Thomas Barroca

Direct optical nanoscopy with axially localized detection

Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of ∼150 nm above the coverslip. Researchers exploit direct stochastic optical reconstruction microscopy and dedicated detection of super-critical-angle fluorescence emission to enable direct optical nanoscopy with axially localized detection.

Full-field supercritical angle fluorescence microscopy for live cell imaging.

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.

Confocal supercritical angle microscopy for cell membrane imaging

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.

Label-free evanescent microscopy for membrane nano-tomography in living cells.

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.

Gold nanocrescents for remotely measuring and controlling local temperature.

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.

Supercritical self-interference fluorescence microscopy for full-field membrane imaging

We present a new technique based on the self-interference of Supercritical Angle Fluorescence (SAF) emission in order to perform full-field cell membrane imaging. We show that our Point Spread Function (PSF) engineering technique allows us to obtain a 100 nm axial sectioning while conserving the original lateral resolution of the microscope. The images are acquired using an optical module that can be connected to any fluorescent microscope to simultaneously monitor in real time both the cell membrane and in-depth phenomena.

Supercritical scattering microscoy for quantitative phase in the vicinity of a lamella

In this paper, we discuss the possibility of making a super-axially-resolved image of a biological sample using supercritical angle diffusion. This labeling-free approach is suitable to any microscope equipped with a NAobj < 1.33 microscope objective and can be used either for conventional intensity imaging or for quantitative phase imaging. We expose some results on beads an cells showing the potential of this method.

Towards STED microscopy with nanometric optical sectioning

Circumventing the limit imposed by diffraction is a major issue in the instrumental development to realize finer resolutions in biological samples. With STED microscopy, we exploit the molecular transitions of the fluorescent marker to image well below the Rayleigh criterion. Also in combination with STED, we propose to use an alternative technique for optically sectioning fluorescent emitters close to the water-glass interface by selectively filtering the supercritical emission at the pupil plane. We discuss the instrumental development of such a system and its combination with other imaging techniques.