104 3.6 Basic Fluorescence Microscopy Illumination Modes
characterized by fast diffusion of one more molecular component but often slow reaction
events with other components (see Chapter 8). Thus, the time scale for the entire process may
be substantially longer than the millisecond diffusional time scale. To overcome this issue,
individual millisecond image frames may be obtained discontinuously, that is, using strobing.
In this way, the limited fluorescence emission photon budget of a single fluorescent protein
molecule may be used over substantially longer time scales than just a few tens of milliseconds
to gain insight into the dynamics of several different processes inside the cell while still enab
ling unblurred fluorophore detection on individual images frames. A technical issue in regard
to strobing is the bandwidth of the shuttering mechanism used to turn the laser excitation
on and off. Typical mechanical shutters are limited to a bandwidth of ~100 Hz, and so the
minimum sampling time window that can be accommodated is ~10 ms, which is too high for
millisecond imaging. Alternative faster shuttering can be implemented directly through elec
tronic modulation of the laser power output on some devices or utilizing acousto-optic-based
technology for shuttering, for example, an acousto-optic modulator that can be shuttered at
>MHz bandwidth using similar physics principles to AODs (see Chapter 6).
KEY POINT 3.5
In fluorescence imaging at the molecular scale, many single-particle tracks are
truncated, often because of rapid fluorophore photobleaching. Strobing can permit the
limited photon emission budget to be stretched out over larger time scales to probe
slower biological processes.
Worked Case Example 3.3: Slimfield
A Slimfield microscope comprised a 488 nm wavelength laser of beam width 0.1 mm
directed into the back aperture of a high numerical aperture objective lens of NA 1.45, focal
length 2 mm, to image a single GFP molecule at room temperature, peak fluorescence
wavelength ~505 nm, tagged to a cell membrane protein such that the protein is integrated
into the phospholipid membrane. The membrane viscosity was measured separately to be
~100 cP, but the GFP molecule itself sits just outside the membrane in the cytoplasm, whose
viscosity was closer to 1 cP. The cell membrane is in contact with the microscope coverslip
and can be considered reasonably planar over a circle of diameter ~5 µm.
a If the frictional drag coefficient of the membrane integrated protein is 1.1 × 10-7 kg/s,
what is the maximum sampling time per image frame which will allow you to observe
the GFP unblurred? Before the protein integrates into the membrane, it must first diffuse
through the 3D volume of the cytoplasm. If you wish to monitor this cytoplasmic diffusion
as molecules come into focus before they integrate into the membrane, what is the max
imum sampling time that could be used?
b In a separate experiment, the average time that a single GFP molecule fluoresces
before photobleaching using identical Slimfield imaging conditions as that for measuring
the cytoplasmic diffusion was measured to be 30 ms. If the membrane protein is integrated
into a lipid raft region of the cell membrane whose effective diameter is 200 nm, do you
think you will be able to also monitor the diffusion of the membrane integrated protein as
it moves from the center to the edge of a raft?
Answers
a
The PSF width for imaging a GFP molecule is given by Equation 3.54 of w=0.61λ/
NA. Here λ relates to the fluorescence emission, not the excitation, so using the
characteristic peak value given indicates that:
w = 0.61 × (505 × 10-9)/1.45 = 212 × 10–9 m (i.e. 212 nm)
KEY BIOLOGICAL
APPLICATIONS:
DIFFERENT
FLUORESCENCE
MICROSCOPY MODES
Investigating cell membrane
processes in live cells; Probing
rapid processes in the cell cyto
plasm of live cells; Rendering
axial as well as lateral localization
information for processes and
components inside cells.