ABSTRACT
We report a new full-field fluorescence microscopy method for imaging live cell membranes based on supercritical near-field emission. This technique consists of extracting the self-interference between undercritical and supercritical light by simple image subtraction. In the objective back focal plane, this is equivalent to adding a virtual mask blocking the subcritical emission. We show that this virtual mask is radically different from a real physical mask, enabling a 100 nm axial confinement and enhancing the image sensitivity without damaging the lateral resolution. This technique is easy to implement and simultaneously provides images of the inner parts of the cell and its membrane with standard-illumination light.
Subject(s)
Image Processing, Computer-Assisted/methods , Microscopy, Fluorescence/methods , Models, Theoretical , Carbocyanines/chemistry , Cell Membrane/ultrastructure , Fluorescent Dyes/chemistry , Fourier Analysis , HEK293 Cells , Humans , Microscopy, Fluorescence/instrumentationABSTRACT
We introduce a full-field fluorescence imaging technique with axial confinement of about 100 nm at the sample/substrate interface. Contrary to standard surface imaging techniques, this confinement is obtained through emission filtering. This technique is based on supercritical emission selectivity. It can be implemented on any epifluorescence microscope with a commercial high numerical aperture objective and offers a real-time surface imaging capability. This technique is of particular interest for live cell membrane and adhesion studies. Using human embryonic kidney cells, we show that one can observe simultaneously the surface and in-depth cell phenomena.
