122 4.2  Super-Resolution Microscopy

where

λ is the depletion beam wavelength with saturating intensity Is (intensity needed to

reduce fluorescence of the excited state by a factor of 2)

I is the excitation intensity at the center of the donut

For large I, there is an ~1/​√I, dependence on w, so w could in principle be made arbitrarily

small. A limiting factor here is irreversible photobleaching of the fluorophores used, resulting

in a few tens of nanometers for most applications at present. Due to the absence of shorter

wavelength UV activation, STED excitation can penetrate deeper with less scatter into large

cells, which has some advantages over PALM/​STORM. Also, maximum image sampling

rates are typically higher than for PALM/​STORM at a few tens of frames per second cur­

rently higher for STED compared to a few per second for PALM/​STORM. However, there

are potentially greater issues of photodamage with STED due to the high intensity of the

depletion beam.

Minimal photon FLUX (MinFlux) microscopy (Balzarotti et al 2016) is a related super-​

resolution tool that combines SMLM and STED while using fewer fluorescence photons

but enabling higher spatial and time resolution. In MinFlux, the STED donut-​shaped bead

is steered to map onto the molecular position itself while eliciting minimum fluorenscent

photon emissions from the dye molecule. In Minflux, the donut beam is scanned across a

sample to minimally acquire emission data sufficient to estimate roughly where a dye mol­

ecule is by using probabilistic triangulation criteria based on the brightness of the fluores­

cence and the spatial position of the donut beam. This estimate is then used to fine-​tune the

position of the donut beam to center it over the dye by shifting the beam over an area of

length scale, L ~50 nm, and then STED as normal is performed. However, since the center

of the donut beam, the zero-​excitation intensity region, is now roughly colocalized with the

dye position, then the dye molecule subsequently emits relatively low numbers of fluorescent

photons.

The spatial precision, instead of scaling with ~λ/​(NA√N) as suggested by Equation 4.6

scales as ~~L/​√N. This results in a spatial precision of 1–​3 nm for as few as a ~500 emitted

photons but can be made significantly smaller by reducing L to nanoscale levels, thus allowing

for true nanoscale spatial resolution but with substantively longer duration acquisitions while

minimizing photobleaching of the dyes. Also, since steering of the donut beam uses rapid

piezoelectric and electro-​optical control, the time resolution for 2D imaging can be as low as

a few hundred microseconds, hence rapid enough to enable single-​molecule dye diffusion to

be tracked unblurred in the cytoplasm of live cells, with tracking really then limited only by

photoblinking of the dyes themselves.

Variants of the technique enabling multicolor 3D MinFlux imaging now exist (e.g. using

a “z-​donut,” i.e., a 3D shell-​intensity depletion beam volume). At the time of writing, basic

SMLM bespoke microscopy can be implemented for as a little as few tens of thousands of

USD with higher throughput compared to MinFlux, whereas the equivalent cost for a basic

MinFlux system is roughly an order of magnitude greater. Although promising developments

are being made with structured illumination to increase MinFlux throughput, the main bar­

rier to its more widespread application is arguably cost.

4.2.11  PATTERNED ILLUMINATION MICROSCOPY

Structured illumination microscopy (SIM), also known as patterned illumination microscopy,

is a super-​resolution method that utilizes the Moiré pattern interference fringes generated

in the focal plane using a spatially patterned illumination (Gustafsson, 2000). Moiré fringes

are equivalent to a beat pattern. When measurements are made in the so-​called reciprocal

space or frequency space in the Fourier transform of the image, smaller length scale features

in the sample are manifested at higher spatial frequencies, such that the smallest resolv­

able feature using conventional diffraction-​limited microscopy has the highest spatial fre­

quency. In generating a beat pattern, spatial frequencies above this resolution threshold are

translated to lower values in frequency space. On performing an inverse Fourier transform,