Article

Cryogenic Colocalization Microscopy for Nanometer-Distance Measurements

Wiley
ChemPhysChem
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Abstract

The main limiting factor in spatial resolution of localization microscopy is the number of detected photons. Recently we showed that cryogenic measurements improve the photostability of fluorophores, giving access to Angstrom precision in localization of single molecules. Here, we extend this method to colocalize two fluorophores attached to well-defined positions of a double-stranded DNA. By measuring the separations of the fluorophore pairs prepared at different design positions, we verify the feasibility of cryogenic distance measurement with sub-nanometer accuracy. We discuss the important challenges of our method as well as its potential for further improvement and various applications.

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... Moreover, we performed nanoscopic distance measurements in order to confirm a physical interaction between the two protomers of SNAP-tagged D 2L receptor dimers using cryogenic localization microscopy 28,29 . This super-resolution microscopy method has recently demonstrated both Angstrom precision and accuracy in resolving nanometer separations. ...
... To investigate the physical interaction of two dopamine receptor protomers in a dimer configuration, we applied cryogenic localization microscopy as a super-resolution technique, where the stochastic blinking behavior of the fluorophores was used to distinguish and localize them individually. The method allows nanometer distance measurement with angstrom accuracy 28,29 . We prepared samples with CHO cells stably expressing SNAP-D 2L receptors adhered to clean fused silica cover slides. ...
... After inducing membrane protrusion formation through exposure of the cells to hypo-osmotic stress conditions, SNAP-D 2L receptors in intact CHO cells were frozen, using cryo-immobilization which preserved the structures in a near native state and their behavior was compared to monomeric CD86 membrane proteins. The decreased photo-bleaching at lower temperature and the resultant increase of the localization precision is a beneficial side effect of this technique 28,29,46 . ...
Article
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G protein–coupled receptors (GPCRs), including dopamine receptors, represent a group of important pharmacological targets. An increased formation of dopamine receptor D2 homodimers has been suggested to be associated with the pathophysiology of schizophrenia. Selective labeling and ligand-induced modulation of dimerization may therefore allow the investigation of the pathophysiological role of these dimers. Using TIRF microscopy at the single molecule level, transient formation of homodimers of dopamine receptors in the membrane of stably transfected CHO cells has been observed. The equilibrium between dimers and monomers was modulated by the binding of ligands; whereas antagonists showed a ratio that was identical to that of unliganded receptors, agonist-bound D2 receptor-ligand complexes resulted in an increase in dimerization. Addition of bivalent D2 receptor ligands also resulted in a large increase in D2 receptor dimers. A physical interaction between the protomers was confirmed using high resolution cryogenic localization microscopy, with ca. 9 nm between the centers of mass.
... Using a reflecting objective, recently a cryo-FM approach achieved an NA of 0.99 (Inagawa et al., 2015). However, to realize localization precisions close to or below 1 nm, more than 10 6 photons have to be collected per fluorophore, still resulting in acquisition times exceeding 1 h with an NA < 1.0 (Weisenburger et al., 2014;Inagawa et al., 2015;Li et al., 2015b). First proof of concept experiments demonstrated the technical feasibility of a cryo-microscope with immersion objectives (Le Gros et al., 2009). ...
... Even if reaching an NA >1.0 in cryo-FM, this will be associated with further challenges for high demanding applications like super-resolution imaging. Under cryo-conditions, structures, but also fluorophores in the sample, are in a spatially fixed state causing the emitted light to be in a distinct polarization, which can result in an asymmetric PSF (Gould et al., 2008;Weisenburger et al., 2014). To avoid this effect leading to single molecule localization errors of ß100 nm when the fluorophore is 200 nm out of the focal plane, an azimuthal polarization filter can be integrated into the detection path of the microscope (Lew and Moerner, 2014). ...
