Sp8 uv visible laser confocal microscope

FLG samples were characterized through several techniques including scanning electron microscopy (SEM, Hitachi S-4800), Raman spectroscopy (Nanophoton Laser Raman Microscope RAMAN-11), and optical transmittance microscopy (Leica SP8 UV/Visible Laser Confocal Microscope). The optical transmittance image was obtained using a 561 nm laser line with constant intensity. Transmittance intensity of a blank glass cover slide was collected as reference (100% transmittance). The intensity at each pixel was then converted to percent transmittance, which is interpreted as a graphene layer number distribution map, by making use of a 2.3% decrease in optical transmittance per layer.^{34 (link)}

IEC-18 cells were grown in Ca²⁺-free medium [20 mM Hepes, 40% (w/v) glucose, 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂] in the presence of 1 μM pluronic acid F-127 (Thermo Fisher) and 5 μM Indo-1 AM calcium probe (Thermo Fisher) for 30 min at 37 °C, washed two times with PBS, and incubated for 30 min at 37 °C in Ca²⁺-free medium. Ratiometric calcium imaging was performed using an inverted Leica SP8 UV/Visible Laser Confocal Microscope equipped with a Leica HC PL APO 63 × 1.4 N.A. oil immersion objective. Cells were subjected to a 355 nm UV laser. Two confocal images in the spectral range 400–440 nm and 470–540 nm were simultaneously recorded every 5 s using HyD detectors in the counting mode. Variation of the intracellular calcium concentration was determined by the intensity ratio between the two emission bands with the FIJI/ ImageJ distribution Software^{39 (link)}.

In the granule cell layer of the DG, exactly 927 BrdU-positive cells were analyzed from animals in the e-stim treatment, and 960 BrdU-positive cells were analyzed from animals in the sham treatment. In CA1/CA2, 50 BrdU-positive cells were analyzed from e-stim and 47 from sham. Each BrdU-positive cell was examined at 630× total magnification using a Leica SP8 UV/visible laser confocal microscope to determine whether the cell was co-labeled with NeuN or S100ß. A z-stack of images throughout the depth of the section was taken so that each BrdU-positive cell could be analyzed at a focal point with greatest expression of the BrdU-tagged fluorescent label. These images were imported into ImageJ and the average intensity of each channel, BrdU, NeuN, and S100ß over the area of the cell was recorded. These data were then analyzed using the “choisy/cutoff” package in R to obtain a threshold intensity for deciding whether a BrdU positive cell was also NeuN positive or S100ß positive.