Name:________________________________________________
Student Number:____________________________________
Biophysical Techniques and Molecular Imaging
Examination
Answer all questions.
Use the space provided for each question. This should be sufficient for
the expected answer.
If you need more space, use the reverse side of the page.
Closed book/notes.
Good luck!
Name:________________________________________________
Student Number:____________________________________
Question 1: Principles of Fluorescence (10 points)
a. Why is fluorescence microscopy so successful in the life sciences? (2 points)
b. Draw the Jablonski diagram for luminescence (fluorescence and
phosphorescence). Include all radiative and nonradiative processes and indicate
their typical timescales. (5 points)
Name:________________________________________________
Student Number:____________________________________
c. Explain the parameters fluorescence lifetime and quantum yield. Give the
formulas connecting both parameters to the radiative and nonradiative decay rate.
(3 points)
Question 2: Fluorophores (10 points)
a. Fluorescence microscopy in the life sciences relies on the use of so called
“labels”. Explain the concept of using fluorescent labels and why these labels are
necessary. (2 points)
b. Immunolabelling is an often used labelling method.
Explain the concept of immunolabelling. Why is immunolabeling an often used
method? (2 points)
Name:________________________________________________
Student Number:____________________________________
c. Draw typical absorbance and emission spectra of a visible fluorescent protein (like
GFP) and a quantum dot (both emission maximum ~ 510 nm). (2 points)
d. Describe qualitatively the effect on the emission (intensity, spectral shape and
position, lifetime) of the fluorescent protein and the quantum dot when changing
the excitation wavelength from 490 nm to 450 nm while keeping the excitation
power constant. (2 points)
Name:________________________________________________
Student Number:____________________________________
e. Two recently developed cyan fluorescent proteins, Cerulean and mTurquoise,
have the following properties:
Excitation Emission Extinction Quantum Fluorescence
maximum maximum coefficient at Yield lifetime (ns)
(nm) (nm) peak (M-1cm-1)
Cerulean 434 475 43000 0.48 3.17
mTurquoise 434 474 34000 0.84 4.04
i. Calculate the natural or radiative lifetimes of Cerulean and mTurquoise.
(1 point)
ii. Calculate and compare the brightness of Cerulean and mTurquoise.
(1 point)
Question 3: Quenching (5 points)
a. Suppose you add a collisional (or dynamic) quencher Q to your fluorophore
solution. Explain collisional quenching. What happens to the fluorescence
intensity and lifetime of the fluorophore upon addition of Q? Why? How will the
lifetime change as you increase the concentration of the quencher Q? (3 points)
b. Explain the differences between collisional (or dynamic) quenching and static
quenching. Explain what would happen to the fluorescence intensity and lifetime
of a fluorophore upon adding a static quencher (S). (2 points)
Name:________________________________________________
Student Number:____________________________________
Question 4: FRET (10 points)
Förster Resonance Energy Transfer (FRET) is often called a molecular ruler because
of its exquisite sensitivity to distance. Suppose you have a FRET sensor composed
of the fluorescent protein mTurquoise and the yellow emitting fluorescent protein
EYFP connected by a functional linker.
a. Describe the role of the two fluorescent proteins using a simplified Jablonski
diagram. (2 points)
b. Describe two necessary conditions for FRET to occur. (1 point)
c. Draw the evolution of FRET efficiency with donor-acceptor distance. Explain the
term “Foerster distance”, mark it in the diagram and give a typical value of a
Foerster distance, e.g. for the above mentioned proteins. (3 points)
Name:________________________________________________
Student Number:____________________________________
d. Describe two ways how FRET in such a system can be detected and quantified. (2
points)
e. When the biosensor is activated the lifetime of mTurquoise (properties see
question 2) changes to 0.8 ns. The characteristic Foerster distance for the FRET
pair is 5 nm. Calculate the energy transfer efficiency and the distance between
FRET donor and acceptor for the activated biosensor. (2 points)
Question 5: Microscopy techniques (10 points)
a. Explain simply the concept of confocal microscopy.
What are the advantages of confocal microscopy over wide-field microscopy?
What are the disadvantages? (3 points)
Name:________________________________________________
Student Number:____________________________________
b. Spinning disk confocal microscopy is aimed to overcome one of the limitations of
traditional confocal microscopy. Which limitation is this? Describe the benefits and
function of spinning disk confocal microscopy.(1 point)
c. Describe the purpose of the filter cube used in fluorescence microscopy. Sketch a
filter cube, name the components and describe their purpose. (3 points)
d. Explain the concept of STED superresolution microscopy.
What determines the spatial resolution that can be achieved using this method? (3
points)
