In this paper, we have designed and fabricated an atomically thin plasmonic sensing substrate based on two-dimensional phase change material Ge2Sb2Te5 and silver (Ag-GST). This substrate offers an ultra-low reflection in the SPR curves and a strong optical phase singularity. A custom-built SPR setup was developed here to directly measure the phase-singularity-induced lateral position shift. We have obtained a SPR sensitivity regarding the lateral position shift of 9.9577 x 10^7 μm/RIU, which is 3 orders of magnitudes higher than current position shift sensing scheme based on hyperbolic metamaterial. Due to the ultra-high SPR sensitivity, the binding processes between peptide and integrins directly from un-purified liposomes were real-time monitored. The concentrations of Mn2+ ions ranging from 1 fM to 1 mM on the binding dynamics have been systematically monitored with our developed phase-sensitive surface plasmon resonance biosensors.
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 (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.
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.
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.
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