Sylabus
Seeing is believing II. – Advanced Microscopy for Everyone.1. Structural details observable using standard fluorescence microscopy are insufficient. Is there away how to “see below the resolution limit”? Yes, and even a little resolution improvement can do miracles. Cellular structures and processes visualised with ‘widefield’ superresolution microscopies:structured illumination microscopy (SIM), lattice light sheet microscopy (LLM). AireScan.
2. What if “a little resolution improvement” is not enough? In case a broad field is required, there isa confocal-based superresolution microscopy: Stimulated emission depletion (STED) microscopy.Alternatively, we can increase the size of our sample. How? Let’s discuss principles of expansionmicroscopy (ExM) and see deserved details with standard microscopes.
3. Still more’s wanted! Can I see individual biomolecules forming a fine three-dimensional structureinside a cell? Single molecule fluorescence, single molecule localisation microscopy (SMLM; PALM,dSTORM, dTRABI), interferometric scattering (iSCAT) microscopy. Photoconversion, photoactivationand photoswitches.
4. Can we follow molecular interactions directly in living cells? What about monitoring cellularenvironment? Főrster (fluorescence) resonance energy transfer (FRET), anisotropy, fluorescencelifetime and ratiometric imaging. Imaging Flow Cytometry.
5. Static snapshots are boring. Can I track individual molecules inside a cell? Dynamics of moleculesin the cytosol, on fibres (the cytoskeleton) and in membranes. Fluorescence recovery afterphotobleaching (FRAP + variants), fluorescence (cross-)correlation spectroscopy (F(C)CS) and singleparticle tracking.
6. Structural details visualised by electron microscopes are amazing. How can I get molecularspecificity with these methods? Specific labelling for the EM (immuno-gold, APEX, APEX-gold).CryoFM. Correlated light and electron microscopy (CLEM).
7. A bull is a powerful animal. However, can a cell exert a force? Does tumour environment differfrom normal tissues? Measuring mechanical forces in cells. Tissue stiffness. Tricks and tips. Atomicforce microscopy, optical tweezers, nanopillars, Brillouin microscopy, …
8. Can we image specific atoms, ions, bonds, structures? Chemical specificity by Raman Spectroscopy, CARS, EDS/EDX (EM mode), pH/ion imaging/voltageimaging.
9. How to get my sample ready to win ‘Imaging contest’? It must be bright, colourful and artifactfree? Advanced labelling methods: 30 colour microscopy (antibody exchange), DNA-PAINT forlabelling without antibodies and ligands, biorthogonal (direct) labelling of molecules, …
Anotace
Rapid development of microscopy techniques and labelling probes enabled observation of cellular physiology at the molecular level and often in living specimens. In Seeing is believing I. course, we focused on basic light and electron microscopy techniques, which enable everyday analysis of biological specimens. In this second part (Seeing is believing II.), we explore application of more advanced imaging methods, including super-resolution microscopy and spectroscopy-based tools. Importantly, non-standard modes of some basic microscopy techniques and the use of specialised probes to monitor cellular processes and analyse local environment is discussed in this course (see Syllabus for more details).
The practical part: hands-on demonstration of selected applications, including sample preparation, data acquisition, data analysis and data management.