Lecture
-
22 Nov 2022 (0h 30m)
Stefano Luin - Corso (attività didattica) - Mista
Presentazione corso.
29 Nov 2022 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
experimental uncertainties, probability distributions, error bars, hints on statistical tests; statistical estimators, error propagation.
01 Dic 2022 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Covariance matrix and correlation. Noise in measurements, examples in spectroscopy. Fundamentals of lenses. Microscopy: objectives, resolution, numerical aperture. Electromagnetic radiation, refraction and diffraction of light, limit of diffraction for resolution.
13 Dic 2022 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Optical path in transmission microscopes, conjugate planes, field diaphragm and aperture. Optical paths in reflection / epifluorescence microscopes. Transmission microscopy: Köhler illumination, 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.
20 Dic 2022 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
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. Dependence of signal/noise ratio and of resolution on the confocal pinhole aperture (recall and completion). Aberrations (spherical, chromatic).
10 Gen 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
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).
12 Gen 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).
17 Gen 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
FRET efficiency measurement by acceptor photobleaching. 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: analytical treatment (diffusive model, compartment model), experimental details (corrections and normalizations).
19 Gen 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Recall: aberrations due to refraction index mismatch in microscopy: geometrical interpretation. 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).
26 Gen 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
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.
31 Gen 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Light-matter interaction: generalities. Quantum mechanics review; time-dependent perturbation theory, Fermi's golden rule. Review of Fourier series and transform, Taylor series; Dirac delta.
06 Feb 2023 (2h 00m)
Francesco Cardarelli - Corso (attività didattica) - In presenza
Case study I
08 Feb 2023 (2h 00m)
Francesco Cardarelli - Corso (attività didattica) - In presenza
Case study I
09 Feb 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
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.
13 Feb 2023 (2h 00m)
Francesco Cardarelli - Corso (attività didattica) - In presenza
Case study II
14 Feb 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Vibronic transitions. Franck Condon factors. Properties, setups and measurement methods for optical absorption (UV-visible-NIR); 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.
15 Feb 2023 (2h 00m)
Francesco Cardarelli - Corso (attività didattica) - In presenza
Case study II
20 Feb 2023 (2h 00m)
Francesco Cardarelli - Corso (attività didattica) - In presenza
Case study III
21 Feb 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Second order perturbation theory, 2 photon absorption, Raman. Raman: Stokes/antiStokes intensity ratio, thermally activated fluorescence. Vibrational spectroscopy: typical energies, vibrations localized on bonds.
22 Feb 2023 (2h 00m)
Francesco Cardarelli - Corso (attività didattica) - In presenza
Case study IV
23 Feb 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Vibrational spectroscopy (also setups): IR absorption (outline) and Raman. Vibrational and Raman modes in fluorescent proteins (continued). SERS and surface resonance plasmons in metal nanoparticles.
27 Feb 2023 (2h 00m)
Francesco Cardarelli - Corso (attività didattica) - In presenza
Case study IV
28 Feb 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
SERS (cont.): electromagnetic and chemical enhancement; 3-photon absorption of molecule bound to Au nanoparticles. Fs-resolved Raman. Organic fluorophores: orbitals (pi, sigma, non-bond); molecular transitions; electronic delocalization. Molecular states of formaldehyde. Molecular transitions in formaldehyde (outline). Examples of organic fluorophores. Band structure of solids (hints): insulators, semiconductors, semimetals, metals, band gaps.
02 Mar 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
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. Transitions in a 2-level system. Solvent relaxation in fluorescence. Life times, measurement methods (photobleaching rate, frequency and time domain measurements); multiexponential decay; temporally resolved microscopy / FLIM; instrumental considerations (streak camera, gated acquisition, TCSPC, IRF, quadrant mode microchannel plates).
14 Mar 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Measurements in frequency-domain FLIM. Phasor method in FLIM with examples in microscopy. Structure of biological molecules: molecular forces and energies, hydrophobic effect. Carbohidrates; stereochemistry (isomerism in general); structures and conformations of monosaccharides.
16 Mar 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Carbohidrates: glycosidic bonds, disaccharides, polysaccharides (cellulose, starch, glycogen). Glycoproteins and glycolipids. Lipids: categories; fatty acids, triglycerides; membrane lipids. Nucleic acids (NA): DNA and RNA (structures, functions). Artificial NA. Polymerization and hydrolisis; interactions amongst bases; different helices, persistence length. Proteins: aminoacids, chirality, peptide bonds.
23 Mar 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - Mista
Proteins: 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: x-ray cristallography and missing phase problem.
30 Mar 2023 (2h 00m)
Stefano Luin - Corso (attività didattica) - In presenza
Crystallography: solutions for the missing phase problem, resolution. PDB files. NMR: hints. Fluorescent proteins: development, structure, characteristics, photophysics, two-photon spectra, chromophore formation. Brainbow: CRE-LoxP system (hints). SplitGFP, GECI, GCaMP. Proton equilibria in GFP and mutants. Example of other fluolrescent proteins. Superresolved microscopy: 4Pi microscopy, STORM and FPALM (only mention and eventually recall); RESOLFT and STED (Concept and generality, saturation, stimulated emission, instrumentation, example of "extinguishing" PSF). Mention of structured illumination (SIM).
02 Maggio 2023 (2h 00m)
Gian Michele Ratto - Corso (attività didattica) - In presenza
In vivo imaging and statistical tools for data analysis
04 Maggio 2023 (2h 00m)
Gian Michele Ratto - Corso (attività didattica) - In presenza
Electrical recordings from the in vivo brain
05 Maggio 2023 (2h 00m)
Gian Michele Ratto - Corso (attività didattica) - In presenza
Molecular crowding and control of homeostasis
06 Maggio 2023 (2h 00m)
Gian Michele Ratto - Corso (attività didattica) - In presenza
In vivo imaging of intracellular chloride
Vai alla Ricerca avanzata