Seeing is believing II. – Advanced Microscopy for Everyone.
1. Structural details observable using standard fluorescence microscopy are insufficient. Is there a
way 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 is
a confocal-based superresolution microscopy: Stimulated emission depletion (STED) microscopy.
Alternatively, we can increase the size of our sample. How? Let’s discuss principles of expansion
microscopy (ExM) and see deserved details with standard microscopes.

3. Still more’s wanted! Can I see individual biomolecules forming a fine three-dimensional structure
inside a cell? Single molecule fluorescence, single molecule localisation microscopy (SMLM; PALM,
dSTORM, dTRABI), interferometric scattering (iSCAT) microscopy. Photoconversion, photoactivation
and photoswitches.

4. Can we follow molecular interactions directly in living cells? What about monitoring cellular
environment? Főrster (fluorescence) resonance energy transfer (FRET), anisotropy, fluorescence
lifetime and ratiometric imaging. Imaging Flow Cytometry.

5. Static snapshots are boring. Can I track individual molecules inside a cell? Dynamics of molecules
in the cytosol, on fibres (the cytoskeleton) and in membranes. Fluorescence recovery after
photobleaching (FRAP + variants), fluorescence (cross-)correlation spectroscopy (F(C)CS) and single
particle tracking.

6. Structural details visualised by electron microscopes are amazing. How can I get molecular
specificity 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 differ
from normal tissues? Measuring mechanical forces in cells. Tissue stiffness. Tricks and tips. Atomic
force 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/voltage
imaging.

9. How to get my sample ready to win ‘Imaging contest’? It must be bright, colourful and artifact
free? Advanced labelling methods: 30 colour microscopy (antibody exchange), DNA-PAINT for
labelling without antibodies and ligands, biorthogonal (direct) labelling of molecules, …