Te 200 u inverted fluorescence microscope

Image acquisition for spectral analysis was performed using the Nuance Multispectral Imaging System (PerkinElmer Inc.; RRID:SCR_015382) (Mansfield, 2014 (link)), as described previously (Wilkerson et al., 2012a (link); Dengler et al., 2014 (link); Noor et al., 2017 (link)). Briefly, lumbar spinal cord dorsal horn images were obtained using a 20X objective with a Nikon TE-200 U inverted fluorescence microscope. Flat-field correction was applied in order to remove artifacts, including uneven field illumination, to produce a uniform illumination during image acquisition. Images of single-labeled control slides (one each for AF488 and DAPI) and a label-free (autofluorescence) slide were used to create a ‘spectral library’. Pure signals for each fluorophore were then computed by separating a known spectral profile (autofluorescence) from a ‘mixed’ spectrum profile (single labeled + autofluorescence). This allowed for the un-mixing of multi-labeled slides to obtain composite images containing only the labels of interest. Composite images were then used for further analysis using Slidebook 6 software (see Section 2.11).

Image acquisition for spectral analysis was preformed using Nuance spectral imaging system (http://www.cri-inc.com/products/nuancew.asp) [62 (link)], as described previously [26 (link),111 (link)]. Briefly, images of dorsal horn spinal cord were obtained using 20X objective with a Nikon TE-200 U inverted fluorescence microscope. Flat-field correction was applied in order to remove artifacts including uneven field illumination to produce a uniform illumination during image acquisition. A spectral library was then created using single-labeled control slides for each fluorophore and a label-free (autofluorescence) slide. The computed spectrum was then obtained by separating the known spectrum (autofluorescence) from mixed spectrum (single labeled) to produce pure labels of each fluorophore. This allowed for the un-mixing of multi-labeled slides to obtain composite images containing only the labels of interest. These composite images were then used for further analysis (see, Slidebook software image analysis). Sixteen spinal images per experimental group (2 adjacent sections plus 2 sections randomly separated by 140 μm totaling 4 sections per animal, with 4 animals per experimental condition) that were ipsilateral and contralateral to the sciatic manipulation were acquired and analyzed.