Chapter 4 – Making Light Work Harder in Biology  121

(to this day, the ability to resolve single-​microtubule filaments of a few tens of nanometer

width in a tightly packed filament is treated as a benchmark of the technique), they have also

now developed into powerful cellular imaging tools.

In STED, this reduction in laser excitation volume is achieved using a second stimulated

emission laser beam in addition to an excitation beam, which is shaped like a donut in the focal

plane with a central intensity minimum of width ~200 nm. This annulus intensity function

can be generated using two offset beams or by phase modulation optics (Figure 4.2a).

This beam has a longer wavelength than the laser excitation beam and stimulates emission

from the fluorescence excited state (Figure 4.2b), resulting in the depletion of the excited state

in the high-​intensity region of the donut, but a nondepleted central region whose volume is

much smaller than the original PSF. In STED, it is now standard to reduce the width w of

the excitation volume in the sample plane to <100 nm, which, assuming an objective lens of

numerical aperture NA, is given by

(4.6)

w

NA

I Is

=

+

λ

2

1

/

FIGURE 4.2  Stimulated-​emission depletion microscopy (STED) and Förster resonance energy transfer (FRET). (a) Relative

sizes and shapes of STED depletion beam and original PSF of a fluorophore; the donut-​shaped depletion beam stimulates

emission depletion from the fluorophore to generate a much smaller point spread function intensity volume, with (b) the

associated Jablonski energy level diagram indicated. (c) Schematic depiction of FRET, here, indicated between a donor

and acceptor fluorescent protein, which are excited by short and long light wavelengths respectively, with (d) associated

Jablonski energy level diagram and (e) schematic indicating the relative orientation of the donor–​acceptor fluorophore

electric dipole moment axes; each respective electric dipole axis lies in a one of two planes separated by angle θT, and

these two planes intersect at a line defined by arrow indicated that makes angles θA and θD with the acceptor and donor

electric dipole axes, respectively.