ABSTRACT
We show that through-the-objective evanescent microscopy (epi-EM) is a powerful technique to image membranes in living cells. Readily implementable on a standard inverted microscope, this technique enables full-field and real-time tracking of membrane processes without labeling and thus signal fading. In addition, we demonstrate that the membrane/interface distance can be retrieved with 10 nm precision using a multilayer Fresnel model. We apply this nano-axial tomography of living cell membranes to retrieve quantitative information on membrane invagination dynamics.
Subject(s)
Microscopy, Fluorescence/methods , Nanotechnology/methods , Tomography, X-Ray Computed/methods , Cell Adhesion , Cell Membrane/metabolism , Cell Membrane/ultrastructure , HEK293 Cells , Humans , Models, Theoretical , Optics and Photonics , Signal Processing, Computer-AssistedABSTRACT
We demonstrate subwavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fluorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modification on the detection channel. Confocal-supercritical angular fluorescence microscopy would be a powerful tool to achieve high-resolution surface imaging, especially for membrane imaging in biological samples.
Subject(s)
Cell Membrane/metabolism , Microscopy, Confocal/methods , Animals , CHO Cells , Cricetinae , CricetulusABSTRACT
We present a novel technique to remotely measure and control the local temperature within a medium. This technique is based on the observation of the rotational Brownian motion of gold nanocrescent particles, which possess a strong anisotropic light interaction due to their plasmonic properties. Rotational scattering correlation spectroscopy performed on a single nanoparticle is able to determine the local temperature with high accuracy. These nano-thermometers can simultaneously play the role of nano-heaters when absorbing the light of a focused laser beam.
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.
