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Prospects for Fluorescence Nanoscopy Chuankang Li,† Cuifang Kuang,*,†,‡ and Xu Liu†,‡ †
State Key Laboratory of Modern Optical Instrumentation, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310027, China ‡ Collaborative Innovation Center of Extreme Optics, Shanxi University, Taiyuan 030006, China ABSTRACT: Overcoming Abbe’s diffraction limit has been a challenging task and one of great interest for biological investigations. The emergence of fluorescence nanoscopy circumvents the diffraction barrier with nearly limitless power for optical microscopy, which enables investigations of the microscopic world in the 1−100 nm range. Proposed variants, such as expansion microscopy (ExM), stimulated emission depletion microscopy (STED), and Airyscan, are innovative in three aspects: sampling, illumination, and detection. These techniques show increasing strength in bioimaging subcellular structures. In this Perspective, we highlight advances in and prospects of fluorescence nanoscopy.
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depletion intensity, molecular-scale resolution can theoretically be achieved.1 In the frequency domain, structured illumination microscopy (SIM)2 employs nonuniform excitation light patterns to extract highly detailed information with a doubled resolution of approximately 100 nm. It is worth noting that the discernment of details is not determined only by simultaneous separation of the regions of interest. In the temporal domain, stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) register some fluorophores stochastically at one time, whereas other fluorophores remain silent.3,4 Because the fluorophores are registered sporadically, only a single fluorescent molecule can be established in the “on” state within the subdiffracted region at a time. By virtue of repetitive registering of the fluorescent molecules and the combination of each record of emitting positions, the image reconstruction configuration visualizes the profiles of the regions of interest. Furthermore, other super-resolved techniques also focus on temporal features. For example, fluorescent molecules present different lifetimes in various environments, and this specificity can be utilized to achieve fluorescence lifetime imaging microscopy. If fluorophores within subdiffracted regions embody different signals and we extract the specificity signals, we can achieve super-resolution imaging. For example, fluorescence correlation spectroscopy correlatively analyzes fluctuations in the emitters’ intensities. We can investigate other novel features of fluorophores, as well, such as whether nanoscopy can be achieved by utilizing mass specificity or electron spin difference of the fluorophores. In this Perspective, we summarize the advancement of fluorescence super-resolved methods and present prospects for those techniques.
esolution is defined by the ability to distinguish two structures as separate entities; it determines the ability of an imaging system to resolve details in the imaged object. However, the existence of the diffraction limit prevents boundless discernment in a conventional optical system; that is, details closer than the diffraction limit are indistinguishable. Abbe’s diffraction limit theory establishes a ceiling in optical resolution such that an optics system can distinguish two structures in the distance of λ/2NA. Currently, subcellular structures in living cells, which are generally in the 1−100 nm range, have been studied extensively by optics implementation, such as superresolution microscopy (or nanoscopy). An important question is how best to achieve super-resolution microscopy to enable less-invasive imaging in living cells with cost-effective and convenient configurations. Fluorescence microscopy is an imaging method that features multicolor and specificity marking. In fluorescence microscopy, fluorophores are label molecules (or nanostructures) that are fused or bound to target protein molecules. After photons are absorbed from illuminating radiation, fluorescence occurs and makes the target proteins detectable. The fluorescent label molecules visualize the whole region of interest, revealing the profile of the subcellular structure. Denser emitters tend to reveal more feature details. However, imaging is challenging when two emitters are closer than the diffraction barrier, which highlights the need for fluorescence super-resolution microscopy or fluorescence nanoscopy. A variety of super-resolution methods have been developed over the past two decades, which overcome Abbe’s criteria in several different ways. In the spatial domain, stimulated emission depletion microscopy (STED) features two beamsan excitation solid beam and a depletion hollow beamto squeeze the effective point spread function (PSF). With sufficiently high © XXXX American Chemical Society
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the swelling of the polymer materials employed in baby diapers could be useful for this purpose. Expansion microscopy (ExM) was proposed in 2015, and the polymer materials utilized in ExM feature topological specificity.5 With the aim of discerning subdiffracted details in conventional microscopes regardless of complex and expensive architectures, ExM is a novel and promising technique. In this approach, immunofluorescence tags are cross-linked to the gel matrix (a kind of polymer material), such that the locations of the tags are frozen. The original cells or tissues are then decomposed by protease. Expansion microscopy can achieve a 4-fold improvement in resolution (10-fold resolution improvement in iterative ExM) by increasing the distance between the tags while swelling the gel matrix.6 Although the inflation of the polymer gel pushes the labels apart from each other, the spatial relativity to one another remains immobilized. In ExM, what is observed by confocal or conventional fluorescence microscopy is not the structures themselves but “ghost” images of the precise places where the epitopes are located in the structures.7 The workflow of sample preparation and the rationale of circumventing the diffraction limit are shown in Figure 1.
