Fluorescence Microscopy Flashcards
Give examples of some of the things discovered by the simple microscope
Bacteria
Sperm cells
Blood cells
When was the first simple microscope invented and who by?
By Van Leeuwenhoek around 1668
When was the first compound microscope invented and who by?
By Zacharias Janssen, probably with the help of his father in 1595 (considered the first microscope)
Who was the first person to use the word ‘cell’ while describing a piece of cork in 1665?
Robert Hooke
What are the types of microscope aberrations?
Axial chromatic aberration
Spherical aberration
In fluorescence microscopy, there is some energy loss (heat) sure to conformational changes and collisions with what?
Neighbouring molecule
Define Stokes shift
Absorption of light with a short wavelength results in the emission of light with a longer wavelength
What can we use the Stokes shift for?
To separate the excitation and emission light in a fluorescence microscope
What do immunolabels tag?
Epitopes
What does FISH stand for?
Fluorescence in situ hybridisation
When was GFP discovered?
1962
Who discovered GFP?
Shimomura et al.
In what year was the Nobel Prize in Chemistry awarded for the discovery of GFP?
2008
What species was GFP isolated from?
The jellyfish Aequoria victoria
What does GFP stand for?
Green fluorescent protein
Who first cloned GFP?
Prasher et al.
In what year was GFP first cloned?
1992
Where can GFP be fused to a protein?
At either ends of the protein or within the protein
GFP folds in all organisms, true or false?
True
What external factors are needed for GFP fluorescence?
No external factors needed except light
What proteins can be tagged by GFP?
Most proteins
Who first tagged GFP in C. elegans and subsequently won the Nobel Prize in chemistry in 2008?
Martin Chalfie
GFP cannot be visualised in live cell imaging, true or false?
False
Is GFP a large or small molecule? What does this mean for its use in protein tagging?
A large molecule
Might change dynamics, localisation, interactions or function of the protein
How many amino acids are there in GFP?
229
What additional control measures could you consider in order to control for the size of GFP?
Western blot Compare N- and C- terminal labelled constructs Compare with antibody labelling Use different tag (His-tag, FiAsH-tag) Protein activity assay
Who created different colours of GFP and subsequently won the Nobel Prize in chemistry in 2008?
Roger Tsien
Define photobleaching
The loss of ability to fluoresce due to photon-induced chemical damage and covalent modification as a result of interaction with other molecules
What does FRAP stand for?
Fluorescence recovery after photobleaching
Define FRAP
Bleaching a region in the cell using a laser and then following the recovery
Why study FRAP in cells?
It can give information about the dynamics/binding of a protein
How are samples viewed using a wide field microscope?
The whole sample is illuminated with light and the whole image can directly be viewed
Describe how a confocal laser scanning microscope works
Scans pixel-by-pixel
Relatively slow
System only detects light from that part of the sample that is in focus (optical sectioning)
What is a voxel?
A 3D pixel
What are the advantages of light sheet microscopy?
Minimal photo toxicity
Fast and sensitive detection using sCMOS camera
Good penetration in scattering tissues
Multi-view acquisition by rotation of the sample
What is the Rayleigh criterion associated with?
Resolution
How can we improve resolution?
Try to use a shorter wavelength
Using electron microscope
Use FRET
What does FRET stand for?
Forster resonance energy transfer
What wavelength do electrons have?
~5pm
In the last decade, many new fluorescence microscope techniques have been developed with an improved resolution up to 10nm, what are these called?
‘Super resolution microscopes’ or nanoscopes
Give an example of a super resolution microscopes
STED
What does STED stand for?
Stimulated emission depletion microscopy
How does STED work?
Fluorescence is completely suppressed by a stimulated emission process; the electrons are forced back to the ground state without emitting light
When was the Nobel Prize in chemistry awarded for super resolution microscopes and who to?
2014
Betzig, Hell, Moerner
What are the advantages of fluorescence microscopy?
Very sensitive (can detect single molecules)
Highly selective using specific probes
Can be used in vivo
Improved resolution
What are the disadvantages of fluorescence microscopy?
Usually requires a fluorescent label
Excitation light can be damaging (phototoxicity, bleaching)
Often time consuming
Quantitative imaging is challenging
