Dmi6000 inverted microscope

For fluorescence studies, CHO M12 cells were grown on coverslips, transfected with Lamp2A-GFP as per manufacturer instructions, and after 48 h, fixed with 4% PFA for 20 min, washed and then stained with filipin (0.05 mg/mL) and mounted in Mowiol as described [39 (link)]. Samples were observed using a Leica TCS SP5 laser scanning confocal microscope equipped with a DMI6000 inverted microscope, blue diode (405 nm), Argon (458/476/488/496/514), diode pumped solid state (561 nm), HeNe (594/633 nm) lasers, and APO 63× oil immersion objective lenses (Leica Microsystems, Wetzlar, Germany). Image analysis was performed with NIH ImageJ software as described below [40 (link)].
The quantification of filipin staining per cell (≥20 cells per group) was performed as described [25 (link)] and served to determine the relative amounts of free cholesterol in enlarged, perinuclear cholesterol-enriched and Lamp2A-positive late endosomal vesicles in NPC1 mutant M12 cells.

Concentrated suspensions of three series of preparation: (1) fresh, (2) 2.5% glutaraldehdye-fixed (Electron Microscopy Sciences, Hatfield, PA, USA) and (3) fixed-Nile Red (Sigma-Aldrich Corp., St. Louis, MO, USA) (5 micrograms/mL) treated cells were deposited onto the coverslip areas in glass bottom microwell dishes (MatTek Corp., Ashland, MA, USA) and examined by confocal microscopy using a model TCS SP5 system coupled to a DMI 6000 inverted microscope equipped with a 100× objective lens (Leica Microsystems, Exton, PA, USA) in the x,y,z imaging mode and fluorescence scanning mode with excitation from the 488 nm line of an Argon laser. Images were collected in data sets of two channels (500–550 nm and 660–720 nm) for fresh- and glutaraldehyde-fixed cells or in three channels for Nile Red-treated cell suspensions (500–550 nm, 570–620 nm and 660–720 nm) and examined as maximum projections (8–12 micrometers deep) in separate and graphically overlaid image channels. Fluorescence emission scans were performed from 500–750 nm using a 15 nm detector window and frame averaging of selected focal planes. DIC images were taken using the same method for laser-scanning confocal microscopy; however, the transmitted light channel (non-confocal) was employed here.

Imaging was performed on a Leica TCS SP8 confocal laser scanning DMI6000 inverted microscope (Leica Microsystems, Wetzlar, Germany) using a 63×/1.40 oil CS2 objective coupled to a Hamamatsu camera. Phospholipids were excited at 590 nm (600–700 nm) and neutral lipids at 488 nm (500–580 nm). In addition, line averaging was used during image acquisition to minimize background noise. Quantification of LDs was performed by assessing the total number of monocytes with LDs per field of view (FOV), as well as the number of LDs per positive monocyte in 5–10 FOVs. In order to visualize all LDs in the cell, resolution was significantly enhanced through a digital magnification of 1.5–2, z-stack images of 0.75 μm per stack (1,024 × 1,024 pixels × 8 bit), and subsequently merged into a maximum intensity projection for quantification. A minimal number of eight images per sample were taken. For intracellular LD assessment, an extensive z-stack was made of NR-stained monocytes. Subsequently, for 3D imaging, the software program, Imaris® (version 7.7.0; Belfast, UK), was used for rendering these extensive z-stack images into a 3D model.