Cfi plan apo vc60xh objective

Cells, seeded at a density of 50,000 per well onto 25-mm glass coverslips, were allowed to grow for 24 h and then infected with mitochondria-targeted green fluorescent protein (GFP) inserted into an adenoviral vector (Ad-mtGFP Ex/Em: 495/515) as previously described (Izzo et al., 2017b (link)). Protein expression was then allowed for 72 h in the presence or absence of PGZ.
The efficiency of infection was comparable in treated and untreated trisomic cells both in terms of percentage of GFP positive cells (about 80%) and of intensity of fluorescent GFP signal.
Single cells were imaged, by using the same settings for treated and untreated cells, with a Nikon Swept Field Confocal microscope (Nikon Instruments Inc.) equipped with a CFI Plan Apo VC60XH objective and an Andor DU885 EM-CCD camera, which was controlled by NIS Elements 3.2. Fifty-one-plane z-stacks were acquired with voxel dimensions of 133 × 133 × 200 nm (X × Y × Z). The mitochondrial network was then described in numbers of objects, total volume and object volume using the 3D object counter available in the software Fiji (http://www.fiji.sc) (Schindelin et al., 2012 (link)). 3D rendering was obtained with the 3D Viewer plugin.

SH-SY5Y cells were seeded and transfected with mtDsRed as described in the “Results” section. Protein expression was allowed for 36 h and then cells were imaged with Nikon Swept Field Confocal, equipped with CFI Plan Apo VC60XH objective (n.a. 1.4) and an Andor DU885 EM-CCD camera, controlled by the NIS-Elements 3.2. Coverslips were placed in an incubated chamber with controlled temperature, CO₂, and humidity and then z-stacks were acquired by 21 planes with 0.6-μm distance, to allow acquisition of the whole cell. After acquisition, images were restored with the Autoquant 3D-blind deconvolution module, installed on NIS-Elements (Nikon Instruments Inc.), using a theoretical PSF. After restoration, images were loaded in Imaris 4.0, then subtracted of background, and used to generate a threshold-based isosurface object group. From each isosurface, the number of objects and the average object volume expressed as voxel number were calculated.