Computational protocol: Optical Imaging of Neuronal Activity and Visualization of Fine Neural Structures in Non-Desheathed Nervous Systems

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Protocol publication

[…] For comparing the fluorescence before and after desheathing, we used a 5 mega pixel color CMOS camera (TCA-5.0C, Ample Scientific LLC, Norcross, GA, USA) and TSView software (Version 7.3.1.7, Tucsen Imaging Technology Co., Fujian, China). Fluorescent excitation light was provided by a CoolLED system (narrowband LED with 535 nm; Yorktown Heights, NY) and fluorescence emission was detected using a 560 nm beam splitter and a 570–640 nm emission filter (Olympus, Center Valley, PA). Excitation light intensities and imaging exposure time varied and were adjusted to the individual preparation. We either used a 20× objective (XLUMPlanFL N, NA 1.0, WD 2.0 mm, cc = water; Olympus Corporation, Tokyo, Japan) or a 10× objective (UMPlanFL N, NA 0.30, WD 3.3 mm, cc = water; Olympus Corporation, Tokyo, Japan) mounted on an upright epifluorescence microscope (modified BX51, Scientifica, East Sussex, UK). For recording fluorescence changes (‘optical imaging’) the MiCam02 imaging system and software (Brain-Vision Analyzer, BV-ANA, Version 11.08.20; SciMedia Ltd, Tokyo, Japan) were used with the HR (High Resolution) camera (6.4×4.8 mm actual sensor size) set at either 384×256 pixel spatial resolution for high resolution photos or at 192×128, 96×64, or 48×32 pixel spatial resolution for optical imaging. A temporal resolution of 2–20 ms was chosen. Typical recordings lasted between 16 and 32 seconds, and were repeated many times in a given experiment. [...] For spreadsheet analysis, Excel (version 2010 for Windows, Microsoft) or SigmaPlot (version 11 for Windows, Systat Software GmbH, Erkrath, Germany) were used. Normally distributed data are given as mean ± SD. “N” denotes the number of animals, while “n” is the number of trials. In all figures significance is indicated using * (p<0.05), ** (p<0.01), *** (p<0.001). Statistical tests for data analysis were t-test, Pearson product-moment correlation coefficients or One Way ANOVA with Holm Sidak posthoc test. Final figures were prepared with CorelDraw (version X3 for Windows, Corel Corporation, Ottawa, ON, Canada). […]

Pipeline specifications

Software tools MiCAM, SigmaPlot, CorelDraw
Applications Miscellaneous, Metagenomic sequencing analysis
Diseases Neoplasms, Ganglion Cysts