... For certain applications of CLEM, structure-resolving super-resolution FM is not necessary and the precise location of a particle, a cellular compartment or a molecule is sufficient (e.g., in cases where rare objects or events are tagged with a fluorescent marker and no structural information is required in the fluorescence image). For isolated fluorescently labelled objects, it is even possible to achieve a much higher localization precision than the resolution of super-resolution FM (compare Weisenburger et al. (2013); Weisenburger et al. (2014); Li et al. (2015b) and Kaufmann et al. (2014b), Liu et al. (2015)). For the latter, only the photons collected per one switching cycle of the molecule can be used for the position determination, whereas for optically isolated objects all photons are available which are emitted by the molecule(s) before irreversible photo-bleaching. ...
Article
Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo-CLEM, the combination of fluorescence cryo-microscopy (cryo-FM) permitting for non-invasive specific multi-colour labelling, with electron cryo-microscopy (cryo-EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence based information for guiding cryo-EM data acquisition and/or verify the identity of the structure of interest. Furthermore, cryo-CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano-environment. However, a major obstacle of cryo-CLEM currently hindering many biological applications is the large resolution gap between cryo-FM (typically in the range of ∼400 nm) and cryo-EM (single nanometre to Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super-resolution cryo-FM imaging and the correlation with cryo-EM. This opened the door towards super-resolution cryo-CLEM, and thus towards direct correlation of structural details from both imaging modalities. This article is protected by copyright. All rights reserved.
... In recent years, cryo-fluorescence microscopy (cryo-FM) has been increasingly applied in the field of correlative electron and optical microscopy [1,2], single molecule fluorescence localization microscopy [3,4], and single molecule spectroscopy [5,6]. Correlative measurements bridging cryo-FM and cryo X-ray or electron microscopy combine the specificity of fluorescent labeling with molecular resolution, thus assisting the identification of structural details in biological samples [2,7,8]. ...
... In combination with super-resolution fluorescence microscopy techniques such as Stochastic Optical Reconstruction Microscopy (STORM) [9] or (Fluorescence) Photoactivation Localization Microscopy ((F)PALM) [10,11], cryo-FM benefits from an increased photon yield due to the enhanced photostability of the fluorophores at cryogenic temperatures [7,12,13]. This results in an exceptional high single-molecule localization accuracy and improved optical resolution [3]. ...
... Vacuum insulated cryostats [6,22] provide higher thermal and mechanical stability, and are thus usually employed for single-molecule fluorescence microscopy [3] and spectroscopy [5,6]. These cryostats are typically cooled by a continuous circulation of a cryogen [3] or by a static cryogen reservoir [6,22] using liquid helium (LHe) or liquid nitrogen (LN2). ...
Article
Full-text available
We developed a stand-alone cryostat with optical access to the sample which can be adapted to any epi-fluorescence microscope for single-molecule fluorescence spectroscopy and imaging. The cryostat cools the sample to a cryogenic temperature of 89 K, and allows for imaging single molecules using an air objective with a numerical aperture of 0.7. An important property of this system is its excellent thermal and mechanical stability, enabling long-time observations of samples over several hours with negligible drift. Using this system, we performed photo-bleaching studies of Atto647N dye molecules, and find an improvement of the photostability of these molecules by more than two orders of magnitude. The resulting increased photon numbers of several millions allow for single-molecule localization accuracy of sub-nanometer.
... Figure 1: Advancement of the optical resolution over time. Data points are taken from references [4][5][6][7][8][9][10][11][12][13][14][15][16]. ...
... The state of the art in resolution for localization microscopy is about 10 nm [14] limited by the total number of photons emitted before photobleaching. However, even Angstrom localization precision has been reported in cases where photobleaching could be delayed by using oxygen scavengers [13] or cryogenic conditions [16]. The latter measurements offer the additional advantage of a more rigid sample fixation than chemical fixatives. ...
... As a result, localization of the individual fluorophores requires a fitting procedure that takes this effect into account [165,166]. In the presence of nearby interfaces, this can become a nontrivial task [16]. However, the PSF asymmetry is much less pronounced if a microscope objective with low numerical aperture is used [166]. ...
