120 4.2  Super-Resolution Microscopy

membrane-​localized protein to generate super-​resolution cell membrane features in a tech­

nique described as super-​resolution by power-​dependent active intermittency and points

accumulation for imaging in nanoscale topography (SPRAIPAINT) (Lew et al., 2011).

Dye blinking of organic dyes has also been utilized in a technique called “blinking assisted

localization microscopy” (Burnette et al., 2011). This should not be confused with binding-​

activated localization microscopy, which utilizes a fluorescence enhancement of a dye when

bound to certain cellular structures such as nucleic acids compared to being free in solution,

which can be optimized such that the typical nearest-​neighbor distance is greater than the

optical resolution limit (Schoen et al., 2011). Potential advantages over PALM/​STORM of

blinking localization microscopy are that the sampling time scales are faster and also that

there is less photodamage to living cells in avoiding the more damaging shorter wavelength

used in UV-​based activation.

Improvements to localization microscopy precision can be made using prior information

concerning the photophysics of the dyes, resulting in a hybrid technique of analytical infer­

ence with standard localization tools such as PALM/​STORM. Bayesian analysis is an ideal

approach in this regard (discussed fully in Chapter 8). This can be applied to photoblinking

and photobleaching observations trained on prior knowledge of both. Bayesian blinking and

bleaching microscopy (3B microscopy) analyzes data in which many overlapping fluorophores

undergo both bleaching and blinking events to generate spatial localization information at

enhanced resolution. It uses a hidden Markov model (HMM). An HMM assumes that the

underlying process is a Markov process (meaning future states in the system depend only on

the present state and not on the sequence of events that preceded it, i.e., there is no memory

effect) but with unobserved (hidden) states and is often used in Bayesian statistical analysis

(see Chapter 8). It enables information to be obtained that would be impossible to extract

with standard localization microscopy methods.

The general issue of photodamage with fluorescence imaging techniques should be viewed

in the following context:

1 All imaging of live cells with fluorescence (and many other modalities) is potentially

damaging, for example, fast confocal scanners often kill a muscle cell in seconds. That

is, however, acceptable, for certain experiments where we are looking at fast biological

processes (e.g., a few milliseconds) and the mindful biologist builds in careful control

experiments to make sure they can put limits on these effects.

2 The degree of damage seen is likely closely connected to the amount of information

derived but can be reduced if light dosage is reduced using hybrid approaches such as

3B microscopy. Some spatial resolution is inevitably sacrificed for time resolution and

damage reduction—​nothing is for free.

3 Many dark molecules in photoactivating super-​resolution methods greatly reduce

absorption in the sample, and the UV exposure to photoactivate is generally very low.

4.2.10  RESHAPING THE PSF

The Abbe diffraction limit for optical resolution can also be broken using techniques that

reduce the size of the PSF. One of these is 4Pi microscopy (Hell and Stelzer, 1992). Here, the

sample is illuminated with excitation light from above and below using matched high NA

objective lenses, and the name of the technique suggests an aspiration to capture all photons

emitted from all directions (i.e., 4π steradians). However, in reality, the capture solid angle

is less than this. The technique improves the axial resolution by a factor of ~5 to more 100–​

150 nm, generating an almost spherical focal volume six times smaller than confocal imaging.

Stimulated-​emission depletion microscopy (STED) (see Hell and Wichmann, 1994)

and adapted techniques called “ground state depletion,” “saturated structured illumination

microscopy” (SSIM), and “reversible saturable optical fluorescence transitions” microscopy

all reduce the size of the excitation volume by causing depletion of fluorescence emissions

from the outer regions of the usual Airy ring PSF pattern. Although these techniques began as

in vitro super-​resolution methods, typically to investigate the aspect of cytoskeletal structure