Physics of the living cell
Prerequisiti
First-, second-, third-, fourth-, and fifth-year undergraduate students from all disciplines. The examination may also be taken by PhD students and is particularly recommended for students enrolled in the PhD programmes in “Quantum Technology and Nanoscience”, “Chemistry”, “Physics”, and “Neuroscience”.
Programma
Physics of the Living Cell
Basic principles, open questions, and advanced methods
Introduction to cellular biophysics
- From Schrödinger to contemporary cellular biophysics: fundamental questions, physical approaches, and applications.
- Spatial and temporal scales of cellular processes.
- The role of quantitative measurements and physical models in the study of living systems.
Fluorescence microscopy for exploring biological systems
Fundamentals of fluorescence
- Basic principles of fluorescence.
- Photophysical properties of fluorophores.
- Excitation and emission spectra, quantum yield, photobleaching, and phototoxicity.
Fluorescent probes and labelling strategies
- Endogenous cellular fluorophores and label-free microscopy: principles and applications.
- Genetically encoded fluorescence: green fluorescent protein and its variants.
- Emerging approaches: organic fluorophores for live-cell imaging.
Fluorescence-based methods for probing molecular interactions
- Förster resonance energy transfer (FRET).
- Fluorescence anisotropy.
- Fluorescence lifetime and its sensitivity to the local nanoscale environment.
- Methods for fluorescence lifetime measurement and data analysis.
Fluorescence-based methods for probing molecular dynamics
Perturbation-based methods
- Fluorescence recovery after photobleaching (FRAP).
- Interpretation of molecular mobility, diffusion, and binding processes.
Fluctuation-based methods
- Principles of fluorescence correlation spectroscopy (FCS).
- From single-point FCS to spatial and spatiotemporal correlation methods.
- Scanning FCS, raster image correlation spectroscopy, and pair-correlation approaches.
Localization- and tracking-based methods
- Single-molecule localization and tracking.
- Quantitative analysis of molecular trajectories.
- Orbital tracking and feedback-based imaging methods.
Fluorescence-based methods for super-resolution imaging
- The diffraction limit and the principles of super-resolution microscopy.
- Expansion microscopy.
- Single-molecule localization microscopy.
- Photoactivatable and photoswitchable fluorophores.
- PALM and STORM microscopy.
- Stimulated emission depletion microscopy (STED).
Biological case studies
Case study 1 — Membrane heterogeneity
Does membrane heterogeneity exist? How can it be measured, and what is its functional significance?
Case study 2 — Organization of the prokaryotic and eukaryotic cytoplasm
Molecular crowding, phase separation, and membrane-less organelles.
Case study 3 — The nuclear pore complex
What is its structure, and how does it regulate selective transport between the nucleus and the cytoplasm?
Case study 4 — Dynamic measurements in dynamic systems
How can molecular dynamics be measured within moving and continuously reorganizing subcellular organelles?
Obiettivi formativi
The course aims to provide students with a quantitative understanding of the physical principles governing the structure, organization, and dynamics of living cells, with particular emphasis on processes occurring at the micro- and nanoscale. Students will become familiar with the main experimental approaches used to investigate cellular and subcellular phenomena, as well as with the criteria required to interpret their results critically. The course also aims to provide an up-to-date overview of key frontier topics in cellular biophysics, linking physical models, advanced investigative methods, and biological questions.
Riferimenti bibliografici
Phillips et al. Physical Biology of the Cell; Principles of Fluorescence Spectroscopy Third Edition Joseph R. Lakowicz