Christian Pieralli

Use of a scanning near-field optical microscope architecture to study fluorescence and energy transfer near a metal

Fluorescence intensity depends strongly on the distance between the emitting molecule and a metallic interface. We show that a scanning near-field optical microscope (SNOM) is a simple and versatile tool for studying such an effect. The fluorescent molecules are embedded in a layer upon a silica substrate, and metal is coated on the SNOM tip. We present variations of fluorescence intensity versus tip-sample distance from 800 to ~80 nm . A simple model is used to explain the experimental results. The proposed setup could be used to study nonradiative transfer at a nanometric scale. It could also yield to a new type of optical near-field profiler that uses fluorescent signal.

Reflection scanning near-field optical microscopy (R-SNOM) in constant height mode with a dielectric probe Image interpretation and resolution for high topographic variations

Abstract The understanding of the correlation between the near-field images that are recorded by scanning near-field optical microscopy (SNOM) and the local optical properties of the sample surface (topography, index, etc.) is a condition for the development of such microscopes. The aim of this paper is to show that the “constant height imaging” (CHI) mode provides useful near-field characterizations, even in case of high-relief samples. Actually, the CHI near-field signal is free from perturbations brought by usual feedback regulation systems. Furthermore, we show a comparison between experimental and theoretical data to explain near-field image formation. Finally, we develop a specific method based on the Fourier spectral analysis to characterize the experimental SNOM setup working in CHI mode.

Image resolution in reflection scanning near-field optical microscopy using shear-force feedback: characterization with a spline and Fourier spectrum

Scanning near-field optical microscopes (SNOMs) actually lead to nanometric lateral resolution. A combination with shear-force feedback is sometimes used to keep the SNOM tip at a constant force from the sample. However, resolutions in shear-force and optical data are different. An estimation of both resolutions is important for characterizing the capabilities of such systems. The basic principle of the measurement is to compare a spline-fitted logarithm of the power spectra calculated with the optical image with that of the shear force image in which resolution is determined a priori. Quantitative results are given in the case of periodic or untested sample and simulated data. Moreover the accuracy and the stability of the method are discussed.

Gold/Silica biochips: Applications to Surface Plasmon Resonance and fluorescence quenching

We report Gold/Silica biochips for low cost biosensor devices. Firstly, the study of biochemical interactions on silica by means of Surface Plasmon Resonance (SPR) is presented. Secondly, Gold/Silica biochips are employed to reduce the strong quenching that occurs when a fluorophore is close to the gold surface. Furthermore, the control of the Silica-like thickness allows optimizing the distance between the metallic surface and the fluorophore in order to enhance the fluorescent signal. These results represent the first steps towards highly sensitive, specific and low cost biosensors based, for example, on Surface Plasmon Coupled Emission (SPCE) techniques.

Scars collaborative telediagnosis platform using adaptive image flow

Telemedicine has been developed to allow practitioners to remotely connect with patients and with other medical staff. We propose a new system hardware and software, named DICODERM COllaborative DIagnosis of DERMatosis, which makes it possible to monitor the evolution of scars after the excision of a tumorous dermatosis like melanoma. The hardware part of this system is composed of a new optical innovative probe with which two types of images can be acquired simultaneously: anatomic with a white light image and functional with a fluorescence image using autofluorescence from the protoporphyrin within the cancer cell. The software part is composed of two components: the image stitching component, and the collaborative/adaptive layer component. Our system creates a panoramic view of these scars obtained by stitching a sequence of small images. We conducted experiments for different image stitching algorithms to define the best solution. We also deployed a second component: a collaborative system layer which allows to remotely share images of scars and to adapt these images. We also made the system adaptive to communicate across different client platforms. We conducted experiments to compare the exchange of images with or without adaptation: these tests showed the efficiency of our layer.

Gold/silica thin film for biosensors applications: Metal enhanced fluorescence

The work described here concerns the fabrication of cost-effective biosensors that permit to amplify a fluorescence signal without a complex nano-structuration of the surface. The idea is to put to profit the natural pseudo nano-structuring that is observed when depositing metallic layers by various micro-fabrication techniques. This new architecture consists of a glass substrate. A gold film is deposited on the top of it and a silica layer onto the gold. A dye (Cy5) is then absorbed onto the surface and the fluorescence intensity is measured. This intensity depends on the distance between the dye and the metal. It also depends on the properties of the metallic film. The goal of the work is to determine which gold deposition method leads to the highest fluorescence amplification and which silica thickness is required to achieve this amplification.

