118 4.2  Super-Resolution Microscopy

it has been employed to monitor the kinetics and mobility of individual protein molecules

in large molecular structures. The fluorescent speckle generates an identifiable pattern,

and movement of the protein assembly as a whole results in the pattern image translating.

This can be measured accurately without the need for any computationally intensive fitting

algorithms and has been applied to the study of microtubular structures in living cells.

4.2.7  GENETIC ENGINEERING APPROACHES TO INCREASE THE

NEAREST-​NEIGHBOR DISTANCE

For FP-​labeling experiments, it may be possible to control concentration levels of the

fluorophore through the application of inducer chemicals in the cell (see Chapter 7). This is

technically challenging to optimize predictably, however. Also, there are issues of deviations

from native biological conditions since the concentration of the molecules observed may, in

general, be different from their natural levels.

Pairs of putatively interacting proteins can satisfy the Clim condition using a technique

called “bifunctional fluorescence complementation” (BiFC). Here, one of the proteins in the

pair is labeled with a truncated nonfluorescent part of a FP structure using the same type of

genetics technology as for conventional FP labeling. The other protein in the pair is labeled

with the complementary remaining part of the FP structure. When the two molecules are

within less than roughly a nanometer of each other, the complementary parts of the FP struc­

ture can bind together facilitated by short alpha helical attachment made from leucine amino

acids that interact strongly to form a leucine zipper motif. In doing so, a fully functional FP

is then formed (Figure 4.1c), with a cellular concentration, which may be below Clim even

though those of the individual proteins themselves may be above this threshold.

4.2.8  STOCHASTIC ACTIVATION AND SWITCHING OF FLUOROPHORES

Ensuring that the photoactive fluorophore concentration is below Clim can also be achieved

through stochastic activation, photoswitching, and blinking of specialized fluorophores.

The techniques of photoactivatable localization microscopy (PALM) (Betzig et al., 2006)

are essentially the same in terms of core physics principles as the ones described for fluores­

cence photoactivatable localization microscopy and stochastic optical reconstruction micros­

copy (STORM) (Rust et al., 2006). They use photoactivatable or photoswitchable fluorophores

to allow a high density of target molecules to be labeled and tracked. Ultraviolet (UV) light

is utilized to stochastically either activate a fluorophore from an inactive into a photoactive

form, which can be subsequently excited into fluorescence at longer visible light wavelengths,

or to switch a fluorophore from, usually, green color emission to red.

Both approaches have been implemented with organic dyes as well as FPs (e.g.,

photoactivatable GFP [paGFP], and PAmCherry in particular, and photoswitchable proteins

such as Eos and variants and mMaple). Both techniques rely on photoconversion to the

ultimate fluorescent state being stochastic in nature, allowing only a subpopulation to be

present in any given image and therefore increasing the typical nearest-​neighbor separation

of photoactive fluorophores to above the optical resolution threshold. Over many (>10⁴)

repeated activation/​imaging cycles, the intensity centroid can be determined to reconstruct

the localization of the majority of fluorescently labeled molecules. This generates a super-​

resolution reconstructed image of a spatially extended subcellular structure.

The principal problems with PALM/​STORM techniques are the relatively slow image

acquisition time and photodamage effects. Recent faster STORM methods have been

developed, which utilize bright organic dyes attached via genetically encoded SNAP-​Tags.

These permit dual-​color 3D dynamic live-​cell STORM imaging up to two image frames per

second (Jones et al., 2011), but this is still two to three orders of magnitude slower than

many dynamic biological processes at the molecular scale. Most samples in PALM/​STORM

investigations are chemically fixed to minimize sample movement, and therefore, the study

of dynamic processes, and of potential photodamage effects, is not relevant. However, the use