Seeing is believing I - Methods of observing vital processes in tissues and organisms - MB100P02

• Title: Vidět znamená chápat a věřit I - Metody pozorování životních procesů v tkáních a organismech
• Czech title: Vidět znamená chápat a věřit I - Metody pozorování životních procesů v tkáních a organismech
• Guaranteed by: Biology Section (31-101)
• Faculty: Faculty of Science
• Actual: from 2020 to 2021
• Semester: summer
• E-Credits: 4
• Examination process: summer s.:
• Hours per week, examination: summer s.:2/1, C+Ex [HT]
• Capacity: 20
• Min. number of students: unlimited
• 4EU+: no
• Virtual mobility / capacity: no
• State of the course: not taught
• Language: English
• Explanation: Observing and quantifying cellular processes at molecular levelOnline teaching Wednesday at 13:00 !!https://meet.google.com/jct-woep-vsm
• Additional information: https://meet.google.com/jct-woep-vsm
• Note: enabled for web enrollment; the course is taught as cyclical
• Guarantor: Mgr. Aleš Benda, Ph.D.; Mgr. Marek Cebecauer, Ph.D.

Annotation

Last update: Mgr. Aleš Benda, Ph.D. (11.01.2024)

Rapid development of imaging techniques and specific probes enabled observation of cellular physiology at the molecular level and live. One can visualise important details of cellular structures and perform direct quantitative measurements of molecules in their native environment, e.g. organelles, cells, tissues. In this course, problem-oriented lectures will guide students through the exhibition of tools - mainly fluorescent probes and imaging techniques – which allow studies of biological structures and processes at the molecular level but also their quantitative analysis. 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 and forces, membrane and virus trafficking and involvement of molecular motors or monitoring of cellular responses to oxidative stress (see the syllabus). The technology required for the use of appropriate probes will be explained, mainly by providing examples where clever combination of the imaging techniques and smartly-designed probes led to the break-through findings. Basic microscopy methods (e.g. rapid wide-field epifluorescence) will be explained in more detail to highlight their simplicity and, at the same time, their capacity to provide temporal and quantitative information about ions, signalling molecules or applied forces in living cells. We will highlight benefits of advanced fluorescence techniques such as super-resolution imaging or variants of correlation spectroscopy but also discuss their limitations and, often, improper use. The lectures will be peppered with discussions about advantages and limitations of chemical or genetically-encoded fluorescent sensors used to uncover rapid changes in levels of intracellular molecules (e.g. secondary messengers).
The practical part will focus on hands-on demonstration of selected applications, including sample preparation, data acquisition, data analysis and data evaluation.

Literature

Last update: Mgr. Aleš Benda, Ph.D. (14.10.2018)

Supporting literature (not required for the exam):

Pawley et al. Handbook of Biological Confocal Microscopy, 3^rd Edition. Springer Science; 2006. ISBN-13: 978-0387259215

Peng Xi. Optical Nanoscopy and Novel Microscopy Techniques. CRC Press; 2014. ISBN 9781466586291

Diaspro et al. Nanoscopy and Multidimensional Optical Fluorescence Microscopy. Chapman and Hall/CRC; 2010. ISBN 9781420078862

http://www.olympusmicro.com

http://www.microscopyu.com

http://micro.magnet.fsu.edu

Requirements to the exam

Last update: Mgr. Aleš Benda, Ph.D. (15.09.2020)

A student needs to participate at minimum 60% of the lectures. For the exam, the student selects 2 papers (from 25-30 available). The exam is in the form of discussion on the technology used and its suitability to generate exciting observation(s) presented in the selected papers.

Active participation at the practical part.

The exam can be in Czech or Slovak in case the student does not feel comfortable with an exam in English.

Syllabus

Last update: Mgr. Aleš Benda, Ph.D. (11.01.2024)

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.

Topics 1-5 are covered in Seeing is believing I, topics 6-10 in Seeing is believing II.

Seeing is believing I.

1.       What is the shape, mass and size of my cell? Can I track motile cells? What about conjugates formation? WHOLE CELL ORIENTED IMAGE ANALYSIS.

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

2.       Can we image cells in whole organisms? What about specificity? Can we distinguish healthy and transformed cells? Can we image unstained tissues and vasculature? ANALYSIS OF TISSUES AND THEIR CELLULAR CONTENT.

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

3.       CYTOSKELETON: What defines the shape of my cell? How to visualise cytoskeleton dynamics in living cells? Is there a Central Park or Velin in the cell? What are the ‘hairs’ surrounding the cell? CYTOSKELETON.

