2.3 GSD nanoscopy with emitters resistant to photobleaching
2.3.6 Restoration of emitter distributions from the GSD image
The GSD microscopy discussed here provides raw-data images in a ‘negative’ modality, as the emitter positions are encoded in the detected signal minima. Mathematically, an imageg(r)formed in a raster scanning GSD microscope is described by the convolution of the effective PSFhGSD(r)and the real emitter positions fobj{δ(r−rj)}in the sample
g(r)=hGSD(r) ∗ fobj{δ(r −rj)}. (2.22)
whererjare the positions of the molecules and∗denotes the convolution operator. In GSD microscopy, the effective PSFhGSDis dependent on the ‘on’↔‘off’ state dynamics and given by
hGSD(r,Iexc)= hPSF(r) ∗hobj(δ,Iexc), (2.23) wherehPSFis the diffraction-limited (linear) excitation PSF andhobjdenotes the response function of the point-like emitter to the given excitation intensityIexc.
A map of the emitters’ distribution fobj(r)can be approximately restored from the raw-data GSD image g(r) by deconvolution. A basic deconvolution algorithm exploits the fact that convolution corresponds to a multiplication in Fourier domain
GGSD(ν,Iexc)= HGSD(ν,Iexc)Fobj(ν) (2.24) whereGGSD,HGSD,Fobj are the frequency representation ofgGSD, hGSDand fobj, respectively. The estimation of the exact object distribution can be described by
fobj(r)=F−1{Fobj(ν)}=F−1
GGSD(ν,Iexc) HGSD(ν,Iexc)
, (2.25)
ifHGSD(ν,Iexc),0 andF−1denotes the inverse Fourier transform. It is very unlikely thatHGSD(ν,Iexc),0 for all frequencies. Thus, presented above simple approach requires additional regularization param-eters to minimize related artifacts. Moreover, both the estimated effective PSFHGSDand registered imageGGSDare affected by presence of the measurement noise. Artifacts are visible as wave patterns (‘ringing’) around edges of the retrieved object positions (see restored image by Wiener deconvolution in Fig. 2.7e). Additional artifacts originate from the image boundary conditions. Signal disconti-nuities, between right-left and top-bottom borders of the image result in horizontal and vertical oscillations (‘stripes’) propagating trough the restored image. The most widely used deconvolution algorithms are Wiener filtering115and Richardson-Lucy deconvolution116,117.
Concluding discussion
This research examined the impact of STED-light photon flux on the photobleaching of several com-mercially available organic dyes (ATTO647N, STAR635P, ATTO590, STAR580) in bulk experiments in thiodiglycol. The observed effects of STED-light induced photodamage varied among the investi-gated dyes. In general, we observed two characteristic bleaching regimes for a constant STED pulse energy, which roughly corresponds to a given factor of resolution enhancement: STED-intensity-dependent photobleaching and STED-intensity-inSTED-intensity-dependent photobleaching. For ATTO647N the (bulk) photobleaching measurements revealed a single effective scaling with the STED intensity
∝ ISTED1.4 , indicating a significant involvement of higher-excited molecular states in STED-light-induced damage. We proposed an intuitive photobleaching model for this dye, which provides a link from the bulk results to STED imaging conditions. This model can predict the attainable resolution and spatially-dependent photobleaching probability at different STED intensity levels. We showed that the high-order photobleaching component is minimized by increasing the STED pulse duration. To main-tain the maximal resolution at a given STED pulse energy, the fluorescence detection gating has to be adjusted to the STED pulse duration. The optimal STED pulse length depends on the photobleaching scaling with the STED-light intensity and has to be optimized for a particular molecular probe and its microenvironment. These results rationalized the evident success of super-resolution imaging demon-strated with∼1 ns STED pulses76. Moreover, this work revealed that recently demonstrated image acquisition strategies as MINFIELD96or DyMIN97 could benefit from using relatively short STED pulses (∼10−100 ps), as the high-order photodamage component associated with excessive intensities present at the STED doughnut crest is already minimized. The application of shorter STED pulses would make it possible to collect more fluorescence signal at the coordinate targeted by the STED beam minimum, since detection gating is not necessary. Signal increase would shorten the necessary STED-light exposure time per pixel to acquire a suitable signal-to-noise ratio and therefore reduce photodamage.
Due to the complexity of the photobleaching process, we have limited the investigations to STED microscope operating at the commonly used de-excitation wavelength of 750 nm. The choice of dyes (listed above) was restricted by the laser spectral properties. All measurements were conducted for a single environment of the molecular probes (thiodiglycol). These limitations should be addressed in follow-up studies. For example, optimization of the stimulated emission spectral window could
35
36 Chapter 3. Concluding discussion
diminish the damage mediated by particular optical transitions of the markers induced by absorption of STED-light photons. Unfortunately, the excited-state absorption spectra (in the singlet and triplet systems) at the STED wavelengths are often unknown for commonly used STED probes. Moreover, at high STED pulse energies, required for currently accessible resolutions of∼20 nm, a significant part of photobleaching is likely mediated by one-photon excitation of ‘hot’ molecules by photons from the STED beam. These events are not apparent in the STED experiment because of efficient de-excitation occurring in parallel, and also due to the confocalized detection system, which rejects a large part of the signal generated outside the targeted coordinate. The photobleaching initiated by one-photon absorption of ground-state molecules can be diminished by shifting the STED operation to a longer wavelength which is further away from the absorption maximum of the fluorophore. This approach reduces in the same time the stimulated emission cross-section. However, the roughly exponential decay of the thermally broadened red edge of the absorption spectral band is typically faster than the decay of the stimulated emission cross-section with STED wavelength. Additionally, the STED-light-mediated excitation of ground-state fluorophores is expected to be diminished at low temperatures. Low temperatures should freeze the Boltzmann broadening of the absorption spectral lines and allow to provide STED photons closer to the maximum of stimulated emission cross-section.
