Andor neo 5.5 scmos camera

Liraglutide^VT750, MECA-32^VT750, and c-Fos brain samples were imaged by LSFM. The scan parameters were chosen to balance data acquisition time and resolution. Image stacks (16 bit tiff) were acquired using an UltraMicroscope II LSFM system (Lavision Biotec, Bielefeld, Germany) equipped with an Andor Neo 5.5 scmos camera (Andor Technology Ltd., Belfast, UK) in 10.32 μm isotropic resolution for the liraglutide^VT750, and MECA-32^VT750 distribution study, and 4.06 μm for the c-Fos activation study. Data acquisition was performed using a 620/60 nm excitation filter and 680/30 nm emission filter (liraglutide^VT750, MECA-32^VT750) or 545/30 nm excitation filter and 620/60 nm emission filter (c-Fos) for imaging auto-fluorescence, and a 710/75 nm excitation filter and a 780/40 nm emission filter (liraglutide^VT750, MECA-32^VT750) or a 620/60 nm excitation filter and 680/30 nm emission filter (c-Fos) for imaging specific signals.

Roots were fixed in PEM buffer (4% paraformaldehyde in 1 M NaOH, 50 mM PIPES, 1 mM EGTA and 5 mM MgSO4), then rinsed three times with 100 mM Na-phosphate buffer (pH 8). The tissues were stained directly before microscopy with 0.1% methyl blue (certified for use as aniline blue; Sigma) in 100 mM Na-phosphate buffer. Images were acquired using a Leica DM 6000 epifluorescence microscope equipped with an Andor Neo 5.5 sCMOS camera (Andor Technology Ltd., Belfast, UK).

The UltraMicroscope II (Bioimaging Facility, Department of Molecular Biology and Genetics, Democritus University of Thrace) is equipped with an Andor Neo 5.5 sCMOS camera (Andor Technology, Belfast, UK), with a pixel pitch of 6.5 μm, a Nikon 16x (0.8 NA) water immersion objective, and a zoom body of 1.8x magnification, for a total of 28.8x magnification. The illumination is achieved by three intersecting light sheets coming from the right side, achieving a uniform illumination across the sample and reducing shadows and stripe artifacts. The detection axis is perpendicular and above the illumination path. The illumination NA was set to 0.156 creating a light sheet with a thickness of 2w₀ = 4.53 μm (as reported from the software; InSpector Pro). Excitation and detection were performed using a 488 nm, 561 nm, or 640 nm laser and 525/50 nm, 620/60 nm, and 680/30 nm filters, respectively. z-stacks were acquired with a 1 or 2 μm step.
Fixed and stained HIOs were enclosed in the top surface of 1% low-melting agarose (in PBS) cubes and were immersed inside the imaging cuvette filled with distilled water. This technique ensures that the HIO structure remains undamaged and unpressurized, and therefore, the images taken depict their actual form. Image analysis, 3D rendering, and slice selection were performed in ImageJ (National Institutes of Health, USA).