Seeing is Believing – Metody pozorování životně důležitých procesů v buňkách a organismech - MB151P109

• Anglický název: Seeing is believing - Methods of observing vital processes in cells and organisms
• Český název: Seeing is Believing – Metody pozorování životně důležitých procesů v buňkách a organismech
• Zajišťuje: Katedra buněčné biologie (31-151)
• Fakulta: Přírodovědecká fakulta
• Platnost: od 2022
• Semestr: zimní
• E-Kredity: 4
• Způsob provedení zkoušky: zimní s.:kombinovaná
• Rozsah, examinace: zimní s.:2/1, Z+Zk [HT]
• Počet míst: neomezen
• Minimální obsazenost: neomezen
• 4EU+: ne
• Virtuální mobilita / počet míst pro virtuální mobilitu: ne
• Stav předmětu: zrušen
• Jazyk výuky: čeština
• Poznámka: povolen pro zápis po webu
• Garant: Mgr. Marek Cebecauer, Ph.D.; Mgr. Aleš Benda, Ph.D.

Anotace

Poslední úprava: Mgr. Aleš Benda, Ph.D. (19.06.2017)

At the molecular level, cellular structures and processes were originally described mainly by biochemical and genetic approaches or indirectly using functional assays and chemical drugs. Rapid development of imaging techniques and specific probes enabled verification and, in the most cases, also highly detailed and direct molecular characterisation in cells and organisms. In this course, problem-oriented lectures will guide students through the exhibition of tools - mainly fluorescent probes – and methodology for studies of biological structures and processes at the molecular level and with focus on quantitation. At the beginning of a lecture, a biological question to solve will be defined. Afterwards, we will describe the available tools and methods used to answer these questions by top scientists. Alternatively, we will suggest different options and discuss advantages and limitations of these diverse approaches. To mention just a few, the topics will cover determination of components of signalling and metabolic pathways and timing of individual steps in cells or small animal (plant) models, quantitative imaging of ions, investigation of cytoskeletal dynamics, membrane trafficking and relocalisation/structural changes in nucleoprotein assemblies or monitoring of cellular responses to oxidative stress. The technology required for the appropriate use of mentioned probes will be explained, mainly by providing examples where clever combination of the right imaging techniques and smartly-designed probes led to break-through findings. Advanced fluorescence techniques such as super-resolution imaging and variants of correlation spectroscopy will be elucidated in more detail to highlight their advantages but also to indicate their limitations and, often, improper use. We will also open the room with label-free techniques (e.g. second and third harmonic imaging) to indicate current state of these methods which fulfil the dream of a biologist to avoid probes and labels.
The practical part will focus on hands-on demonstration of selected applications, including sample preparation, data acquisition, data analysis and data evaluation.

Sylabus

Poslední úprava: Mgr. Aleš Benda, Ph.D. (22.08.2017)

The aim of this course is to teach attendants how to observe physiological and molecular processes directly in cells. A number of biological structures and processes were first demonstrated in vitro. Dramatic development of imaging techniques over last decades enabled single cell/single molecule analysis in real time in living cells. Molecular and cellular processes became accessible for everyday life of biologists, including those in Prague.

In this course, lectures will be problem based; i.e. a question will be stated at the beginning of a lecture and, afterwards, we’ll try to find the best solution for an answer. We will suggest a combination of selected tools (probes), techniques and analytical methods to answer defined question(s). If possible, we will suggest two or three analytical approaches to answer a single question. We will also mention places where such instruments (and frequently expertise) are available in Prague or nearby.

Below, we present a list of questions planned for the lectures. A couple of additional lectures will be dedicated to some excellent science published at the time of the course or suggested directly by students.

1.       What is a shape and size of my cell? Where does it go? Is there any partner cell next to it? WHOLE CELL ORIENTED IMAGE ANALYSIS.

Simple but useful quantitative wide-field epifluorescence microscopy. Interference reflection microscopy. Quantitative phase imaging.

2.       Where is my cell? Is it hidden inside a tissue? Does it cooperate with the surrounding cells? Is it healthy or transformed? ANALYSIS OF TISSUES AND THEIR CELLULAR CONTENT.

Light sheet microscopy. Two-photon microscopy. SHG/THG. CARS.

3.       What keeps the shape of my cell? What happens when a cell responses to chemotactic signal or adhesion to a substrate? Can we predict cellular function from cytoskeleton reorganisation? CYTOSKELETON. CORTICAL SKELETON.

Lattice light sheet microscopy and super-resolution variants. Fluorescent speckle microscopy of cytoskeleton dynamics.

4.       Can we track chromatin remodelling? What about DNA damage? Is my gene transcribed? What is the speed of its transcription? How is it with my RNA on its way to the cytoplasm? Will it survive there? NUCLEUS, NUCLEIC ACIDS AND ASSOCIATED PROCESSES.

Laser-induced DNA damage. FISH and modified techniques. Energy transfer/quenching. Single particle tracking (SPT). Fluorescence correlation spectroscopy (FCS).

5.       Where is my protein of interest translated? Neurons can be pretty long. Is the translation efficient?  What about protein folding? Is it posttranslationally modified? Where and how? Is a protein properly localised? Oups, isn’t it degraded?

PROTEINS AND THEIR LIFE (AND DEATH) IN CELLS.

Immunofluorescence vs. GFP and variants – positives and limitations. Click chemistry. FRET and FRET probes.

6.       My cells are looking bad. Is their energy source in a good shape? Where are those toxic agents coming from? Can I follow stress processes in a cell which is still living? MITOCHODRIA AND THEIR HEALTH. MITOPHAGY. ER STRESS.

SOFI (super-resolution). pH-, redox-, NADPH- and some other metabolic probes. Fluorescence lifetime imaging (FLIM).

7.       Where is this molecule going on? It is lost? How? Can pathogen or dangerous agent enter my cell? Where will it end up? VESICULAR TRAFFICKING. EXO/ENDOCYTOSIS. EXTRACELLULAR VESICLES IN HUMAN PATHOLOGY. VIRAL INFECTION.

SIM (super-resolution). Live cell imaging in microfluidics and other microdevices. Curvature detection.

8.       My protein (lipid) reached cell surface. Is that enough for its function? Or is it somehow organised there and I cannot ‘see’ it with my confocal microscope? What regulates organisation of molecules on cell membranes? What is it good for? PLASMA MEMBRANE AND ITS TOPOGRAPHY. MEMBRANE CONTACT SITES AND TRANSPORT.

SMLM and STED (super-resolution). TOCCSL (just another SPT – but better).   

9.       Both sides are salty, I like sweets. Can I follow specific ions or small molecules helping cells in their business? There is rapid calcium influx, can I investigate the rapid changes it caused?

IONS AND ION CHANNELS. CALCIUM SIGNALLING.

Small molecule probes. Opto-switches and light-controlled tools for biology. Fast imaging. ICS and derivatives

10.   Why is vesicular transport so fast in cells? Can we play a traffic police with speed cameras in cells? Can I follow different forces applied within/at a cell or tissue? CELLULAR AND MOLECULAR MOTORS. FORCES AND MECHANICAL ENERGY.

Atomic force microscopy. Single molecule microscopy. Fluorescent rotors. Anisotropy.