Principles of S TED Microscopy

In STED microscopy, proposed in 1994 by Stefan Hell and Jan Wich-mann [136] and implemented by Thomas Klar and Stefan Hell in 1999 [173], a focused laser beam excites fluorescent markers from the elec-tronic ground state S₀ to the first excited state S₁. The laser beam is scanned relative to the sample, as in conventional laser scanning microscopy. The image is usually assembled one pixel after the other by recording the fluorescence emitted by the sample at any place se-quentially with a point detector, although parallelization should be pos-sible and point detection is not mandatory [130]. Switching off the fluorescence ability of the fluorophores in the outer part of the ex-citation focus generates a smaller effective exex-citation volume. The switching of the molecules into a non-fluorescing state is achieved by de-exciting the fluorophores via stimulated emission [86, 216] with a second, red-shifted, laser beam (“STED beam”, “de-excitation beam”,

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Figure 2.1:Jabło ´nski diagram of the molecular states and transitions in-volved in STEDmicroscopy. The fluorophore is excited from the lowest vi-bronic level of the electronic ground state S₀to a higher vibronic level of the first excited electronic state S₁by absorption of a photon of the excitation light. After relaxation to the lowest vibronic level of S₁, the fluorophore emits spontaneously a fluorescence photon and goes to a higher vibronic level of S₀. Stimulated emission forces the transition from S₁to S₀. This transition can go to a different vibronic level, i. e. it can occur at a different wavelength than the fluorescence emission. After spontaneous or stimu-lated emission, the fluorophore returns to the lowest vibronic level of S₀.

“depletion beam”). Figure 2.1 shows the involved energy levels and transitions with a Jabło ´nski diagram [157, 294]: the fluorophores are excited from the ground state S₀ to higher vibronic states of the first excited electronic state S₁. Within picoseconds relaxation to the low-est vibronic state occurs, from where a fluorescence photon is emitted spontaneously within nanoseconds. This spontaneous emission can be suppressed via stimulated emission which can deplete the state S₁ by optically forcing the molecule into S₀immediately after excitation.

With increasing intensity of the de-excitation beam, the excited state S₁is more and more likely depleted; the molecule is spending almost no time in this state. Hence the fluorophore is essentially confined to its ground state S₀, which is equivalent to switching the fluorescence abil-ity of the molecule off [126]. To stimulate emission, a wavelength at the red end of the emission spectrum is used for two reasons: First, the

exci-Figure 2.2: Configuration of foci in a STED microscope. Overlaying a Gaussian excitation focus (left) with a toroidal de-excitation focus (mid-dle) results in a small region of remaining fluorescence (right). This small region is the effectivePSF(point spread function) of a STEDmicroscope.

tation cross section must be small at the de-excitation wavelength, oth-erwise the de-excitation light would also excite the dye instead of only stimulating the S₁to S₀transition. For most dyes, the excitation spec-trum overlaps with the emission specspec-trum, the excitation probability vanishes only towards the red end of the spectra. Second, stimulating the S₁ to S₀ transition at the red end of the emission spectrum allows the spectral separation of spontaneous and stimulated emission.

To use the stimulated emission for increasing the resolution in a STEDmicroscope, a de-excitation focus of toroidal (“donut”) shape [338, 341] as shown in Fig.2.2(other shapes of the de-excitation focus are pos-sible [174,172] but less common) is overlaid onto the excitation focus of Gaussian shape. Thus, the fluorophores are switched off via stimulated emission in the periphery of the excitation focus. Using a high intensity of the de-excitation beam, the S₁state of the fluorophores is almost com-pletely depleted also in those regions where therelativeintensity of the beam is low, i. e. close to the midpoint. Only in the very center, where the de-excitation focus has zero intensity, the fluorophores remain in the fluorescing (“on”) state (Fig.2.3).

The area wherein the fluorophores are not switched off and accord-ingly the minimally resolvable distance shrink to zero with increasing intensity of the depletion beam [128]. The lateral resolution follows the relationship [117]

d≈d_c^.q1 +d_c²a²I/I_sat, (2.1)

Figure 2.3: Interaction of the excitation and depletion foci to generate a small fluorescing region. The lower panels are enlargements of the upper panels. The blue horizontal line indicates the saturation intensity (the in-tensity at which half of the molecules are switched off by stimulated emis-sion). Cross sections through the overlaid foci are plotted: blue dotted:

excitation focus, red: depletion focus, black: effectivePSF. Left: low deple-tion intensity. Only in the outer part of the focus is the depledeple-tion intensity above the saturation intensity of the dye. The effectivePSFis slightly nar-rowed. Right: high depletion intensity. Although theshapeof the depletion focus is unchanged, the depletion intensity is above the saturation inten-sity also close to the center. The effectivePSFis strongly narrowed because all molecules in the periphery are kept in the off-state. Note that the dif-ferent intensities are not drawn to scale: the depletion intensity (red line) is usually much higher than the excitation intensity (blue dotted line).

wheredis theFWHM⁽¹⁾ of the fluorescence intensity distribution in the area that remains fluorescing, dc the FWHM of the corresponding con-focal PSF⁽²⁾ [≈ λ/(2NA), with λ the excitation wavelength and NAthe numerical aperture⁽³⁾], aa factor describing the shape of the depletion pattern,I the maximal intensity of the depletion focus andIsatthe satu-ration intensity, a fluorophore-characteristic constant; at this intensity half of the excited dye molecules are forced to the ground state (i. e.

switched off) by stimulated emission.

Increasing the depletion intensity decreases the fluorescing area (the “effective PSF”): No lower limit exists for the size of d. With the depletion-beam turned off, the microscope operates as a standard con-focal laser scanning microscope.