Over the past 10-15 years, new technologies have gone beyond the diffraction limit of optical microscopy. These “super-resolution” microscopies rely on fluorescence imaging and encompass multiple techniques that achieve higher resolution than traditional light microscopy giving exciting opportunities for scientists to ask entirely new levels of questions. The differences between these individual technologies result in a trade-off between spatial resolution, temporal resolution and phototoxicity applied to a specific biological question.
Are you ready to test your knowledge on these microscopy methods used in some of the NCCR labs, such as the
Manley lab at the EPFL?
This quiz was created by Victoria von Glasenapp (University of Geneva) and Luis Hernandez Ramirez (EPFL).
Thanks a lot to both of them!
What is the smallest distance between two points that a classical light microscope can distinguish?
The Nobel Prize for the development of super-resolved fluorescence microscopy was given to:
Jacques Dubochet, Joachim Frank and Richard Henderson in 2017.
Eric Betzig, Stefan W. Hell and William E. Moerner in 2014.
Ernst Ruska, Gerd Binnig, Heinrich Rohrer in 1986.
Any microscopy technique that overcomes the resolution limit of conventional light microscopy by at least a factor of two is considered to be a super-resolution technique.
To label the protein of interest in nanoscopy antibodies and immunocytochemistry techniques should be avoided because:
The IgG complexes are too large as they are in the range of the resolution attainable with super-resolution techniques.
The fluorescence is too strong.
The labelling is not specific.
The following are super-resolution techniques:
Total Internal Reflection Fluorescence (TIRF) and Two-Photon Excitation (2P) microscopy.
Stimulated Emission Depletion (STED) microscopy, Structured Illumination Microscopy (SIM), Stochastic and Localization Microscopy (PALM/STORM).
Spinning disk confocal microscopy and Light sheet microscopy.
What does Single Molecule Localization Microscopy (SMLM) consist of?
The separation of the signal of individual emitters in time to determine their positions with nanometer precision.
Imaging of single molecules and the study of the low-level molecular interactions at the subcellular level.
Exploiting stochastic blinking or binding phenomena.
All of the above answers.
The following is true about photoactivatable, photoconvertible, and photoswitchable fluorescent proteins, EXCEPT:
Photoactivatable fluorescent proteins can be switched "on" from a non-fluorescent state to a fluorescent state by irradiation with light in the blue/violet spectrum.
Photoconvertable fluorescent proteins are able to change their fluorescence emission spectrum from one maximum to another.
Photoswitchable fluorescent proteins can be switched "on and off" with the help of light pulses of two different wavelengths.
Photoactivatable proteins emit fluorescence already in their non-converted state.
Which of the following statements about Expansion Microscopy (ExM) are true?
ExM allows nanoscale imaging of biological specimens with conventional microscopes.
ExM combines the physics of swellable polyelectrolyte hydrogels, which vastly increase in size when immersed in a solvent, and the embedding of preserved biological specimens in polymer hydrogels for imaging purposes.
ExM workflow consists in fixation, anchoring, gelation, mechanical homogenization and expansion.
All of the above answers.
What is the major advantage of Structured illumination microscopy (SIM)?
SIM is particularly well suited for live cell imaging as it is not very phototoxic and allows for a high temporal resolution.
The resolution in lateral dimension is below 40 nm.
It combines fluorescence microscopy and electron microscopy allowing to study small structures in great detail.
Super-resolution microscopy: ready to test your knowledge?
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