All myofiber imaging was carried out on a Zeiss LSM 880 AxioObserver microscope with Airyscan using a Plan-Apochromat 1.3 NA 40x oil objective (Fig. 1, 2, 3, 4, 5, S1, S2, S3, S4, S5, S6, Video S2, Video S3), a Plan-Apochromat 1.4 NA 63x oil objective (Fig. S1A, Video S1), or a Plan-Apochromat 1.4 NA 100x oil objective (Fig. S3B andS3C). Airyscan processing was performed on all images in Zeiss ZEN software. Z-stacks were acquired at 0.5 μm intervals. Myoblast and myotube imaging was carried out on a Zeiss LSM 880 AxioObserver microscope using wide field fluorescence illumination, a Plan-Apochromat 1.4 NA 100x oil objective, and Zeiss AxioCam monochrome camera (Fig. 3I). Z-stacks through myoblasts and myotubes were acquired at 0.5 μm intervals.
Lsm 880 axioobserver microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 880 AxioObserver microscope is a confocal laser scanning microscope designed for high-resolution imaging of biological samples. It features a modular design and is equipped with a set of advanced optics and detection systems for capturing detailed images of fluorescently labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam monochrome camera, by Zeiss (2 mentions)
Jf646 halotag ligand, by Promega (2 mentions)
Plan apochromat 1.3 na 40 oil objective, by Zeiss (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Zen black software, by Zeiss (1 mentions)
Eclipse te300 inverted microscope, by Nikon (1 mentions)
Fv1000 mpe microscope, by Olympus (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Plan apochromat 63 1.4 oil dic m27 objective, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
4 well chamber slide, by Thermo Fisher Scientific (1 mentions)
D6w5b, by Cell Signaling Technology (1 mentions)
Plan apochromat 1.4 na 100x oil objective, by Zeiss (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Mouse anti α smooth muscle actin, by Merck Group (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
13 protocols using lsm 880 axioobserver microscope
1
Myofiber Imaging Protocols Using Zeiss LSM 880
2
High-Resolution Confocal Microscopy Protocol
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Cells were imaged on a Zeiss LSM 880 AxioObserver microscope equipped with a Plan-Apochromat oil 63×/1.4 NA differential interference contrast M27 objective. Z-stacks were obtained using a step size of 2 μm.
3
Quantitative Colocalization Analysis of MBNL1 and KIF in Neurons
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Results of HCR FISH in C2C12 myoblasts and N2A cells were imaged on a Zeiss LSM 880 Axio Observer microscope with Airyscan using a Plan-Apochromat 1.46 NA x100 oil objective and processed using Zeiss ZEN Black software (Version 14.0.15.201). Fixed cell imaging for centrosome recruitment assay was done with widefield illumination using a Plan-Apochromat 1.3 NA ×40 oil objective. Fixed cell imaging of the split kinesin assay in N2As and MBNL in primary neurons was performed using widefield illumination on a Nikon Eclipse TE300 inverted microscope with a Plan-Neofluar 1.4 NA x60 oil objective. For quantitative colocalization experiments, 12 images were taken in a z-series at 2 um steps and deconvolved using a 3-D blind constrained iterative algorithm (AutoQuant, CyberMetrics). Imaris imaging software (Bitplane), namely the ‘Coloc’ module, was then used for analysis on the deconvolved images. Quantitative colocalization analysis involved creating a 3-D mask of the MAP2 channel from select neurites and excluding background signal from outside this volume. Within the MAP2 masked volume, two measures of colocalization, Pearson’s Correlation Coefficient and Mander’s Overlap Coefficient, were calculated between HA-MBNL1 and the respective endogenous KIF signals.
4
Multimodal Microscopic Imaging of Spheroids
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Imaging was conducted using two microscopes based on availability and access, but care was taken to compare images only within the same system and settings:
Olympus FV1000 MPE microscope and excited by a MaiTai DeepSee Ti:Sapphire laser, with a 25× water dipping objective lens optimized for TPM (XLPLN25XWMP) with an NA of 1.05 and a working distance of 2 mm for all imaging, combined with 495–540 nm (green) and 576–630 nm (red) filters.
Zeiss LSM 880 AxioObserver microscope with Plan-Apochromat 20×/0.8 M27 and N-Achroplan 10×/0.25 Ph1 M27_b lenses, with the laser set at 405, 488 and 594 nm. The spheroids were transferred to a 96-well plate with flat cover glass #17 before imaging. Unless specified, all the images are taken using this microscope.