Expansion microscopy achieves super-resolution imaging with classic or conventional hardware, is optically aberration-free, and enables biological reaction spacing and multi-epitope labeling. Sampling. A remarkable, alternative approach for nanoscopy consists of adjusting the sample while keeping the microscope technology unchanged. With this approach, the idea is to extend the size of the structures under examination but to maintain the resolution limit intact. As an illustration, if a sample volume experienced a 64-fold expansion, a distance of 50 nm between structures would become a distance of 200 nm. Therefore, subcellular structures with dimensions below the optical diffraction barrier could be resolved via classic microscopes. However, we need to determine how to “swell” a sample without inducing spatial distortion or structural damage. Interestingly, understanding
Figure 1. Schematics of the sample preparation workflow in expansion microscopy (ExM) and its rationale for circumventing the diffraction limit. (a) Neuron tissues. (b) Certain substances (pink colored, fluorochrome-conjugated antibodies) anchored to target biomolecules (cream colored). (c) Neuron tissues are embedded into a polymer gel, which is the heat-activated cross-linker agent. The polymer matrix also binds to the anchoring substances. (d) Protease is then added so that most of protein molecules are degraded, including the target biomolecules, which mechanically homogenizes the cells. (e) By exchanging with deionized water, the volume of the tissues extends, and the hydrogel matrix is also enlarged (water will make up ∼99% of the content of the inflated sample, which makes the subsequent observation aberration-free). (f) Expanded sample is eventually labeled with fluorophores (red colored). Therefore, the structures previously indiscernible by a conventional microscope can be separated. (g−i) Diagrams of the ExM principle. Adapted with permission from ref 7. Copyright 2017 The Spanish Society of Anatomy. B
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emotions. Rather than magnifying these invisible structures with a microscope, they succeeded in physically enlarging them and making them easier to observe. This novel and increasingly used method conspicuously circumvents the diffraction limitation, enabling scalable super-resolution imaging to be achieved with diffraction-limited microscopes. Illumination. Stimulated emission depletion microscopy selectively deactivates fluorophores to increase imaging resolution. The developers of STED proposed that only a fraction of the molecules should be excited: first, all of the fluorophores are lit up, and then most of them are extinguished. This selective deactivation is the fundamental strategy of the STED modality, which features a phase-modulated doughnut-shaped focal spot. The doughnut focal spot is characterized by intensity minima (ideally intensity zero) in the center, which enables on-switching (fluorescence) of the center region and off-switching (without fluorescence) of the peripheral region, thereby narrowing the PSF. The full width at half-maximum of the effective PSF is routinely far below the diffraction limit. During the point-scanning process, each coordinate of the target area experiences on−off switching (Figure 2). The reverse protocol is also worthy of
In addition to swelling, a sample could also be shrunk back; a 2-fold or 3-fold decrease in size can be obtained by the addition of salt. Expansion microscopy offers several benefits: it achieves super-resolution imaging with classic or conventional hardware, in contrast to other expensive and complex techniques, with limitations in imaging speed and number of colors; it is optically aberration-free as 99% of the inflated sample content is water, simultaneously overcoming the problem of scattering in depth imaging; and the decrowding of the biomolecules or labels enables biological reaction spacing and multi-epitope labeling. However, ExM faces technical challenges, as well. It remains unclear whether the expanded cellular structures truly represent the native conformation in cells below the resolution limit. An increasing number of ExM studies in organelles such as mitochondria, centrioles, and motile cilia, however, show accurate matches with the results obtained by other optical super-resolved and electron microscopy techniques. The determination of the extent to which the inflated structures resemble the true nature of the cellular interior is still at the proof-of-principle stage. Data have shown distortions in length of 1−4%, corresponding to errors of 5−10 nm, which are acceptable for most purposes. Another challenge is that ExM is unable to characterize regions with clustered protein molecules, like synaptic cleft, because fluorophores are of nonzero size, and the overcrowding of labels leads to poor staining and uneven anchoring or pulling in overpopulated circumstances. Currently, the highest resolution achieved by ExM is 25 nm. In addition, labeling can be performed before or after the expansion, the advantages and disadvantages of which need to be investigated further experimentally. Some epitopes may be lost during the procedure of denaturation and degradation of most of the proteins by protease, so the labels should be sufficiently protease-resistant. A crucial weakness of ExM is its limitation in monitoring dynamics. However, because ExM is often more convenient and cost-effective than electron microscopy, this limitation motivates combinations with other super-resolution techniques. As an example, ExM and SIM provided a successful combination.8 In this issue of ACS Nano, Gao et al. combine ExM and STED microscopy (ExSTED) to take advantage of creativity in sample manipulation as well as light modulation in illumination.9 The problems that Gao et al. address include the following: (1) oil immersion objectives tend to yield spherical aberrations with whole-cell imaging; therefore, they used water immersion lenses instead, which have lower collection efficiencies yet higher signal-to-noise ratios (SNR); (2) the multiepitope labeling approach overcomes the problem of signal loss during the isotropic three-dimension (3D) expansion; (3) longterm observations and large field-of-view scanning takes hours to complete and induces loss of water, which could lead to sample drift; therefore, Gao et al. immersed the cover-glassmounted gels in two-component silicone, thereby extending the imaging duration to days.
