Fundamentals of Biophysics at the Nanoscale

Registro delle lezioni

Anno accademico 2023/2024
Docente Francesco Cardarelli, Stefano Luin, Gian Michele Ratto

Lecture

  • 16 Nov 2023 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Experimental uncertainties, probability distributions (Gaussian, Lorentzian, Poissonian), error bars, hints on statistical tests; statistical estimators (average, standard deviation, standard error, confidence level).

  • 23 Nov 2023 (2h 00m)

    Stefano Luin - Corso (attività didattica) - A distanza

    Error propagation. Probability distributions convolution. Covariance matrix and correlation. Noise in measurements, examples in spectroscopy. Fundamentals of lenses.

  • 29 Nov 2023 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Microscopy: objectives, resolution, numerical aperture. Electromagnetic radiation, refraction and diffraction of light, limit of diffraction for resolution. Optical path in transmission microscopes, conjugate planes, field diaphragm and aperture. Optical paths in reflection / epifluorescence microscopes. Transmission microscopy: Köhler illumination. Contrast definitions.

  • 30 Nov 2023 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Contrast techniques (introduction, polarization contrast, dark field); optical path and phase differences. Contrast techniques (phase contrast, DIC). Alignment of a transmission microscope. Contrast techniques in reflection microscopy. Epifluorescence microscopy. Outline of fluorophores (and immunofluorescence) and dichroics / optical filters. Introduction to confocal microscopy

  • 06 Dic 2023 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Confocal microscopy: implementations, Point Spread Function, convolution; magnification and resolution; dependence on signal/noise ratio, variable with laser intensity and detector gain. Dependence of signal/noise ratio and of resolution on the confocal pinhole aperture.

  • 07 Dic 2023 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Aberrations (spherical, chromatic). 2 photon microscopy; TIRF microscopy. Fluorescence microscopy: labelling and types of fluorophores (fluorescent proteins, Qdots, organic fluorophores); chemical tags (ACP and its derivatives): methods of insertion into proteins (transfection, vectors, plasmids, promoters).

  • 13 Dic 2023 (2h 00m)

    Stefano Luin - Corso (attività didattica) - Mista

    More on preparation, insertion, transcription of genes, transcription in mRNA, its translation for the expression of (exogenous) proteins (lentiviral vectors, promoters, controlled expression, insertional mutagenesis, hints on PCR, primers). Advanced methods in fluorescence microscopy: colocalization and its quantifications; FRET and efficiency measurement methods (sensitized emission and its corrections, introduction to FLIM).

  • 20 Dic 2023 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    FRET efficiency measurement by acceptor photobleaching. Calculations on FRET sensitized emission corrections. FLIM (hints). FRET-FLIM with examples. Laws of diffusion, sum over microtrajectories, Stokes-Einstein equation; solution of the diffusion equation starting with a Dirac Delta for the concentration. FRAP: experimental details (corrections, normalizations, geometries, examples).

  • 10 Gen 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    FRAP: analytical treatment (diffusive model, compartment model). Other "FRAP-like" microscopy techniques based on photobleaching: FLIP; iFRAP, photoactivation (PA), FLAP, photoconversion (PC). FCS: (auto)correlation function, its fit for diffusion phenomena, other contributions (dark states, molecules rotation, antibunching). Spectroscopy of single molecules (outline and techniques).

  • 11 Gen 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Single molecules tracking: analysis; types of trajectories, MSD; anomalous diffusion, transient arrest of diffusion. Hints on experiments on membrane receptors and neurotrophins: 2D-distributions of couple of parameters, counting fluorescent molecules in tracked spots. Anomalous diffusion by transient confinement zones and by diffusion-reaction; log-log graphs and power dependencies. Deconvolution. Light-matter interactions: introduction, generalities. Quantum mechanics review; perturbation on a Hamiltonian and transforming the Schrödinger equation to differential equations for the coefficients in the linear combination of the unperturbed hamiltonian eigenfunctions.