5
Confocal Imaging of Gephyrin.FingR Neurons
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Confocal image stacks centered around Gephyrin.FingR-expressing soma were collected with an LSM 880 AxioObserver Microscope (Zeiss) using a 63× oil-immersion objective lens (Plan-Apochromat, 1/40 Oil DIC M27) with the zoom factor set to 1, and the pinhole set at 1.0 Airy disk unit for each fluorescence channel. Laser intensities for each channel were independently set to avoid pixel saturation for each field of view. Fluorescence acquisition setting were as follows: EGFP (excitation λ488, detection λ489–512), tdTomato and Oyster® 550 (excitation λ561, detection 566–579). Maximum image size was 1024 × 124 pixels to collect 135 × 135 × ≤40 µm images, with a corresponding voxel dimension of 0.13 µm (x, y) and 0.3 µm (z).
6
Super-resolution Imaging using Zeiss Airyscan
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Super-resolution imaging was performed using a Zeiss LSM 880 Axio Observer microscope with Airyscan detection system in SR mode using a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss, Jena, Germany). Excitation was achieved using the 488 nm line from an argon laser and a 561 nm diode laser or 594 nm HeNe laser and emitted light was collected through appropriate filters to eliminate any spill-over. Raw images were immediately Airyscan processed using the default settings and analysed using ZEN 2.1 software.
7
Multimodal Fluorescence Microscopy Protocols
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Standard fluorescence
imaging in the UV–visible emission range was performed on the
hyperspectral microscope by using an XCite Series 120Q lamp as the
light source and a QiClick CCD camera (QImaging) directly attached
to a c-mount on a separate port of the microscope. Fluorescence filter
sets from Chroma Technology and Semrock were used. Confocal imaging
was performed on a Zeiss LSM 880, AxioObserver microscope equipped
with a Plan-Apochromat 63× oil 1.4 NA differential interference
contrast M27 objective in a humidified chamber at 37 °C. Z-stacks were obtained using a step size of 198–220
nm.
imaging in the UV–visible emission range was performed on the
hyperspectral microscope by using an XCite Series 120Q lamp as the
light source and a QiClick CCD camera (QImaging) directly attached
to a c-mount on a separate port of the microscope. Fluorescence filter
sets from Chroma Technology and Semrock were used. Confocal imaging
was performed on a Zeiss LSM 880, AxioObserver microscope equipped
with a Plan-Apochromat 63× oil 1.4 NA differential interference
contrast M27 objective in a humidified chamber at 37 °C. Z-stacks were obtained using a step size of 198–220
nm.
8
Macrophage Lipid Imaging Protocol
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RAW264.7 macrophage-like cells were incubated with Alexa546–agLDL for 1 h, labeled with Alexa555–CtB (10 μg/ml) for 5 min on ice, fixed with 3% PFA for 20 min, washed with phosphate buffered saline (PBS), incubated with filipin (50 μg/ml) for 1 h and then washed 3 times with PBS. Images were acquired on a Zeiss LSM 880, AxioObserver microscope (Thornwood, NY) equipped with a Plan-Apochromat 63× Oil 1.4 NA differential interference contrast (DIC) M27 objective. Z-stacks were obtained using a step size of 170 nm.
9
Live imaging of differentiated myotubes
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Immediately before live imaging, differentiated myotubes were stained with JF646 HaloTag Ligand (Promega) according to manufacturer specifications. Imaging was performed in Fluorobrite phenol-free culture medium at 37 °C, 5% CO2 in a stage mounted incubator using a Zeiss LSM 880 AxioObserver microscope with Plan-Apochromat 1.46 NA ×100 oil objective, widefield fluorescence illumination, and Zeiss AxioCam monochrome camera (Fig. 8 ). For five fast imaging movies, images were captured continuously for 30 s to 2 min with exposure times between 100 and 500 ms. For long imaging movies, images were captured at 1.0 s exposure time with a 15 s time lag over the course of ~50 min.
10
Advanced Microscopy Imaging of Myofibers
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All myofiber, myotube, and myoblast fixed sample imaging was carried out on a Zeiss LSM 880 AxioObserver microscope with Airyscan using a Plan-Apochromat 1.3 NA ×40 oil objective (Figs. 1 –7 , Supplementary Figs. 1 –6 , and Supplementary Movie 2 and 3 ), a Plan-Apochromat 1.4 NA ×63 oil objective (Supplementary Movie 1 ), or a Plan-Apochromat 1.4 NA ×100 oil objective (Fig. 3 E, F ). Airyscan processing was performed on all images in Zeiss ZEN software. Z-stacks were acquired at 0.5 μm intervals.
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