Syllabus
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!
Annotation
Rapid development of imaging techniques and specific probes enabled observation of cells, tissues and organisms in their native environment. In this course, problem-oriented lectures will guide students through the exhibition of tools - mainly fluorescent probes and imaging techniques – which allow characterisation of physiological and transformed state of cells and tissues in organisms but also definition of their physical properties. 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 label-free intravital imaging of cells and tissues, non-invasive tracking of cells in tissues or small animal (plant) models, monitoring of changes of nucleoprotein assemblies during transcription and translation as well as investigation of cytoskeletal dynamics (see the syllabus for more details). The technology required for the use of appropriate probes will be explained, mainly by providing the examples where clever combination of imaging techniques and smartly-designed probes led to break-through findings. Basic microscopy methods (e.g. phase contrast) will be explained in more detail to highlight their simplicity and, at the same time, their capacity to provide a broad information about the observed specimen. 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. We will open the room for label-free techniques (e.g. second and third harmonic imaging) to indicate a current state of these methods which fulfill the dream of a biologist to investigate cellular or molecular processes without influencing the sample by the application of specific (but always imperfect) 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.