154 Questions
~1 Hz per acquired spectrum (Scarcelli and Yun, 2007). BLS has been used in a number of
studies to investigate the biomechanical properties of a variety of tissues, which are discussed
in Chapter 6.
4.8 SUMMARY POINTS
◾There are several super-resolution methods, characterized by techniques that use
light microscopy to determine the location of a single dye molecule better than the
diffraction-determined optical resolution limit.
◾An array of nonlinear methods is useful for studying biological processes deep into
tissues, especially two-photon excitation fluorescence imaging.
◾Tools to correct for optical inhomogeneity in deep tissues exist, such as AO.
◾Interactions between two different biomolecules can be monitored using FRET,
down to the level of single molecules.
◾FCS is a robust tool to determine diffusion rates, which complements single-
particle tracking methods.
◾Inelastic scattering methods such as Raman and Brillouin scattering are “label-
free” and facilitate monitoring of a range of biomolecule types beyond just
the primary focus of fluorescence microscopy, which is fluorescently labeled
proteins.
QUESTIONS
4.1
A high-power oil-immersion objective lens has an NA or 1.45, transmitting 80% of
visible light, total magnification 300.
a
What is the photon capture efficiency of the lens?
In a TIRF experiment using the objective lens method with a laser excitation of
wavelength 561 nm, a protein is labeled with the FP mCherry.
b
Using PubSpectra (http://www.pubspectra.org/) or equivalent, select a suit
able dichroic mirror and emission filter for exciting and visualizing mCherry
molecules and estimates the maximum proportion of fluorescence photons that
can in principle reach a camera detector.
c
If the camera detector converts 90% of all incident visible light photons into a
signal and has a pixel size of 18 μm and a background noise level (b) equivalent to
~200 counts, what is the best spatial precision with which one can estimate the
position of a single mCherry molecule stuck to the surface of a glass coverslip?
In an actual experiment, the best precision was measured to be approximately 10
times worse than the theoretical estimate.
d
What is the most likely cause of this discrepancy?
4.2
Derive formulas for the relation between sigma width and w for the full width at half
maximum value to a Gaussian approximation to the PSF.
4.3
X-ray diffraction data (see Chapter 5) suggest that a certain protein found in the
cell membrane can be present as both a monomer and a dimer. A single-molecule
yellow-color FP called YPet was fused to the end of this protein to perform live-cell
fluorescence imaging. TIRF microscopy was used on live cells indicating typically six
detected fluorescent spots of full width at half maximum intensity profile ~180 nm per
cell in each illuminated section of cell membrane. TIRF microscopy here was found
to illuminate only ~15% of the total extent of the cell membrane per cell, with the
total membrane area being ~30 μm2 per cell. On the basis of intensity measurements
of the fluorescent spots, this indicated that ~75% of the spots were monomers, ~15%
were dimers, and the remaining 10% were consistent with being oligomers. Explain
with reasoning if you think the oligomers are “real” or simply monomers or dimers,
which are very close to each other. (Hint: if two fluorescent molecules observed using
KEY BIOLOGICAL
APPLICATIONS:
I NELASTIC
SCATTERING TOOLS
Label-free molecular identifi
cation; Monitoring mechanical
stress in tissues.