BIOPHYSICS

Discussion of the biophysical methods based on fluorescence spectroscopy and microscopy (confocal microscopy, FLIM, FRET, FCS, superresolution) and their application to the investigation of the cell as a complex physical system.

Critical understanding of the most advanced developments of Modern Physics, both theoretical and experimental, and their interrelations. Knowledge and understanding of advanced biophysical methods based on fluorescence spectroscopy and microscopy. (knowledge and understanding)

Ability to identify the essential elements in a phenomenon, in terms of orders of magnitude and approximation level, and being able to perform the required approximations. Ability to use analogy as a tool to apply known solutions to new biophysical problems (problem solving). Ability to apply physical methods to biological problems. (applying knowledge and understanding)

Ability to convey own interpretations of physical phenomena, when discussing within a research team. Developing one's own sense of responsibility, through the choice of optional courses and of the final project. (making judgements)

Ability to discuss about advanced physical concepts, both in Italian and in English. Ability to present one's own research activity or a review topic both to an expert and to an non-expert audience. (communication skills).

Ability to acquire adequate tools for the continuous update of one's knowledge. Ability to access to specialized literature in the Biophysics field and in closely related fields. Ability to exploit databases and bibliographical and scientific resources to extract information and suggestions to better frame and develop one's study and research activity. (learning skills)

Should the circumstances require online or blended teaching, appropriate modifications to what is hereby stated may be introduced, in order to achieve the main objectives of the course.

Exams may take place online, depending on circumstances.

Frontal lectures. Class attendance is mandatory.

Introduction: what is Biophysics? The variety of topics in Biophysics research. Cellular Biophysics and microscopy. Other topics: Cryo-Electron Microscopy, Intrinsically disordered proteins, Optogenetics, Single-Molecule Biophysics, virus entry into cell.

Biophysics of the cell. The building blocks of life. Model Building in Biology, Quantitative Models and the Power of Idealization. Cells and structures within them: Macromolecular assemblies. Temporal scales in biological processes: the central dogma of biology, the cell cycle. Mechanical and chemical equilibrium in the cell: energy in the cell, free-energy and structure. The Statistical Mechanics of Gene Expression. Random walks and structure of macromolecules: size of genomes, DNA packing, chromatin. Biological membranes: the nature of membranes, vesicles in the cell. Diffusion in the cell, crowded environments.

Fundamentals of fluorescence spectroscopy. Fluorescent probes. Absorption of UV-Visible light. Fluorescence excitation and emission spectra. Extinction coefficient (EC) and quantum yield (QY). Fluorescence lifetime. Polarization (anisotropy) of the emission. Quenching/Photobleaching. Time-resolved fluorescence spectroscopy. Steady state vs time-resolved. Time-domain vs frequency-domain. Instrumentation for lifetime measurements.

Fundamentals of optical microscopy. Image acquisition in optical microscopy. Contrast. Optical Resolution limit. Point Spread Function (PSF) of a microscope. Optical sectioning. Confocal microscopy.

Advanced Fluorescence microscopy methods. Fluorescence Lifetime Imaging Microscopy (FLIM). FLIM acquisition and data analysis. Forster Resonance Energy Transfer (FRET). FRET imaging and applications. Fluorescence-based sensors. Fluorescence methods to measure mobility of molecules: Single-Particle Tracking (SPT), Fluorescence Recovery after Photobleaching (FRAP), Fluorescence Correlation Spectroscopy (FCS) and related techniques.

Optical Super-resolution microscopy. Breaking the diffraction limit (Nobel Prize in Chemistry 2014). Stimulated Emission Depletion (STED) microscopy. Stochastic Optical Reconstruction Microscopy (STORM) and Photoactivatable Localization Microscopy (PALM). Structured Illumination Microscopy (SIM) and related techniques.

Phillips et al. Physical Biology of the Cell, CRC Press 2013

D. Jameson, Introduction to Fluorescence, CRC Press 2014

Valeur, Molecular Fluorescence: Principles and Applications. Wiley-VCH Verlag GmbH 2001

Pawley, Handbook of Biological Confocal Microscopy, Springer 1995