Z-axis stacks of images of neuromuscular junctions (NMJs) were obtained at sequential focal planes (1.0 μm separation) using an upright microscope equipped with a motorized stage (Leica). Stacks were deconvolved using a commercially available inverse filter algorithm (ImagePro). In some cases, stacks of confocal images were obtained.
Motorized stage
Manufactured by Leica
The Motorized Stage is a precision-engineered component designed to provide accurate and controlled movement of specimens or samples within a microscope or imaging system. It allows for automated and programmable positioning of the sample, enabling efficient and repeatable data collection.
Automatically generated - may contain errors
Lab products found in correlation
Dmi6000 inverted microscope, by Leica (1 mentions)
Dmi6000 inverted fluorescence microscope, by Leica (1 mentions)
Axioplan 2 imaging fluorescence microscope, by Zeiss (1 mentions)
Hcx pl apo 63 1.4 oil objective, by Leica (1 mentions)
Plan neofluar 100 1.30 oil objective, by Zeiss (1 mentions)
Dmi6000, by Leica (1 mentions)
5 protocols using motorized stage
1
Imaging Neuromuscular Junctions in 3D
2
Fluorescent Microscopy Imaging of hES Cells
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Fluorescent microscopy images for hES cells were taken on a fluorescence DMI-6000 inverted microscope with a motorized stage (Leica), equipped with a charge-coupled device (CCD) HQ2 camera (Roper Scientifics) and an HCX PL APO ×64-100 oil objective (numerical aperture, 1.4; Leica) using the MetaMorph software (version 7.04, Roper Scientifics). Approximately 40 optical z-sections were collected at 0.3-μm steps at different wavelengths depending on the signal (DAPI, 360-470 nm; FITC, 470-525 nm; Cy3, 550-570 nm; Cy5, 647-668 nm). We computed the dispersion of XIST RNA by comparing the cumulative volume of the signal to the theoretical spherical volume it could occupy on the basis of the maximal radial distance. Embryo confocal immunofluorescence images were acquired with an A1-SIM Nikon confocal microscope and a ×20 oil objective. Whole embryos were imaged with 0.5-1-μm optical sections. Stacks were processed using ImageJ and are represented as a two-dimensional 'maximum projection' throughout the manuscript.
3
Imaging Neuromuscular Junctions via Deconvolution
4
Fluorescence Microscopy Imaging Protocol
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Images were taken on an Axioplan 2 Imaging fluorescence microscope (Zeiss) with a cooled Coolsnap camera (Roper Scientifics) or a DMI-6000 inverted fluorescence microscope with a motorized stage (Leica) and a charge-coupled device camera HQ2 (Roper Scientific), both controlled by the MetaMorph 7.04 software (Roper Scientifics), using a Plan-NEOFLUAR 63×/1.25 oil objective (Zeiss), a Plan-NEOFLUAR 100×/1.30 oil objective (Zeiss), or a HCX PL APO 63×/1.4 oil objective (Leica). Optical sections were collected at 0.2-mm steps through each nucleus at different wavelengths (in nanometers) {Zeiss: DAPI (345, 455), FITC (488, 507), and CY3 (625, 660); Leica: DAPI (360, 470), FITC (470, 525), and CY3 (550, 570)}. Approximately 40 optical sections per nucleus were collected. Stacks were processed using Icy (http://icy.bioimageanalysis.org ), and the images are represented as two-dimensional projections of the stacks (maximum projection).
5
3D Fluorescence Microscopy Imaging
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All images were acquired with a fluorescence DMI-6000 inverted microscope with a motorized stage (Leica) using a HCX PL APO ×100 oil objective and a CCD Camera HQ2 (Roper Scientifics) using the Metamorph software (version 7.04, Roper Scientifics). Approximately 40 optical Z-sections were collected at 0.5 µm steps across the nucleus for each wavelengths (DAPI [360 nm, 470 nm], fluorescein isothiocyanate [470 nm, 525 nm], and Cy3 [550 nm, 570 nm]). Stacks were processed using ImageJ. Throughout the manuscript, the three-dimensional FISH experiments are represented as a two-dimensional projection of the stacks (maximum intensity projection).
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