  • 24 Gen 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Time-dependent perturbation theory, Fermi's golden rule. Review of Fourier series and transform, Taylor series; Dirac delta. Interactions with radiation. Einstein coefficients; operating principles of lasers. Jablonski diagrams and absorption. Electronic and vibrational spectroscopy: Born Oppenheimer approximation, IR absorption. Vibrational modes and selection rules. Vibronic transitions. Franck Condon factors. Properties, setups and measurement methods for optical absorption (UV-visible-NIR).

  • 25 Gen 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Time evolution of combination of two stationary states; aberrations due to refraction index mismatch in microscopy: geometrical interpretation. Setup for measuring optical absorption (continue): spectrometers and grating. Lambert-Beer law. Fluorescence properties, and measurements methods and setups. Spectrofluorimeter interface. Non-linear light-matter interactions. Differences amongst 2-photon absorption and second harmonic generation. Second order perturbation theory, 2 photon absorption, Raman.

  • 31 Gen 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Raman (continued): Stokes/antiStokes intensity ratio, thermally activated fluorescence. Vibrational spectroscopy: typical energies, vibrations localized on bonds. Vibrational spectroscopy (also setups): IR absorption (outline) and Raman. Vibrational and Raman modes in fluorescent proteins, presentation of experiments in photochromic ones and their chromophores.

  • 01 Feb 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Phoptochromic proteins studied through Raman (continued): photophysics of wtGFP, preresonance conditions, reversible switching mechanisms. Example of stimulated Raman spectrum. SERS and surface resonance plasmons in metal nanoparticles; electromagnetic and chemical enhancement; 3-photon absorption of molecule bound to Au nanoparticles. Fs-resolved Raman (hints). Organic fluorophores: orbitals (pi, sigma, non-bond); molecular transitions; electronic delocalization. Molecular states of formaldehyde.

  • 07 Feb 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Single-double bonds alternation in organic molecules; resonance structures and molecular orbitals. Molecular transitions in formaldehyde (outline). Examples of organic fluorophores. Organic fluorophores: notes on the use of organic fluorophores in fixed or living cells and in vivo. AM loading. Fluorescent quantum dots: structure, spectra, functionalizations. Band structure of solids (hints): insulators, semiconductors, semimetals, metals, band gaps. Transitions in a 2-level system.

  • 08 Feb 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Solvent relaxation in fluorescence. Life times, multiexponential decays, measurement methods (frequency and time domain measurements); temporally resolved microscopy / FLIM; instrumental considerations (gated acquisition, TCSPC, IRF, quadrant mode microchannel plates). Measurements in frequency-domain FLIM. Phasor method in FLIM with examples in microscopy. Structure of biological molecules: molecular forces and energies.

  • 22 Feb 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Hydrophobic effect. Carbohidrates: stereochemistry (isomerism in general); structures and conformations of monosaccharides; glycosidic bonds, disaccharides, polysaccharides (cellulose, starch, glycogen). Glycoproteins and glycolipids. Lipids: categories; fatty acids, triglycerides; membrane lipids. Plasmamembrane structure.

  • 29 Feb 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Nucleic acids (NA): DNA and RNA (structures, functions). Artificial NA. Polymerization and hydrolysis; interactions amongst bases; different helices, persistence length. Secondary and tertiary structures; chromatin structure, histones, nucleosomes, epigenetics (hints). Aptamers and ribozymes. Proteins: amino acids, peptide bond.

  • 06 Mar 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Proteins: amino acids and peptidic bond (cont.); posttranslational modifications; dihedral angles in the backbone and Ramachandran plots; secondary structures: helices, beta sheets, turns, loops; tertiary stucture and its classification; quaternary structure; protein folding, stability and denaturation, flexibility and function. Protein data bank. Experimental determination of structures: introduction

  • 07 Mar 2024 (2h 00m)

    Stefano Luin - Corso (attività didattica) - In presenza

    Experimental determination of structures: x-ray cristallography, missing phase problem and solutions, resolution. PDB files. NMR: hints. Fluorescent proteins: development, structure, characteristics, photophysics, photobleaching, chromophore formation. Proton equilibria in GFP and mutants.