Fig. 1: Schematics and characterization of the optical setup.

a The main components of a confocal laser scanning microscope (CLSM), which is extended to perform iSCAT microscopy in both wide-field and confocal modes. OBJ objective, BS beam splitter, PH pinhole, DC dichroic mirror, EF emission filter, PMT photomultiplier tube. Inset: Wavefronts of laser illumination (dashed lines) and sample radiation (solid lines) for the three modalities. Lateral and axial point spread functions (PSF) of a 100 nm fluorescence-labeled polystyrene bead for wide-field iSCAT (b, c), confocal iSCAT (d, e), and confocal fluorescence (f, g) modalities. The focus was scanned over 4 μm in steps of 30 nm in c, e, and g. The background was accounted for in each z plane. Curves on the right-hand side depict the intensity profiles along the cross sections shown in each figure. Horizontal and vertical scale bars are 200 nm and 500 nm, respectively. h Scanning electron micrograph of a nanofabricated test sample consisting of two chromium pillars of diameter 45 nm, height 45 nm and center-to-center separation 130 nm. Scale bar is 200 nm. i C-iSCAT image of the sample recorded with a pinhole setting of 0.3 AU at a wavelength of 445 nm. Scale bar is 200 nm. j Cross sections along the white dotted lines from (h, orange) and (i, blue). The green curve also shows a cross section from a C-iSCAT image recorded with a pinhole setting of 1.2 AU. k–n The plasma membrane of a HeLa cell simultaneously imaged in W-iSCAT (k), C-iSCAT (l) and confocal fluorescence (m) modes. The plasma membrane was fluorescence-labeled with GFP-GPI. The W-iSCAT image is flat-fielded, whereas the C-iSCAT image is presented in its raw form. n An overlay of the images in l and m. Scale bars in k–n are 2 μm.