Las x software platform

Images of SH-SY5Y cells were obtained with a non-confocal fluorescence microscope (Leica DMI6000 B) using a 60×/1.4 NA oil immersion lens and the LAS X software platform (Leica). Images of hiPSCs and hiPSC-derived motor neurons were taken with a non-confocal Eclipse Ti-2 epifluorescence microscope (Nikon) using the NIS-Elements AR software (Ver 5.01) and either a 20×/dry or 60×/1.4 NA oil immersion lens. Confocal images of hiPSC-derived motor neurons and mouse spinal cord were obtained with a super-resolution VT-iSIM microscope (Nikon) using a 100×/1.49 NA oil immersion lens. Deconvolution was performed with the NIS-Elements AR software (Ver 5.01) using the Richardson/Lucy algorithm with 20 iterations. For printing, brightness and contrast of individual channels were linearly enhanced using the Fiji software^{69 (link)}.

Transfected cells were grown to confluency on 35 mm glass-bottomed dishes (Corning). Cells were washed in HBSS and incubated in HBSS imaging buffer [HBSS supplemented with 5 mM glucose, 10 mM HEPES, 1 mM MgCl₂, 1.26 mM CaCl₂] with 25 nM tetramethylrhodamine (TMRM) for 30 min at 37°C. Cells were imaged immediately, with TMRM retained in the buffer, using a Leica SP8 confocal microscope and LAS X software platform, at 37°C.

Images of the root tips from plants grown vertically for 6 days, then placed at a 45° angle from the vertical for a further 3 days, were taken using a Leica DFC365FX camera attached to a Leica M205FA stereo microscope (Leica Microsystems, Cambridge, UK) with a Planapo × 1.6 objective set to magnification of × 80.5. Images were stitched using the LAS X software platform (Leica Microsystems). Following Wang et al. (2011), CFR was defined as the number of epidermal cell files that crossed a 1‐mm‐long straight line drawn down the longitudinal axis of the root from 1.5 to 2.5 mm from the root apex. Using the same images as for CFR measurements, root diameter was measured approximately 2 mm from the root apex using ImageJ (Schneider et al., 2012), three measurements were done per individual root.

Cells were plated on No. 1.5 square 22x22 mm coverslips (Corning). Primary antibodies used for immunofluorescence are: anti-Cx43 (Sigma-Aldrich C6219) and anti-EGFR (Santa Cruz sc-373746). Secondary antibodies are Alexa Fluor 488 (Thermo-Fisher Scientific) and Alexa Fluor 594 (Thermo-Fisher Scientific). Imaging was performed using 63X oil immersion objective (total magnification 630X) on a Leica TCS SPE confocal microscope and processed using the LAS X software platform (Leica Microsystems Inc., Buffalo Grove, IL).

Colocalization analysis of immunofluorescence images was conducted through a LAS X Leica software platform (Version 3.3.0.16799) in accordance with the manufacturer’s protocol. The data used were respective data collected over an interval of 500 nm in the Z axis in individual cells, from 3 cells per each condition. For analysis of cellular uptake of Alexa-TiO₂-PEG NPs to measure the size and the number, the raw data were analyzed by Python Script that functions using Anaconda (Anaconda, Inc., Austin, TX, USA) and Spyder. The results of the image analysis were further investigated using LAS X software (Leica Microsystems, Bensheim, Germany)). The amounts of Alexa-TiO₂-PEG NPs in cells were calculated using the spherical volume from the analyzed size, and TiO₂ material density (4.23 g/cm³). We assumed the shape of single tumor cell as conical, then calculated the volume of cell using their length and height from the image data taken in Section 4.7. The amounts of Alexa-TiO₂-PEG NPs was divided by the volume of cell to determine the intracellular concentration of NPs.

After dewaxing and rehydration, samples were subjected to heat-induced epitope retrieval (HIER) in 10 mM citrate buffer pH 6.0, then probed in a sequential manner with appropriate antibodies as indicated in Supplementary Table 4. The relevant antigen–antibody complexes were detected using secondary antibodies conjugated with fluorescent dyes (Invitrogen, Paisley, U.K). Cell nuclei were stained with DAPI. After mounting, images were captured with a Leica AF6000 microscope (Leica, Milton Keynes, UK) and processed using the standard LASX Leica software platform (Version 1.9.013747). For quantification studies, randomly selected insulin-containing islets (ICIs) from individuals with or without diabetes were imaged, in addition to insulin-deficient islets (IDIs) from individuals with diabetes. Thirty ICIs were analyzed from 6 independent individuals (5 islets per individual), 20 IDIs were analyzed from 4 independent individuals (5 islets per individual) and 30 ICIs were analyzed from 6 independent control individuals (5 islets per individual). The mean fluorescence intensity (MFI) arising from detection of HLA-E was measured using LASX Leica quantification software.