Advanced methods in biological physics and soft matter

A (not fixed) list of potential experiments :

• Droplet-based microfluidic towards cell biology.

   

• Magnetic tweezers : this micromanipulation tool will be used to determine single DNA molecule elastic properties and to observe single molecule enzymatic activity.

   

• Fluorescence Correlation Spectroscopy : in situ measurement of absolute concentrations and diffusion coefficients : The students will built a optical microscope using 2 photons excitation. The fluctuations of the fluorescence from the excitation volume will be used to measure diffusion coefficients of nanoparticles or absolute fluorophore concentrations.

   

• Super-resolution microscopy on fixed cells , 2D and 3D :
  Thanks to its specificity and ability to image living cells, fluorescence microscopy is widely used to observe cellular structures. However, due to the diffraction property of light, only structures above 250nm can be resolved with light microscopy. This resolution limit conceals many cellular complexes and structures.
  Super-resolution regroups several optical microscopy techniques that allow breaking the diffraction barrier. Single-molecule based techniques rely on separating the emission of individual molecules in space and in time in order to localize their centers with very high precision.
  The purpose of this practical is to introduce the principle of super-resolution single-molecule localization microscopy (SMLM). The different parameters that affect the quality of obtained images and precision of localization will be discussed. The students will have the possibility to acquire 2D SMLM images of labeled structures in cells. The analysis will be performed using an ImageJ plugin.
  In the second part of the practical the principle of SMLM will be expended to the third dimension. We will introduce the 3D point spread function of the microscope and show how to localize single-molecules based on the distortion of the PSF using astigmatic lens. The final aim is to define and compare the resolution in widefield images and super-resolution images acquired in the same cell.

• Total internal reflection fluorescence microscopy : Brownian motion, near-wall velocimetry and particle-surface interactions

   

• Fluorescence microscopy and nanoscopy : Anatomy of a "home made" fluorescence microscope based on total internal reflection fluorescence and visualisation of supercritical angle fluorescence (SAF). Live cells acquisition with SAF imaging. In a second part, we will see how the same microscope can be used for single molecule imaging (SMLM), acquisition on fixed cells and discussion will illustrates the different keys prameters to reach nanometric resolution. Spectral unmixing of dyes with close emission spectra will be imaged to obtained 3D multicolor imaging of multi proteins in fixed cells.

• Cell mechanics : force measurement on artificial cells.

   

• Microfluidics 2 : In this lab, students will be introduced to the fundamentals of microfluidics, with an emphasis on biomedical applications. We will briefly discuss micro-scale fluid mechanics, and the fabrication of standard microfluidic devices. In the experimental part, students will use a microfluidic device that enables loading, trapping and on-chip attachment of biological specimens for high-throughput single-molecule imaging.

   

• Small Angle scattering : during the experimental classes, the participants will prepare samples and characterized them using Small Angle X ray Scattering on the apparatus available at Laboratoire Léon Brillouin,(CEA Saclay). The formalism presented during the lectures will be then used in order to interpret the collected experimental data and determine the structural features of the measured samples.

   

• Probing sensorimotor computations in the vertebrate brain using whole-brain functional imaging

   

• Label-free virus detection with full field interferometric microscopy : In this practical course, we will introduce a new interferometric and non-destructive optical approach to detect, count and sort different types of label-free, biotic and non-biotic single nanoparticles (NPs). We will measure the light scattered by the nanoparticles and obtain an interferometric signal related to their size and refractive index ; this measurement will be complemented with single particle tracking of their Brownian motion in order to obtain the diffusion coefficient and size of the light-diffusing NPs. The ability of classifying virus in a given environment according to their size and structure is an essential tool in applications ranging from environmental to medical studies. During this course, the participants will learn how to build such an optical set-up, the nature of the signal from in an interferometric microscope, and understand the differences with fluorescence microscopy. You will also learn about single-molecule detection in a non-fluorescence context, as well as the basics of Brownian diffusion and its application for analyzing a data set of single-particle traces.

   

• 3D super-resolution imaging in single-molecule localization microscopy (SMLM) using adaptive optics.

Evaluation will be based on the articles the students will present , a poster they will make to present one of their experiment 

Good knowledge in Optics, Statistical physics and basic notions in biology.