Nanometer-Scale Resolution Achieved with Nonradiative Excitation

Abstract

Nonradiative Excitation Fluorescence Microscopy (NEFM) is a promising technique allowing the observation of biological samples beyond the diraction limit. By coating a substrate with an homogeneous monolayer of quantum dots (QDs), NEFM is achieved through a nonradiative energy transfer from QDs (donors) to dye molecules located in the sample (acceptors). The excitation depth of the sample is then given by the Förster radius, which corresponds to few nanometers above the surface. The powerful axial resolution of NEFM is highlighted by observing the adhesion of Giant Unilamellar Vesicles (GUVs) on strong interaction with coated surfaces. In this paper, we demonstrate that the QD-quenching level is valuable to calculate and map the distance between the membrane and the surface with a high precision along the optical axis. By tuning the electrostatic interactions between the membrane and the substrate, we have been able to measure a height displacement of ≈ 1 nm of the lipid membrane. The experimental results were discussed according to simulations, which take into account all the common forces appearing between lipid membranes and surfaces. 1 Keywords Biophotonics, Förster Resonance Energy Transfer, Membrane, Nanoscopy Förster Resonance Energy Transfer (FRET) is now a standard technique, widely employed in biophysics and biophtonics. FRET relies on a nonradiative energy transfer from excited donor molecules to acceptor molecules in their ground state. This energy transfer is ecient as long as the distance between the two molecules is less than 10 nm. FRET is usually considered as a nanoscale ruler with a broad area of applications that includes structural biology, 1 biosensing, 2,3 binding measurement between molecules 4 or structure of intermembrane junction. 5 The signal obtained from FRET is typically examined by spectra, uorescence pictures or by time-resolved investigations. In this paper, we propose an original imaging method, based on donor quenching analysis, to measure, with a nanoscale accuracy, distances involved in the adhesion of membranes on a surface.

Micropatterning of Quantum Dots for Biofunctionalization and Nanoimaging

“…First, S1813 G2 was exposed to the desired patterns. After the S1813 G2 development, the surface is chemically modified with a silanization process, 11 creating an adhesion layer on which QDs grafting happens through the formation of disulfide bridges. This method allows one to obtain a monolayer.…”

“…An axial nanoscale resolution can be achieved, allowing outstanding observation on vesicles or living cells. 11 Besides NEFI, several different optical tools have been developed to study cells and their interactions with various substrates. For example, 3d-RICN (Reflection Interference Contrast Nanoscopy) was developed in order to reconstruct lamellipodia in 3D with nanometric axial resolution.…”

“…This technique is based on Förster Resonance Energy Transfer (FRET) between a surface coated with a monolayer of QDs and fluorescent probes present in the sample. An axial nanoscale resolution can be achieved, allowing outstanding observation on vesicles or living cells . Besides NEFI, several different optical tools have been developed to study cells and their interactions with various substrates.…”

“…Colloidal quantum dots of semiconductor nanocrystals (QDs) have been developed to exhibit narrow emission spectra and tunable photostable emission by changing their size and composition. − They are used in a wide variety of applications such as biosensing, − integrated photonics, , light-emitting diodes, , or fluorescence imaging. , In microscopy, QDs are mostly used as fluorescent probes in replacement of usual organic dye molecules. Several years ago we have introduced Nonradiative Excitation Fluorescence Imaging (NEFI), which involves QDs beyond their usual use in fluorescence labeling.…”

“…1−3 They are used in a wide variety of applications such as biosensing, 4−6 integrated photonics, 7,8 light-emitting diodes, 9,10 or fluorescence imaging. 11,12 In microscopy, QDs are mostly used as fluorescent probes in replacement of usual organic dye molecules. Several years ago we have introduced Nonradiative Excitation Fluorescence Imaging (NEFI), 13 which involves QDs beyond their usual use in fluorescence labeling.…”