Colibri 2 led light source

Sections co-labelled for hemoglobin, lectin, and SMI-32; or hemoglobin, lectin, and barrier integrity markers; were imaged across the entire section with a MetaSystems VSlide slide scanner at 10×magnification (0.45 NA), using MetaCyte acquisition and stitching software. The Colibri 2 LED light source (Zeiss) was used to acquire DAPI (excitation band 375/38 nm), AlexaFluor 488 (484/25 nm), Cy3 (580/23 nm), and Cy5 (631/22 nm), while the X-Cite light source was used for AlexaFluor 594 (560/40 nm). Phospho-TDP-43 (pTDP-43) pathology in the spinal cord anterior horn was imaged with a Nikon Eclipse NiE microscope with a Nikon DS-Ri2 camera using NIS elements (Nikon, Version 4.20) at 20×magnification (0.50 NA). All sections (ALS and control) were imaged with the same settings for each staining combination. Vessel-associated pTDP-43 inclusions were imaged on an Olympus FV1000 confocal microscope at 60× (1.35 NA) magnification.

The hamster cheek pouch was prepared for intravital microscopy as reported by our group [8 (link),20 (link),21 (link),54 (link),89 (link)], using a digital camera (AxioCamHRc, Carl Zeiss, Oberkochen, Germany). The microcirculation was observed with an Axioskop 40 microscope, using objectives of 4×, 20× (water immersion lens) and oculars 10× (Carl Zeiss, Oberkochen, Germany). The microscope was equipped with appropriate filters (490/520 nm, FITC-dextran, and 540/580 nm, TRITC-dextran), a mercury arc lamp, and a Colibri 2 LED-light source (Carl Zeiss, Oberkochen, Germany). The image resolution is 1388 × 1040 for an area of approximately 5 mm² (objective 4×), which yields pixel spacing of 1.862 µm, and therefore a pixel area of 3.467 µm². We used two macromolecular markers of equal molecular masses, FITC-dextran, and TRITC-dextran (150 kDa, 100 mg/kg b.w., TdB Labs, Uppsala, Sweden). TRITC-dextran was used to identify intracellular GFP-T. cruzi and localize microvessels by combining IVM to confocal microscopy. A digital camera, AxioCamHRc, and a computer with the AxioVision 4.4 Software program (Carl Zeiss, Oberkochen, Germany) were used for image recording and measurement of fluorescence (Relative Fluorescent Units, RFU).