Images of fixed nota were acquired with a Leica SPE confocal microscope and a Zeiss Airyscan microscope (LSM 880 with AiryScan module). Live imaging of NiGFP with Par3::Scarlet was performed with a Leica SPE confocal microscope. Live imaging of NiGFP in wild-type SOP and in strat SOP expressing pnr-GAL4>AP-47dsRNA (Fig. S2 ) was performed with a Zeiss Airyscan microscope (LSM 880 with AiryScan module). All images were processed and assembled using ImageJ 1.48 and Adobe Illustrator.
Airyscan microscope
Manufactured by Zeiss
Sourced in Germany
The Airyscan microscope is a high-resolution imaging system developed by Zeiss. It utilizes a specialized detector to capture more information from the sample, improving image quality and resolution compared to conventional confocal microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (10 mentions)
Lsm 800, by Zeiss (3 mentions)
Spe confocal microscope, by Zeiss (2 mentions)
Spe confocal microscope, by Leica camera (2 mentions)
Eclipse ti s microscope, by Nikon (2 mentions)
Lightsheet z 1 microscope, by Zeiss (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
A1r eclipse ti microscope, by Nikon (1 mentions)
D8417, by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
4 6 diamidino 2 phenylidone dapi, by Merck Group (1 mentions)
High precision cover glass, by Zeiss (1 mentions)
Lsm880 system, by Zeiss (1 mentions)
Confocal dishes, by NEST Biotechnology (1 mentions)
Ab53219, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Pm063, by Medical & Biological Laboratories (1 mentions)
A2547, by Merck Group (1 mentions)
Ab232589, by Abcam (1 mentions)
Lsm 980, by Zeiss (1 mentions)
30 protocols using airyscan microscope
1
Confocal and Airyscan Microscopy Protocol
2
Confocal and Airyscan Imaging of Cellular Processes
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Images of fixed nota were acquired with a Leica SPE confocal microscope and a Zeiss Airyscan microscope (LSM 880 with AiryScan module). Live imaging of NiGFP with Par3::Scarlet was performed with a Leica SPE confocal microscope. Live imaging of NiGFP in wild-type SOP and in strat SOP expressing pnr-GAL4>AP-47 dsRNA (Fig. S2) was performed with a Zeiss Airyscan microscope (LSM 880 with AiryScan module). All images were processed and assembled using ImageJ 1.48 and Adobe Illustrator.
3
Visualizing Viral Particle Internalization
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The cell lines were grown overnight on glass cover slips (25,000 cells per well) placed in an untreated 12-well plate in 200 µl of 10% DMEM. The cells were washed twice with Dulbecco's PBS (DPBS) to remove unbound and dead cells before addition of A488-SeMV or SeMV-Cy5.5 particles (5×10 6 particles per cell, corresponding to ~ 1 µg/ well) and incubated for 0.5, 2 and 6 h. The cells were washed three times with DPBS to remove unbound particles and fixed with 4% paraformaldehyde in PBS for 5 min at RT. The nucleus was stained with DAPI (10 ng/ml) for 5 min at RT and washed twice with PBS. The coverslips were mounted on glass slides using Fluoroshield and sealed with nail polish. Confocal images were captured on a Zeiss Airyscan microscope, and the images were processed using the Zen software.
For live cell imaging, 2×10 5 MDA-MB-231 cells were seeded into a 35-mm glass-bottomed Petri dish and incubated overnight. One µg of SeMV-Cy5.5 particles was added, and images were captured every 5 min, while recording the events continuously using a Zeiss Airyscan microscope for a period of 75 min.
For live cell imaging, 2×10 5 MDA-MB-231 cells were seeded into a 35-mm glass-bottomed Petri dish and incubated overnight. One µg of SeMV-Cy5.5 particles was added, and images were captured every 5 min, while recording the events continuously using a Zeiss Airyscan microscope for a period of 75 min.
4
Live Zebrafish Cardiac Morphology Imaging
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Live zebrafish embryos were imaged on a ZEISS Lightsheet Z.1 microscope. To assess cardiac morphology at 50hpf and 72hpf embryos were anesthetized by immersion in 8.4% Tricaine (Merck 10521) before mounting in 1% low melting point agarose in E3 with 8.4% Tricaine. To stop the heart the imaging chamber was filled with E3 with 8.4% Tricaine and the temperature maintained at 10°C. All samples were imaged using a 20× lens and 1.0 zoom at 0.47–0.65 µm z-step size, with sufficient z slices to capture the entire heart. Dual side lasers with dual side fusion and pivot scan were used for sample illumination.
Embryos injected with ssNcan-GFP mRNA were fixed overnight in 4% PFA with 4% sucrose, and the GFP signal amplified by immunohistochemistry. Dissected embryos were imaged using a Zeiss Airyscan microscope, z stacks were obtained with a step size of 1 µm.
Detailed image quantification methodology is included inSupplementary material online .
Embryos injected with ssNcan-GFP mRNA were fixed overnight in 4% PFA with 4% sucrose, and the GFP signal amplified by immunohistochemistry. Dissected embryos were imaged using a Zeiss Airyscan microscope, z stacks were obtained with a step size of 1 µm.
