Real-time confocal microscopy analysis was used to image and analyze the entire diaphragm. Confocal images were obtained with a Leica DM LFSA microscope with an Ultraview confocal scanner (PerkinElmer). The images were captured and processed using Metamorph software (Universal Imaging). Starting from the beginning of one hemi diaphragm, consecutive images were taken without overlap to the end. Z-stack images with a depth encompassing the entire depth of the motor endplates were used. For each whole diaphragm, the sum of α-bungarotoxin (Btx) stained endplates was quantified using ImageJ. Also using ImageJ, the bandwidth of innervation was configured by measuring a series of straight lines drawn using the software from one side of the band to the other and averaged per genotype. The individual area was calculated by tracing the circumference of individual motor endplates. ImageJ quantification in pixels was converted to micrometer using standard conversion equations.
Ultraview confocal scanner
Manufactured by PerkinElmer
Sourced in United States
The Ultraview confocal scanner is a high-performance microscopy system designed for live-cell imaging. It utilizes confocal technology to provide optical sectioning and improved image contrast. The system is capable of capturing high-speed, high-resolution images and videos of dynamic cellular processes.
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2 protocols using ultraview confocal scanner
1
Quantifying Diaphragm Motor Endplates
2
Measuring Intracellular Calcium Dynamics
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After 24 to 30 hours of culture, the ICC cultured on coverslips (25 mm) were well grown and ready for measurement of [Ca2+]i concentrations. The medium in culture dishes was removed. The cells were washed twice and incubated at 37℃ for 10 minutes with bath solution. Next, the cells were stained by Fluo-4 acetoxymethyl ester (AM) (1 mM, Invitrogen) at 37℃ for 15 minutes and then rinsed for 3 times with normal solution. After mounting on the perfusion chamber, the cells were scanned every 0.4 seconds with Nikon Eclipse TE2000-U (Nikon Inc, Melville, NY, USA) inverted microscope equipped with a PerkinElmer Ultraview confocal scanner (PerkinElmer Inc, Waltham, MA, USA) and a Hamamatsu Orca ER 12-bit CCD camera (×200; Hamamatsu Instrument, Hamamatsu, Shizuoka, Japan). Fluorescence was excited at a wavelength of 488 nm, and emitted light was observed at 515 nm. The temperature of perfusion chamber remained at 30℃ during the experiment. Relative alterations of [Ca2+]i fluorescence emission intensity was expressed as the ratio (F1/F0), where F0 was taken from the fluorescence of the first image.
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