Principles of GSD microscopy

Ground state depletion (GSD) microscopy is a superresolution method complementary to STED microscopy, proposed by Stefan Hell in 1995⁴⁵. Several years later, the applicability of this concept to organic fluorophores was proven experimentally¹¹². The imaging scheme is very similar to STED

28 2.3. GSD nanoscopy with emitters resistant to photobleaching

microscopy, however, separation of the states is mediated by the process of optical excitation rather than by stimulated emission. The doughnut-shaped GSD beam is applied to deplete the ground state by optical shelving into an excited state, everywhere except at the targeted coordinate, where the beam intensity ideally equals zero. Originally, the GSD concept was proposed to utilize the transfer of fluorescent emitters from the ground state S₀ to a metastable long-lived dark state, as for instance the first excited triplet stateT₁ naturally occurring in molecular dye probes. The molecular shelving intoT₁ can be indirectly controlled by the excitation light, seeing that with each optical transition a small fraction of the excited molecules will eventually undergo intersystem crossing (S0→S₁ T₁).

Thus, the efficiency of this process depends on the intensity of the excitation GSD beam, which populates S₁ and effectively provides the ground state depletion. Upon separate illumination by a second weaker Gaussian-shaped probe beam operating at the same wavelength, molecules trapped in T₁ cannot generate fluorescence signal (‘off’ state). Only molecules nearby the coordinate targeted by the minimum of the GSD beam remain in the singlet system (‘on’ state) and can interact with the probing light. These emitters can be promoted to the first excited singlet state (S₀→S₁) and emit

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Figure 2.7: GSD nanoscopy. (a)Scheme of the GSD microscope. (Obj: objective lens,λ/4: quarter-wave plate, DM: dichroic mirror, HPP: helical phase plate, L: tube lens, MMF: multimode fiber, APD: avalanche photodiode). (b)Jabłoński diagram of an organic molecule showing the relevant electronic states: excitation (Exc) and spontaneous relaxation by fluorescence (Fluo). (c)Point spread function of the excitation beam in lateral (xy) and axial (xz) directions, as measured by scattering of a 80 nm gold bead. The scattering signal was measured on a photomultiplier tube (path not shown in (a)). The excitation wavelength was 720 nm. (d)Raw data image of red-emitting fluorospheres (715 nm/755 nm; maximal absorption and emission, respectively) with a diameter of 80 nm. The average excitation power equals∼500 µW at 80 MHz. (e)Bead positions from image (d) restored by Wiener deconvolution with PSF (inset) estimated from a single bead image. Scale bars:

500 nm (c), 1 µm (d,e).

fluorescence signal. The volume of the effective ‘on’-state emitters, which governs the resolution, can be significantly smaller than the diffraction limit, depending on the intensity of the GSD beam.

The general concept of GSD microscopy is not restricted to the metastable dark state. Any excited molecular state which population depletes the ground state can be employed to increase the optical resolution. The very same idea was applied later in saturated structured illumination microscopy (SSIM)¹¹³, where ground state depletion occurs by optical shelving of emitters in the first excited singlet stateS₁in a spatially parallelized manner. Employment of a long-lived dark state has the benefit of a more intuitive imaging modality which, unlike SSIM, does not necessitate any post-acquisition processing of data. The drawback is a limited speed, as for the point-to-point image acquisition markers trapped in the triplet state have to be able to return to the singlet system.

For some fluorophores, the triplet state cannot be efficiently populated due to a low probability of ISC. In this case, ground state depletion can be realized by shelving the emitters in the first excited singlet stateS₁. Such a simple realization of the GSD concept is presented in Fig.2.7. The experimental setup is very similar to that presented previously for STED microscopy (see Fig.2.1).

The doughnut-shaped excitation (GSD) focal spot is generated by a helical phase plate (HPP, Fig.2.7a).

To ensure a high-quality doughnut ‘zero’ and uniform interactions of photons with randomly oriented molecular dipoles, the beam features circular polarization in the focal plane. The image is created by point-by-point scanning and collection of the fluorescence signal map. Confocal detection is realized by a multimode optical fiber (MMF) to increase the image contrast in the axial direction.

The possible molecular energy states are simplified to the ground stateS₀and the first excited singlet stateS₁ (Fig.2.7b). For an excitation rate significantly higher than the fluorescence rate, depletion of emitters from S₀ to S₁ is possible. At such high excitation intensities, the molecular response (fluorescence) to the excitation light is no longer linear. In this case, the fluorophore image represents the convolution of a molecular response function at the given excitation intensity and the diffraction-limited doughnut-shaped excitation focal spot (Fig. 2.7c). An example of a GSD raw-data image of red-emitting fluorescent beads is presented in Fig. 2.7d . The GSD variant discussed here has a ‘negative’ modality as the information about the molecular position is encoded in the minimum of the detected signal. The effective width of the dip, i.e. resolution, depends on the properties of the utilized optical transition. A more intuitive representation of the emitter positions can be retrieved after deconvolution (Fig.2.7e, Wiener filtering). It is important to note that emitters at the doughnut crest will be subjected to significantly higher intensities than are necessary for shelving them in S₁. The success of this strategy in practice depends on how well the emitters can tolerate the light exposure. This method, even though experimentally simpler than STED, is usually strongly limited by photobleaching. Thus, the GSD concept has been realized only in special cases of low or negligible photobleaching, with NV centers in the diamond lattice^9,114, some organic fluorophores at room temperature¹¹²and organic fluorophores at liquid helium temperatures¹⁶.

30 2.3. GSD nanoscopy with emitters resistant to photobleaching