective. Linear adjustments to images were made using ImageJ 1.53 (NIH).
5
EGFR Labeling and Imaging Protocol
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After the transfection of N-terminal SNAP and C-terminal eGFP-tagged EGFR, the cells on a cover glass (16 mm in diameter) were treated with a solution of AdA-BG (10 μM) in a serum-free cell culture medium (1.0 ml) and incubated for 30 min at 37 °C. Then, the cells were washed with PBS three times and treated with a solution of Cy3-CB[7] (200 nM) in a blocking buffer (1.0 ml) for another 30 min at room temperature. After five times washings with PBS, the cells on a cover glass were examined under confocal laser scanning microscope (FV1000, Olympus) with 60×/1.42NA Oil objective.
6
Super-resolution Imaging of Centrioles and SAPs
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Images were acquired on a DeltaVision OMX V3 Blaze microscope (Applied Precision, GE Healthcare) equipped with a 60×, 1.42 NA oil objective (Olympus) and four different PCO Edge 4.2 sCMOS cameras (PCO AG) for detecting different colour channels. At least six z-planes with a 0.125 μm step size were acquired. For each plane, five phases and three angles were acquired. Images were processed with SoftWorx 6.1 software (GE Healthcare) using channel- and filter-specific measured Optical Transfer Functions. For multicolour imaging, the images were aligned using 1 μm to 200 nm diameter TetraSpeck Microspheres (ThermoFisher Scientific) and the OMX Editor software. Images were processed further in Fiji ImageJ and their quality assessed using the SIM-Check ImageJ plugin (Ball et al., 2015 (link)). ImageJ was used to calculate area and total fluorescence intensity of centrioles and SAPs (the Otsu filter was used to threshold the image). ImageJ was used to profile the radial distribution of PCM and centriole proteins around centrioles or SAPs, as previously described (Conduit et al., 2014a (link)). Ten centrioles or SAPs were analysed in each condition and averaged together; graphs were plotted using Prism 6.0 for Mac OSX, GraphPad Software.
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