Lattice light sheet microscopy. Fluorescent speckle microscopy of cytoskeleton dynamics. TIRF. Spinning disk.

4.       How to visualise nucleus and nucleolus? Can we track chromatin remodelling? What about DNA damage? Is my gene transcribed? What is the speed of its transcription? What is the lifetime of my RNA and where in a cell does it go? NUCLEUS, NUCLEIC ACIDS AND ASSOCIATED PROCESSES.

Single molecule fluorescence. Particle tracking. Kymograph. Laser-induced DNA damage. FISH and modified techniques. Energy transfer/quenching. Fluorescence correlation spectroscopy (FCS). DNA origami.

5.       Can I ‘see’ cellular membranes? Can I detect membrane subclasses? Can I track membrane protein (lipid) sorting. What about small-scale membrane organisation? Can I monitor membrane-associated processes? Is there a way to measure membrane potential? What about membrane topology? PLASMA MEMBRANE AND ITS TOPOLOGY. MEMBRANE CONTACT SITES AND TRANSPORT.

Seeing is believing II.

6.       Can I see cellular proteins – not all are fluorescent? What about protein folding? Is it alone or in an assembly? Proteins are often post-translationally modified, can I see where? Is my protein properly localised? Oups, is it degraded? What is phase separation of proteins? PROTEINS AND THEIR LIFE (AND DEATH) IN CELLS.

More on single molecule fluorescence, photoconversion and labelling: Immunofluorescence vs. GFP and variants – positives and limitations. Click chemistry. SMLM (super-resolution). TOCCSL (just another SPT – but better).

7.       My cells are looking bad. Is their energy source (mitochondria) in a good shape? Where are those toxic agents coming from? Can I follow stress processes in a cell which is still living? Is it possible to measure temperature in cells? MITOCHODRIA AND THEIR HEALTH. MITOPHAGY. ER STRESS.

SOFI (super-resolution). pH-, redox-, NADPH- and some other metabolic probes. Inorganic probes for live cell imaging (e.g. nanodiamonds).

7.       Cell are really busy. Can I track all those molecules? Can I do it individually or just following vesicles? Can I ‘see’ a virus entering or leaving my cell? Could extracellular vesicles (exosomes) be transferred to a neighbouring cell or throughout a tissue/organism? Can we quantitatively analyse exosomes or other micro-vesicles? VESICULAR TRAFFICKING. EXO/ENDOCYTOSIS. EXTRACELLULAR VESICLES IN HUMAN PATHOLOGY. VIRAL INFECTION.

SIM (super-resolution). Live cell imaging in microfluidics and other microdevices. Curvature detection. Clinical diagnosis using fluorescence imaging tools.

9.       Both sides are salty, I like sweets. Can I follow specific ions or small molecules helping cells in their business? Can I follow activity of ion channels? How to control ion flow trough the membrane using light? Sugars look similar. Are there tools to image their life on cells? IONS AND ION CHANNELS. CALCIUM SIGNALLING. GLYCOSTRUCTURES ON CELLS.

Small molecule probes. Opto-switches and light-controlled tools for biology. Super-fast fluorescence (or not) imaging: ICS and derivatives. Dendrimers. STED (super-resolution).

10.   Be careful – physics in biology: Can I measure speeds, forces and other physical parameters of tissues? Or in cells?? Or in organelles??? Within macromolecules???? Of small molecules such as water????? CELLULAR AND MOLECULAR MOTORS. FORCES AND MECHANICAL ENERGY. OTHER PHYSICAL PROPERTIES OF BIOLOGICAL MATERIALS.

Atomic force microscopy. More on quantitative fluorescence imaging. Intramolecular FRET. Fluorescent rotors. Anisotropy. Probes to monitor forces, viscosity, or electric field. Statistics for dummies.

Bonus: DATA PROCESSING AND PRESENTATION!

Entry requirements

Last update: Mgr. Aleš Benda, Ph.D. (14.10.2018)

The lectures will be taught in English.

Prerequisite:

Biologie buňky (Biology of the cell) - MB150P31(E)

Recommended:

Fluorescenční mikroskopie v buněčné biologii (Fluorescence microscopy of cells) - MB151P96

Landmarks/Milestones of Cell Biology - MBCPLUS002

Registration requirements

Last update: Mgr. Aleš Benda, Ph.D. (14.10.2018)

The course is designed for Master and PhD students.