Figure 2. Schematics of stimulated emission depletion microscopy (STED). (a) Comparison between confocal microscopy (left) and STED microscopy (right). In a conventional microscope, point scanning captures signals from mixed targets that are located closer than the diffraction limit. In the STED method, the narrowed focal spot can separate structures within subdiffracted regions. (b) Comparison between coordinate-targeted nanoscopy (left) and coordinate-stochastic nanoscopy (right). Stimulated emission depletion microscopy is a typical targeted nanoscopy, registering fluorescent molecules one by one in a predefined path. Stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) are typical modalities, stochastically activating probes to fluoresce. Reprinted with permission from ref 10. Copyright 2016 Elsevier.
In this issue of ACS Nano, Gao et al. combine ExM and STED microscopy (ExSTED) to take advantage of creativity in sample manipulation as well as light modulation in illumination.
implementation, with on-switching at the peripheral region and off-switching at the minima. Significant progress has been made over the past two decades in STED microscopy, in both hardware and software, with (1) selection on continuous wave lasers or pulse lasers, (2) selection of wavelengths, and (3) modulation of depletion beam. Recently,
At the beginning of ExM development, the developers aimed to reveal how the tiny biomolecules in our brains generate C
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The smaller the size of the pinhole, the larger the enhancement in resolution. However, too small sizes decrease the pass-through and admission of photons, consequently reducing SNR. Airyscan, which is a type of two-dimension detector array that was commercialized in 2014, can also be utilized.24 Compared with the single detector in a conventional wide-field microscope, a detector array with 32 channels yields 1.7-fold resolution enhancement and lowers the intensity requirement. Virtual k-space modulation optical microscopy,25 a modality that combines the detector array and the virtual modulation, shows promise for applications in bioimaging. In addition to pinholes or Airyscan (so-called space gating in detection), the aforementioned time-gating detection also improves the resolution of an imaging system. Time-gating detection enables early fluorescent photons emitted by fluorophores (with short lifetimes) that are insufficiently irradiated by the illumination to be abandoned so that fluorescent molecules with longer lifetimes can be addressed, thereby increasing the effective resolution and avoiding a trade-off between the resolution and SNR. Although cost-prohibitive at present, we believe that detector arrays will eventually be widely used in combination with fluorescence super-resolution microscopy.
