ECTS - University of Pisa
| Modules | Area | Type | Hours | Teacher(s) | |
| FUNDAMENTALS OF BIOPHYSICS AT THE NANOSCALE | ING-IND/34 | LEZIONI | 48 |
|
Fornire le basi (anche teoriche) per comprendere quantitativamente esperimenti in biofisica molecolare e cellulare alla nanoscala; dare un linguaggio comune in biofisica a studenti con background diversi.
Providing (also theoretical) bases for quantitatively grasping experiments in molecular and cellular Biophysics at the nanoscale; giving a common language in Biophysics to students with different backgrounds.
Esame orale
Oral exam
Principi basilari in ottica, in chimica organica ed inorganica, in meccanica quantistica.
Basic principles of Optics, of Organic and Inorganic Chemistry, and of Quantum Mechanics.
Frontal lessons.
Misure in microscopia e spettroscopia [1-4]
Rumore nelle misure, incertezze sperimentali, cenni su distribuzioni di probabilità, propagazione degli errori.
Microscopia in trasmissione, riflessione, epifluorescenza. Ingrandimento e risoluzione; tecniche di contrasto; aberrazioni (sferica, cromatica); cenni su dicroici e filtri ottici.
Microscopia confocale: implementazioni, funzione di allargamento del punto, cenni su deconvoluzione, confronto con microscopia a 2 fotoni e TIRF.
Interazione luce-materia: fondamenti (anche quantomeccanici) e strumentazioni per assorbimento, fluorescenza, Raman, eccitazione a più fotoni. Diagrammi di Jablonski e proprietà della fluorescenza. Fluorofori organici: struttura chimica ed utilizzo in microscopia a fluorescenza. Cenni sui quantum dots. Proteine fluorescenti della famiglia della GFP.
La diffusione ed il moto browniano. Tecniche di microscopia a fluorescenza: colocalizzazione, FRAP e tecniche analoghe, FRET, FLIM (fondamenti, strumenti, metodo dei fasori), FCS, superamento del limite di diffrazione (RESOLFT, STED, F-PALM, SIM), spettroscopia e tracking di singole molecole.
Introduzione alla struttura di molecole biologiche [6]. Proteine fluorescenti e loro fotofisica [4]
Basi di biologia molecolare e cellulare [5]
Procarioti vs eucarioti. Organizzazione generale della cellula eucariotica. Citoplasma: struttura e trasporto di membrana, compartimenti intracellulari, il citoscheletro, trasduzione del segnale. Nucleo: DNA cromosomico e sua organizzazione, il Nuclear Pore Complex e trasporto nucleo-citoplasma. Ciclo e divisione cellulare. Morte cellulare. Tecniche di laboratorio in biologia (Metodi di isolamento delle cellule e crescita in coltura; metodi di purificazione e analisi di proteine, DNA e RNA: metodi di trasfezione; studio dell’espressione e della funzione dei geni; marcatura di molecole di interesse: strategie e limiti)
Basi di elettrofisiologia e microscopia in-vivo
Measurements in microscopy and spectroscopy [1-4]
Noise in measurements, experimental uncertainties, basics of probability distributions, propagation of uncertainties.
Transmission, reflection and epifluorescence microscopy.
Magnification and resolution; contrast techniques; spherical and chromatic aberrations; hints on optical filters and dichroics.
Confocal microscopy: set-up, point spread function, hints on deconvolution, comparison with TIRF and 2-photon microscopy.
Light-matter interaction: fundamentals (also quantum mechanics) and setups for absorption, fluorescence, Raman, and multiphoton excitation. Jablonski diagrams and properties of fluorescence. Organic dyes: chemical structures and exploitation in fluorescence microscopy.
Hints on fluorescent quantum dots. Fluorescent proteins, GFP family.
Diffusion and Brownian motion. Techniques in fluorescence microscopy: colocalization, FRAP-like techniques, FRET, FLIM (fundamentals, instruments, phasors), FCS, super-resolution (RESOLFT, STED, F-PALM, SIM), single molecule spectroscopy and tracking.
Introduction to the structure of biological molecules [6]. Fluorescent proteins and their photophysics. [4]
Basis of molecular and cellular biology [5]
Prokaryotes vs eukaryotes. General organization of the eukaryotic cell. Cytoplasm: membrane structure and transport, intracellular compartments, cytoskeleton, cell signalling. The nucleus: chromosomal DNA and its organization, the Nuclear Pore Complex and nucleus-cytoplasmic transport. Cell cycle and cell division. Cell death. Biology laboratory techniques (Methods for cells growth and analysis; purification and analysis of proteins; DNA and RNA: methods for transfection; analysis of genes expression and function; labelling of molecules of interest: strategies and limits)
In-vivo microscopy and electrophysiology
[1] "Introduzione all'Analisi degli Errori", J. R. Taylor (Cap. 1-4, 9, 11)
[2] "Microscopy from the very beginning", Dr. H. G. Kapitza, © Carl Zeiss Jena GmbH, 1997, 2nd revised edition, disponibile on-line
[3] "Introduction to Confocal Fluorescence Microscopy", Michiel Müller, edited by SPIE press (WA, USA), second edition (2006)
[4] "Fluorescence Applications in Biotechnology and Life Sciences", Ewa M. Goldys ed. (2009), pubblicato da John Wiley & Sons (Hoboken, NJ, USA). Cap. 1-6, 9-11, 16.
[5] "Biologia Molecolare della Cellula", B. Alberts et al. (estratti)
[6] "Biophysical Chemistry", Cantor and Schlimmel; Part I
[1] "An Introduction to Error Analysis", J. R. Taylor (Ch. 1-4, 9, 11)
[2] "Microscopy from the very beginning", Dr. H. G. Kapitza, © Carl Zeiss Jena GmbH, 1997, 2nd revised edition, on-line available
[3] "Introduction to Confocal Fluorescence Microscopy", Michiel Müller, edited by SPIE press (WA, USA), second edition (2006)
[4] "Fluorescence Applications in Biotechnology and Life Sciences", edited by Ewa M. Goldys (2009), published by John Wiley & Sons (Hoboken, NJ, USA), Ch. 1-6, 9-11, 16.
[5] "Molecular Biology of the cells", B. Alberts et al. (chosen parts)
[6] "Biophysical Chemistry", Cantor and Schlimmel; Part I
Esame orale (vedi versione in inglese per dettagli)
The exam is made up of one oral test.
The oral test consists of an interview between the candidate and the lecturer and usually two other experts or teachers; the average length of the interview is 1 hour, and it will usually start with 4-5 questions on the different point of the program (uncertainties in measurement, microscopy, biochemistry, cell biology, and/or spectroscopy/quantum mechanics). More than the notions, the language and the reasoning of the candidate, in particular in linking different parts of the program, will impact on the final result.
https://www.sns.it/it/corsoinsegnamento/fundamentals-biophysics-nanoscale