The low-temperatures conditions, however, are less relevant in the biological context. Moreover, the photophysical properties of the molecules are different than at room temperature (e.g., longer lifetime of the lowest triplet state) and may require the modification of currently existing imaging schemes.
Our experiment did not discriminate singlet-system photobleaching from triplet-system photo-bleaching. Additional data on the photobleaching scaling in singlet and triplet systems with STED intensity would be of great interest. Techniques such as fluorescence correlation spectroscopy could bring new insights. The triplet-mediated damage can be significantly reduced by application of low repetition rate lasers29,30, fast scanning over a large field of view91or chemical triplet quenchers20. After reduction of the triplet-mediated bleaching, any considerable high-order photobleaching could be lowered by application of relatively long STED pulses.
Further photobleaching studies of organic dyes and fluorescent proteins, especially in aqueous environments, could advance the capabilities of STED microscopy in biological systems. Research on the photobleaching of fluorescent proteins revealed the involvement of higher excited states in photobleaching under conventional confocal microscopy conditions89, indicating that long STED pulses could reduce photobleaching also for many of these emitters. However, little is known about the photobleaching of fluorescent proteins induced by light at the STED wavelengths, and further studies are necessary.
Reducing photobleaching of the fluorophores would make it possible to provide more STED photons to the sample and, therefore, increase the currently achievable resolution. Further increases require a better spatial definition of the STED doughnut minimum. At the currently applied STED energies, a significant part of the signal generated at the targeted coordinate is affected by the residual STED intensity in the minimum (causing stimulated emission). New experimental protocols to vali-date and improve the STED beam quality would, therefore, be valuable.
The second part of this thesis demonstrated the use of GSD nanoscopy to image emissive GaInP segments within a non-emissive GaP nanowire. We have shown a 5-fold lateral resolution enhancement over conventional confocal microscopy. By using just a single laser beam at a wavelength of 700 nm (featuring low cellular toxicity) with a moderate power of∼3 mW (pulse durationτ=5 ps, repetition rate 80 MHz), we resolved different geometries of GaP–GaInP nanowire barcodes with diameters of 20−80 nm. We put an emphasis on studying the influence of barcode geometry on the GSD image contrast. These findings should be relevant for applications of heterostructured nanowires in biological systems, as well as in other fields.
The combined super-resolution imaging of biological structures labeled with fluorophores and GSD imaging of heterostructured nanowires entails several challenges. A careful choice of the fluorescent markers is required. The GSD beam should ideally interact only with NWs and not with other molecular probes present in the sample. The spectral properties of the fluorophores should avoid one- and two-photon excitation events from GSD pulses of relatively short duration. Even in case of molecular excitation by GSD-light, the photoluminescence signal from nanowires can be decoupled from the fluorescence signal by lifetime differentiation using detection gating. The NWs investigated in this study featured emission lifetimes of∼10 ps, which is several orders of magnitude shorter than the fluorescence lifetime of common fluorescent probes (∼1−5 ns). In addition, time gating can be employed to image fluorophores and nanowires with overlapping emission spectral composition.
The limiting factor to further resolution improvements in the GSD imaging demonstrated here is related to the light-induced damage of photoluminescent GaInP segments. At certain GSD powers of ∼3−7 mW, we observed an abrupt and irreversible loss of the photoluminescence signal. The mechanism of photodamage is not yet understood. Further quantitative studies of the NWs’ photo-damage could enable the development of new procedures for nanostructure synthesis. For example, axial coating of GaP–GaInP nanowires by a higher bandgap semiconductor (core-shell engineering) could protect the GaInP electrons from chemical reactions with the local environment. Quantitative studies, however, require more detailed information on the GaInP crystal structure before and after the GSD-light-induced damage. Energy dispersive X-ray spectra and low-temperature photoluminescence spectra may bring new insights. Such additional data would make it possible to study the influence of dopants and surface defects on GaInP photostability for different nanowire geometries. In the current work, we were not able to address these issues, as even for identical growth parameters the consecutive GaInP segments within a single nanowire exhibited significant differences in the photoluminescence strength, likely indicating a difference in the segments’ material composition.
The imaging of GaP–GaInP nanowires with diameters <20 nm constitutes another challenge.
With the decrease of nanowire diameter we noticed an increase of random fluctuations of the emitted photoluminescence signal. For ∼10 nm diameter NWs, we have observed relatively strong signal intermittencies. This severe drawback did not allow us to resolve the GaInP segments using GSD nanoscopy in this case. Signal fluctuations can be possibly reduced by core-shell engineering, similarly as it has been done to reduce blinking in quantum dots103,104. As signal flickering occurs when the
38 Chapter 3. Concluding discussion
nanostructure size is approaching the characteristic excition size (Bohr radius) of a semiconductor material, imaging of thinner nanowires could be realized by choosing a NW material with a smaller exciton Bohr radius.
The point-by-point image acquisition scheme applied here is not time-efficient for large fields of view. However, the moderate power requirements of GSD imaging would allow for parallelization of the image collection, in a similar manner to the scheme demonstrated for RESOLFT nanoscopy60.
Many applications involve nanowire arrays, where nanowires are oriented in parallel to the objec-tive axis of the microscopy system. An axial resolution enhancement for heterostructured nanowires has not been demonstrated yet, however, this should be relatively straightforward for GSD nanoscopy, as the strategy can be replicated from that previously applied in STED nanoscopy23.
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