STED has reached an advanced level and has been commercialized, featuring 3D super-resolution, higher resolution, faster imaging, imaging in depth, lower architecture cost, improvement in system stability, and multicolor imaging. However, crucial limitations of STED microscopy are the high intensity required for the short lifetime of fluorophores and escape of secondary excitation. The ultrahigh light doses required can cause severe photodamage, such as photobleaching and phototoxicity. Therefore, various modalities have been proposed to overcome this problem. By lengthening the duration time to match the triplet-state lifetime, fast scanning11 and triplet-state relaxation12 are convenient and portable strategies. For the requirement of low photodamage in tissues in depth, two-photon excitation13 is superior. The background suppression technique is superior in terms of the trade-off between low photodamage and high SNR.14 Alternatively, time-gating detection is an efficient approach for STED microscopy to reduce its lightinduced negative impact.15 Adaptive optics, or adaptive illumination, aims to require the lowest photon budget to reach the highest imaging effect.16 Overall, STED microscopy, the creators of which were recognized with the Nobel Prize in Chemistry in 2014, is entering a new phase of development with the goals of being readily utilized by life scientists and of requiring only moderate light intensity. The doughnut-distributed or hollow beam enables other super-resolution imaging methods, as well, such as reversible saturated optical linear fluorescence transitions microscopy,17 ground-state depletion microscopy,18 coordinates of a molecule with minimal emission flux (MINFLUX),19 charge state depletion microscopy (CSD),20 nonlinear focal modulation microscopy,21 fluorescence emission difference microscopy,22 and saturated competition microscopy (SAC).23 It would be worthwhile to investigate all of these approaches in combination with ExM. In CSD, a solid beam and a hollow beam act as excitation radiation and depletion radiation, respectively, and the roles of the two beams are interchangeable to obtain super-resolved imaging of nitrogen-vacancy (NV) centers. Resolution of 4.1 nm of the NV centers has been achieved by CSD, which shows promise for the measurement of spin-state dynamics of NVs. As for SAC, which has a low requirement for fluorescent labels, the competition of two incident beams, a solid beam and a hollow beam, is utilized to produce narrower PSFs. The obtained resolution is ∼100 nm, thus SAC has the potential to reveal ultrafine structural details in hybridization with ExM. Alternately, MINFLUX utilizes the advantages of two types of illumination-modulated methods,19 STORM/PALM and STED; STORM/PALM is classified as the coordinate-stochastic illumination method, and STED is classified as the coordinatetargeted illumination method. In MINFLUX, doughnut-shaped beams are utilized to locate the fluorophores, and the localization process is stochastic. The registering process is targeted for an economical use of photons in imaging. With minimal photon flux, a high resolution of ∼1 nm can be achieved. It is worth investigating whether a sub-nanoresolution at the atomic level can be achieved by combining ExM and MINFLUX. The hollow-beam-based optics is promising owing to its beam specificity of on−off switching. Hybrids of ExM and doughnutfocal-spot illumination methods may show great potential. Detection. Detection is of paramount importance in fluorescence nanoscopy. The pinhole is an early method of detection modulation for improving the system resolution, which is important for confocal microscopy. A pinhole enables read-out of in-focus photons by the detector, filtering out-of-focus photons.
CONCLUSIONS AND PROSPECTS Fluorescence super-resolution methods aim to circumvent Abbe’s criterion. Current super-resolution methods can be modulated and improved further in terms of setup, hardware, and software. In this Perspective, we highlighted super-resolution techniques and their progress in sampling, illumination, and detection. Expansion microscopy, a sampling-modulated method, is useful in deciphering subdiffraction features of cells. It enables 4-fold or even 10-fold resolution improvement compared to that with confocal or conventional microscopes. Stimulated emission depletion microscopy is a coordinate-targeted system based on point scanning. The focal spot has a doughnut distribution, featuring center minima and leaving the hollow center size significantly smaller than the diffraction limit, narrowing the effective PSF. Many methods have been developed recently based on doughnuttype focal spots. Regarding detection, detector arrays are promising for lowering the required light intensity, which will improve SNR. The combination of ExM, doughnut-focal-spotbased techniques, and detector arrays could provide solutions in bioimaging with greater resolution, cost-effectiveness, portability, and less invasive characteristics. AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS This work was financially supported by grants from the National Key Research and Development Program of China (2017YFC0110303); National Basic Research Program of China (973Program) (2015CB352003); National Natural Science Foundation of China (NSFC) (61335003, 61427818); Natural Science Foundation of Zhejiang province (LR16F050001); and the Fundamental Research Funds for the Central Universities (2017FZA5004). D
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Fluorescence Emission Difference Microscopy. Sci. Rep. 2013, 3, 01441. (23) Zhao, G. Y.; Kabir, M. M.; Toussaint, K. C.; Kuang, C. F.; Zheng, C.; Yu, Z. Z.; Liu, X. Saturated Absorption Competition Microscopy. Optica 2017, 4, 633−636. (24) Huff, J. The Airyscan Detector from ZEISS: Confocal Imaging with Improved Signal-to-Noise Ratio and Super-Resolution. Nat. Methods 2015, 12, 1−2. (25) Kuang, C.; Ma, Y.; Zhou, R.; Zheng, G.; Fang, Y.; Xu, Y.; Liu, X.; So, P. T. Virtual k-Space Modulation Optical Microscopy. Phys. Rev. Lett. 2016, 117, 028102.
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