BIOPHYSICS

Critical discussion of the biophysical methods based on fluorescence spectroscopy and microscopy (confocal microscopy, FLIM, FRET and FCS techniques, superresolution microscopy techniques) and their application to the investigation of biological macromolecules and 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)

The teaching will be carried out through lectures.  150 hours of total commitment of which 108 of individual study and 42 of frontal lessons

Knowledge of Physics (optics and structure of matter) acquired during the three-year degree.

Class attendance is mandatory

Introduction: what is Biophysics? Motivation. 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. Cells and 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.

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.

Optical microscopy. Image acquisition in optical microscopy. Contrast. Optical Resolution limit. Point Spread Function (PSF) of a microscope. Optical sectioning techniques. Confocal and multiphoton excitation microscopy. Imaging in multiple dimensions: 3D imaging, multi-color imaging, time-lapse. Image processing and quantitative image analysis.

Advanced fluorescence microscopy techniques. Fluorescence Lifetime Imaging Microscopy (FLIM). Phasor analysis of FLIM. 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). Principles and applications of FCS. Image correlation and cross-correlation spectroscopy (ICS and ICCS) and related techniques.

Super-resolution microscopy techniques. 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. Separation of photons by lifetime tuning (SPLIT) and related techniques. Applications to the imaging of cells and macromolecules.

 

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

Materials provided during the course.

Oral examination on the topics of the course. Students should start the exam with the description of a topic of choice. The topic of your choice should be exposed through a presentation (e.g. powerpoint), in order to also evaluate their communication skills. All the topics covered during the course will be examined. The assessment of learning can also be carried out online, if necessary.

Physical origin of the spectral properties of fluorescence

Optical sectioning techniques

Detection and analysis of FLIM images

Analysis of intensity fluctuations in FCS and ICS

Description of a super-resolution microscopy method

Description of an application to biological imaging