Microsensors and image processing for single oocyte qualification: toward multiparametric determination of the best time for fertilization

During intracytoplasmic sperm injection (ICSI) attempts, oocytes reaching metaphase II are microinjected. A morphological examination under a microscope is the usual method for determining oocyte maturity. The level of oocyte maturity is based on the meiotic status (Germinal Vesicle, metaphase I and metaphase II) of the oocytes with respect to their increasing maturity. In this letter, we summarize the studies conducted to analyze cytoplasm maturity using various microsystems and image processing. Optical microsystems are used to measure the transmission spectra and refractive index of the oocytes. We compared the transmission spectra measurements to the transmission electron microscopy results. Karhunen–Loeve transform is also used to evaluate the maturity of the oocytes. To summarize, optical analysis techniques are a minimally invasive technology allowing cytoplasm maturity to be assessed. Oocytes should not only be qualified in terms of GV, MI or MII, but also regarding their temporal evolution over the course of these maturation stages. The ultimate aim of this work is to describe the maturation of the oocytes by a trajectory in a multidimensional space and to determine when would be the best time for successful fertilization.

ESTIMATION OF POINT-SPREAD FUNCTIONS AND MODULATION-TRANSFER FUNCTIONS OF OPTICAL DEVICES BY STATISTICAL PROPERTIES OF RANDOMLY DISTRIBUTED SURFACES

The point-spread function a(PSF) and the modulation-transfer function (MTF) are important tools to characterize the information transfer through optical devices. They give useful information about the resolution. Several methods have already been achieved to calculate the PSF and the MTF from theoretical aspects of wave propagation or from experimental results. I present a novel way of estimating these two functions. It deals with statistical considerations for a randomly distributed surface involving a statistical determination of the PSF and the MTF. Indeed, in this case the theoretical shape of the autocorrelation function of such surface profiles is known. It is a decaying exponential function α[exp(-β|x|)]. Comparingthe theoretical autocorrelation-function profile with the experimental one and deconvolving in Fourier space leads to an estimation of the MTF of the imaging device. Applying the inverse Fourier transform to the MTF involves the computation of the PSF, assuming that the latter has no imaginary part and is symmetrical. The two-dimensional images are regarded as an iteration of one-dimensional ones according to the orthogonal direction. The MTFs and PSFs are therefore one-dimensional. Different results are presented. The first result proceeds from investigation with scanning near-field microscopy and illustrates the method step by step. The tunneling effect is detected assuming that the information transfer is linear. The last result concerns an optical profilometer, and the influence of the microscope objective is studied.

Determination of the resolution in scanning near-field optical images with the help of shear force feedback

One of the challenges for newly born probe microscopies is the estimation of their capabilities. The latter is closely related to the reachable resolution. We present a method adapted to these microscopies for determining resolution. It is applied to Reflection Scanning Near-field Optical Microscopy combined with shear-force (ShF) feedback but the method is quite general. The resolution is deduced from the comparison of the optical data spectrum to the shear force data one. Indeed, it is well known that spatial resolution of shear force is better than that of optical microscope and the optical resolution can thus be estimated through this comparison. Actually, the considered set-up uses ShF feedback. Quantitative results are given either with periodic or non periodic objects.

Modeling of C-SNARF-1 pH Fluorescence Properties: Towards Calibration Free Optical Fiber pH Sensing for in Vivo Applications

Organic functions of the human body are related to biological constants. Variations of these constants, among them pH, induce pathological troubles. The general goal of our work is to fabricate a fluorescent pH sensor at the end of an optical fiber for in vivo pH measurements. One difficulty using fluorescence indicators is the need to perform an accurate calibration. In this communication, we present methods used to simplify and potentially avoid calibration procedures of fluorescence indicators. The first method concerns the simplification of calibration procedures making them independent of the indicator’s concentration, path length and equipment used. The second method concerns modelling the fluorescence emission of the molecules as a function of pH only. This model is used to fit the exact shape of C-SNARF-1 fluorescence spectra obtained at any pH. Subsequently, the pH of a solution can be computed with an accuracy of 0.1 pH unit without the calibration procedure employed up to now. These methods constitute the first steps toward calibration free pH measurements. They can be applied to any fluorescent indicator exhibiting a dual emission peak. As a conclusion, this is the first time that fluorescence properties of C-SNARF-1 are fully mathematically