Detailed image quantification methodology is included in
5
High-Resolution Imaging of Pom1 Clusters
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To achieve maximum resolution and sensitivity, fluorescence intensity of Pom1 clusters on both cell sides and cell tips was measured using a Zeiss Airyscan microscope (Figure 1—figure supplement 1C,D,E ), composed of a Zeiss LSM-880 laser scanning confocal microscope (Zeiss, Oberkochen, Germany) equipped with 100X alpha Plan-Apochromat/NA 1.46 Oil DIC M27 Elyra objective, Airyscan super-resolution module and GaAsP Detectors, and Zen Blue acquisition software using the Super-resolution mode with pin-hole size of 1.2 airy-units to prioritize resolution. Z-volumes of 16 slices with 0.19 µm spacing for high spatial resolution in all dimensions were centered on the cell cortex closest to the coverslip. Airyscan images were processed in Zeiss Zen Blue software, and quantification was performed on sum projections of Airyscan reconstructed stacks.
6
Labeling Drosophila Egg Chambers
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Egg chambers were dissected out of the muscle sheath and incubated in Schneider's medium containing 0.5 µM JF646–Halo for 20 min. The egg chambers were then removed to Schneider's medium and imaged in an 8-well µ-slide with poly-lysine coating (Ibidi) using a Zeiss Airyscan microscope.
7
Imaging and Quantifying Drosophila Fat Body
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Images were collected using a Nikon A1R Eclipse Ti microscope and 40x or 60x objectives. We measured the size of fat body cells when the optical section bisected the nucleus, by outlining the cell boundary (as defined by phalloidin) with the freehand selection tool. We measured LD density within each cell by determining the percentage of pixels that had LD‐associated fluorescence. LD size was measured using an optimized macro that detected single LD particles (analyze particles size = 5‐infinity) after smoothening and applying a threshold that was manually verified to specifically detect LD. Nuclear size was measured from images of anti‐LaminDm0‐labeled tissue imaged on a Zeiss Airyscan microscope at 63× magnification, with the Z‐plane bisecting the widest part of a nucleus. The area of each nucleus in an image (> 30 nuclei in total for each age) was measured by manual outlining.
8
Astrocyte Morphological Analysis in GFAP-mVenus Mice
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Mice were stereotactically injected with AAV expressing membrane-targeted mVenus under the control of the GFAP promoter at P21 and then perfused, as described in the immunohistochemistry section, at P35. Brains were post-fixed in 4% PFA for 24 hours at 4°C. Brains were then washed 3 times in PBS and then vibratome sectioned at 100μm in PBS. Sections were counterstained with DAPI (D8417, Sigma) and mounted on gelatin-coated slides (FD Neurotechenologies, Cat# PO101) in 10% PBS. Once sections were dry, slides were dipped in water and again allowed to dry. Coverslips (#1.5, VWR) were affixed with Fluoromount-G (Southern Biotech). Images were taken on a Zeiss AiryScan microscope using the same imaging parameters for controls and Astro-Nlgn123 cKO animal. While blinded, astrocyte morphologies were reconstructed with 3D rendering in Imaris and astrocyte volumes were measured.
9
Super-resolution Airyscan Microscopy Protocol
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Super-resolution Airyscan images were acquired on a Zeiss LSM 880 with Airyscan microscope (Carl Zeiss). Data were collected using a 63 x 1.4 NA objective for the majority of experiments, although some were acquired using a 40 x 1.3 NA objective. 405nm, 561nm and 640 nm laser lines were used, in addition to a multi-line argon laser (488nm) and images acquired sequentially using the optimal resolution determined by the Zeiss ZEN software. When acquiring z-stacks, the software-recommend slice size was used. Live-cell experiments were performed in an environmental chamber at 37°C and 5% CO2. Airyscan processing was performed using the Airyscan processing function in the ZEN software, and to maintain clarity some images have been pseudocloured and brightness and contrast altered in FIJI (ImageJ v2.0.0).
10
Bimolecular Fluorescence Complementation in Protoplasts
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The Gateway compatible destination vectors used were pDH51-GW-YFPN and pDH51-GW-YFPC (Zhong et al., 2008 (link)), enabling the fusion of the N terminus or C terminus of the yellow fluorescent protein (YFP) moieties, respectively, to the C terminus of the protein of interest. Control vectors were pDH51-YFPC and pDH51-YFPN (Zhong et al., 2008 (link)). Protoplasts were isolated as described, transformed with 1 μg of DNA for each plasmid, and incubated in the dark at 25°C for 16 h before subsequent analysis. Fluorescence of YFP was analyzed with a Zeiss LSM 880 with Airyscan microscope. Yellow fluorescence was excited with 488 nm laser light and collected at 497-546 nm wavelength range. Chlorophyll autofluorescence was excited with 640 nm laser light and collected at 656-700 nm wavelength range. Nuclei were stained with 4',6-diamidino-2-phenylidone (DAPI, Sigma-Aldrich), fluorescence was excited at 405 nm and collected at 410-470 nm.
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