0.8 na air objective

Cells in 96-well plates were imaged using a Zeiss LSM780 AxioObserver Z1 confocal microscope and a Plan-APO 20× 0.8 NA air objective. Images were saved as raw .lsm files and exported to FIJI(ImageJ) for analysis including maximum intensity projections, threshold adjustment and fluorescence intensity quantifications. After thresholding, FIJI’s Analyze Particles function was used for quantifying integrated cell fluorescence. Background fluorescence was measured in five unique and non-overlapping areas for every image and averaged for obtaining the average background fluorescence for a given image. Corrected total cell fluorescence (CTCF) was calculated using the formula CTCF = Integrated Density – (Area of selected cell × mean fluorescence of background readings). Scatter dot pots were generated using GraphPad Prism. Images acquired from three independent replicate cultures were used for quantification and statistics were performed using a two-tailed, non-parametric t test with Mann-Whitney correction, p values were calculated and indicated for each comparison.

Coated Nanopatches were visualised on a Zeiss 510 confocal microscope, using a 20 × 0.8 NA air objective (Zeiss). Dylight 647, 550 and 488 signals were acquired at 5 µm intervals with 1.28 µs pixel/dwell and 12-bit depth. All three channels were collected sequentially, using the following settings: red 633 helium neon laser with a HFT 458/543/633 splitter and filter BP 650–710; blue 488 argon laser with a HFT 488/KP700 and filter LP505; green 543 helium neon laser with a HFT 488/543 and meta detector set to 553–692 nm. Image analyses including 3D reconstruction of the images were performed with Imaris software x64 6.3.1 (Bitplane AG, Zurich, Switzerland).