Chapter 4 – Making Light Work Harder in Biology  135

diffusing molecule types. However, the main weakness of FCS is its relative insensitivity to

changes in molecular weight, Mw. Different types of biomolecules can differ relatively mar­

ginally in terms of Mw; however, the “on” time τ scales approximately with the frictional

drag of the molecule, roughly as the effective Stokes radius, which scales broadly as ~

/

Mw

1 3 .

Therefore, FCS is poor at discriminating different types of molecules unless the difference in

Mw is at least a factor of ~4.

FCS can also be used to measure molecular interactions between molecules. Putatively,

interacting molecules are labeled using different colored fluorophores, mostly dual-​color

labeling with two-​color detector channels to monitor interacting pairs of molecules. A variant

of standard FCS called “fluorescence cross-​correlation spectroscopy” can then be applied.

A modification of this technique uses dual-​color labeling but employing just one detector

channel, which captures intensity only when the two separately labeled molecules are close

enough to be interacting, known as FRET-​FCS.

4.4.3  FCS ON MORE COMPLEX SAMPLES

FCS can also be performed on live-​cell samples. By scanning the sample through the confocal

volume, FCS can generate a 2D image map of mobility parameters across a sample. This has

been utilized to measure the variation in diffusion coefficient across different regions of large

living cells. As with scanning confocal microscopy, the scanning speed is a limiting factor.

However, these constraints can be overcome significantly by using a spinning-​disk system.

FCS measurements can also be combined with simultaneous topography imaging using AFM

(see Chapter 6). For example, it is possible to monitor the formation and dynamics of putative

lipid rafts (see Chapter 2) in artificial lipid bilayers using such approaches (Chiantia, 2007).

Worked Case Example 4.3: Localization Microscopy

A time-​correlated PALM experiment was performed on live E. coli bacteria stuck to cover­

slip to track a protein known to incorporate into liquid–​liquid phased separated droplets

in the cytoplasm whose formation was triggered by stressing the cell using a toxin.

The droplet protein was tagged with a red photoactivatable fluorescent protein called

PAmCherry, which was stochastically activated using low intensity 405 nm wavelength

laser excitation and imaged using a high intensity 561 nm wavelength laser excitation in

narrow-​field mode, peak fluorescence wavelength 595 nm, using a 1.4 NA oil immersion

(refractive index =​1.515) objective lens to track individual molecules with sampling time of

5 ms per consecutive image frame. Once fluorescence light from the PAmCherry had been

captured by the objective lens, only ~30% was transmitted through all of the imaging

system to the entrance of the camera detector. The camera detector had a quantum effi­

ciency of 80% at a wavelength of 595 nm. The average effector diameter of the droplet

protein tagged with PAmCherry is ~2–​3 nm.

a

If the PAmCherry emits ~10⁵ fluorescent photons prior photobleaching on average

resulting in single-​molecule tracks of average duration ~50 ms under these narrow-​

field conditions, estimate the localization precision when using a 2D Gaussian fitting

to track single molecules in the microscope focal plane, if you assume that the effect

from non-​photon sources on localization precision is negligible.

b

By increasing the intensity of the 405 nm wavelength activation laser the number

of tracks detected increased, but also in some cases the brightness of tracks was

twice that detected at the lower excitation intensity. Offer an explanation for these

observations and estimate the smallest diameter of droplet that could be detected

under these conditions. What does that imply in terms of the smallest droplet we can

detect and the maximum number of proteins within it?

c

Under the lower intensity activation laser setting, a plot of the average mean squared

displacement versus time interval for all detected tracks was initially a straight

KEY BIOLOGICAL

APPLICATIONS: FCS

Quantifying molecular mobility;

Determining molecular

interactions.