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To obtain 3D reconstruction of CTb-594-labeled axons in the optic nerve, image stacks were stitched using the XuvTools software and the resulting macro-stacks were exported to the Imaris Software (Bitplane, Zürich, Switzerland) to create 3D projections (Videos 1, 2).
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Prior efforts to exploit this technique were impaired by a lack of superimposability for sequential optic nerve images.
Stereochronoscopy, a technique previously explored but abandoned for glaucoma diagnosis, viewed optic nerve images acquired at separate points in time as if a stereo pair.
Sections were taken at the 2-mm level of optic nerve, imaged, and photographed at 25K magnification.
For measurement of docked mitochondria, total numbers of mitochondria that remained stationary during the entire duration of observation were counted and divided by the length of nerve imaged to yield docked mitochondria/µm.
Corneal nerve images, seen in Fig. 1, were obtained and analyzed by methods previously described (18).
Comparisons between automatic (fully computerized) and semiautomated morphometric methods performed by the same computer software using the same nerve images were also performed.
Fluorescence was clearly visible in all nerves imaged, allowing for easy visualization of major nerve trunks and their branches.
In Stage 36 embryos, of 22 nerves imaged in CTL MO animals, one had abnormal morphology and of 14 nerves imaged in morphants, 12 were abnormal (Fisher's exact test P < 0.0001).
In Stage 40 embryos, of 17 nerves imaged in CTL MO animals, three were abnormal and of 18 nerves imaged in morphants, all were abnormal (Fisher's exact test P < 0.0001).
In Stage 36 embryos, of 22 nerves imaged in CTL MO animals, two were abnormal and of 30 nerves imaged in morphants, 26 were abnormal (Fisher's exact test P < 0.0001).
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