Cyrille Vézy

Axial nanoscale localization by normalized total internal reflection fluorescence microscopy

We present a simple modification of a standard total internal reflection fluorescence microscope to achieve nanometric axial resolution, typically ≈10  nm. The technique is based on a normalization of total internal reflection images by conventional epi-illumination images. We demonstrate the potential of our method to study the adhesion of phopholipid giant unilamellar vesicles.

Nanoscale characterization of vesicle adhesion by normalized total internal reflection fluorescence microscopy.

We recently proposed a straightforward fluorescence microscopy technique to study adhesion of Giant Unilamellar Vesicles. This technique is based on dual observations which combine epi-fluorescence microscopy and total internal reflection fluorescence (TIRF) microscopy: TIRF images are normalized by epi-fluorescence ones. By this way, it is possible to map the membrane/substrate separation distance with a nanometric resolution, typically ~20 nm, with a maximal working range of 300-400 nm. The purpose of this paper is to demonstrate that this technique is useful to quantify vesicle adhesion from ultra-weak to strong membrane-surface interactions. Thus, we have examined unspecific and specific adhesion conditions. Concerning unspecific adhesion, we have controlled the strength of electrostatic forces between negatively charged vesicles and various functionalized surfaces which exhibit a positive or a negative effective charge. Specific adhesion was highlighted with lock-and-key forces mediated by the well defined biotin/streptavidin recognition.

Membrane-substrate separation distance assessed by normalized total internal reflection fluorescence microscopy

As a consequence of the recent progress in nanoscale technology, more and more sensitive methods are developed to characterize and understand the dynamic of cell membrane adhesion process. In this paper we present a new quantitative method to measure the separation distances between the membrane and the substrate. This technique is based on a normalization of Total Internal Reflection Fluorescence (TIRF) images by usual epi-illumination images. This simple method allows to achieve a nanometric axial resolution, typically 10 nm. We demonstrate the potential of our technique through the study of phospholipids membranes such as Giant Unilamellar Vesicles (GUVs), which are usual biomimetic systems to investigate membrane-substrate interactions.

Variable-Angle Total Internal Reflection Fluorescence Microscopy: Exploring Integrin-mediated Adhesion

accessibility, spatiotemporal confinement and oligomerization states from a single analysis of a few seconds in living cells.The combination of fast sampling speed (down to 1.23 ms per frame) and super-resolution capability opens the doors to the investigation of spatiotemporal dynamics that are not accessible with any other method, at present. We will demonstrate the possibility and the advantages of having access to several methods in a single acquisition and will show several examples of application in live cells, for instance by analyzing EGFP diffusion in NIH-3T3 cells.

Vesicle adhesion depicted at nanoscale with normalized total internal reflection fluorescence microscopy

We propose a novel fluorescence microscopy technique to study the adhesion of Giant Unilamellar Vesicles. Their adhesion is assessed through the normalization of Total Internal Reflection Fluorescence (TIRF) images by epi-fluorescence ones. This can be achieved by using a motorized rotatable mirror to switch from epi-fluorescence to TIRF. This method allows us to study the vesicle adhesion from ultra-weak to strong membrane surface interactions and to measure the absolute distance between the vesicles and various chemically functionalized glass substrates at the nanoscale.

Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy(Conference Presentation)

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a widespread technique to study cellular process occurring near the contact region with the glass substrate. In this field, determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters more and less well known (local concentration of dyes, absorption cross section, angular emission pattern…). The strategy to get around this problem is to exploit a series of TIRF pictures recorded at different incident angles in evanescent regime. This technique called variable-angle TIRF microscopy (vaTIRFM), allowing to map the membrane-substrate separation distance with a nanometric resolution (10-20 nm). vaTIRFM was developed by Burmeister, Truskey and Reichert in the early 1990s with a prism-based TIRF setup [Journal of Microscopy 173, 39-51 (1994)]. We propose a more convenient prismless setup, which uses only a rotatable mirror to adjust precisely the laser beam on the back focal plane of the oil immersion objective (no azimuthal scanning is needed). The series of TIRF images permit us to calculate accurately membrane-surface distances in each pixel. We demonstrate that vaTIRFM are useful to quantify the adhesion of living cells for specific and unspecific membrane-surface interactions, achieved on various functionalized substrates with polymers (BSA, poly-L-lysin) or extracellular matrix proteins (collagen and fibronectin).

Non-radiative excitation fluorescence microscopy

Non-radiative Excitation Fluorescence Microscopy (NEFM) constitutes a new way to observe biological samples beyond the diffraction limit. Non-radiative excitation of the samples is achieved by coating the substrate with donor species, such as quantum dots (QDs). Thus the dyes are not excited directly by the laser source, as in common fluorescence microscopy, but through a non-radiative energy transfer. To prevent dewetting of the donor film, we have recently implemented a silanization process to covalently bond the QDs on the substrate. An homogeneous monolayer of QDs was then deposited on only one side of the coverslips. Atomic force microscopy was then used to characterize the QD layer. We highlight the potential of our method through the study of Giant Unilamellar Vesicles (GUVs) labeled with DiD as acceptor, in interaction with surface functionalized with poly-L-lysine. In the presence of GUVs, we observed a quenching of QDs emission, together with an emission of DiD located in the membrane, which clearly indicated that non-radiative energy transfer from QDs to DiD occurs.

Application of fluorescence fluctuation spectroscopy to the measurement of the concentration of molecules deposited on solid substrates

Fluorescence fluctuation spectroscopy is applied to study molecules, passing through a small observation volume, usually subjected to diffusive or convective motion in liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate or imbedded in a thin film, with a resolution of a few micrometers. The principle is to translate the solid substrate in front of a confocal fluorescence microscope objective and to record the subsequent fluctuations of the fluorescence intensity. The validity of this concept is investigated on model substrates (fluorescent microspheres), DNA-chips, and dye-stained histidine molecules anchored on silanized glass surfaces.