Article
Full-text available
Optical microscopy is one of the oldest scientific instruments that is still used in forefront research. Ernst Abbe's nineteenth century formulation of the resolution limit in microscopy let generations of scientists believe that optical studies of individual molecules and resolving sub-wavelength structures were not feasible. The Nobel Prize in 2014 for super-resolution fluorescence microscopy marks a clear recognition that the old beliefs have to be revisited. In this article, we present a critical overview of various recent developments in optical microscopy. In addition to the popular super-resolution fluorescence methods, we discuss the prospects of various other techniques and imaging contrasts and consider some of the fundamental and practical challenges that lie ahead.
... Alternatively, the molecule can be localized with scanning confocal arrangements (17), but also in this case the need for large photon numbers remains. Therefore, improving localization has so far concentrated on increasing molecular emission, particularly through anti-bleaching agents (18), special fluorophores (19), cryogenic conditions (20), transient (fluorogenic) labels (21,22), and fluorophore-metal interactions (23). However, apart from the fact that all these remedies entail restrictions when applied to (living) cells, none of them have addressed the problem of limited emission budget fundamentally. ...
... Two dimensional trajectories with 1200 localizations were generated for free isotropic Brownian motion with diffusion constants in the range of ∈ [0.01,1]µ 2 / and count rates of Γ ∈ [20,200] . 100 trajectories were generated for each parameter combination. ...
Preprint
We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. A 22-fold reduction of photon detections over that required in popular centroid-localization is demonstrated. In superresolution microscopy, MINFLUX attained ~1 nm precision, resolving molecules only 6 nm apart. Tracking single fluorescent proteins by MINFLUX increased the temporal resolution and the localizations per trace by 100-fold, as demonstrated with diffusing 30S ribosomal subunits in living E. coli. Since conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.
... However, conventional SR microscopy performed at room temperature is still not considered as a contestant in the arena of structural biology, where Angstrom-level information about the molecular architecture of proteins and protein complexes is sought after. To push the limit of fluorescence microscopy, one can perform measurements under cryogenic conditions (Böning et al., 2021, Hoffman et al., 2020Dahlberg et al., 2020;Moser et al., 2019;Wang et al., 2019;Hulleman et al., 2018b, Xu et al., 2018Weisenburger et al., 2017;Furubayashi et al., 2017;Li et al., 2015;Weisenburger et al., 2014;Weisenburger et al., 2013). In addition to slowing down photochemistry, which allows each fluorophore to emit several orders of magnitude more photons than at room temperature (Li et al., 2015;Weisenburger et al., 2014;Weisenburger et al., 2013), a key advantage of cryogenic temperatures is in offering superior sample preservation and high stability for Angstrom-scale structural studies. ...
... To push the limit of fluorescence microscopy, one can perform measurements under cryogenic conditions (Böning et al., 2021, Hoffman et al., 2020Dahlberg et al., 2020;Moser et al., 2019;Wang et al., 2019;Hulleman et al., 2018b, Xu et al., 2018Weisenburger et al., 2017;Furubayashi et al., 2017;Li et al., 2015;Weisenburger et al., 2014;Weisenburger et al., 2013). In addition to slowing down photochemistry, which allows each fluorophore to emit several orders of magnitude more photons than at room temperature (Li et al., 2015;Weisenburger et al., 2014;Weisenburger et al., 2013), a key advantage of cryogenic temperatures is in offering superior sample preservation and high stability for Angstrom-scale structural studies. In one implementation, cryogenic optical localization in 3D (COLD) was introduced, where the stochastic intensity blinking of organic dyes gave access to the positions of up to four labeling sites on a single protein (Weisenburger et al., 2017). ...
Article
Full-text available
Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.
... Some success has been achieved with fluorescent proteins, which can switch on an off at cryogenic temperatures with a photon yield of ∼10 000 photons [12]. This is, however, a small fraction of the photon yield of organic dyes at cryogenic temperatures, where more than 1 000 000 photons are routinely achieved in sparse samples [7,13,14]. This hundredfold difference in photon yield translates to a ten times difference in localization precision [15]. ...
... This amount of photons is 1-2 orders of magnitude below reported average photon yields of sparse single molecules at cryogenic temperatures [7,[13][14][15]. One of the main bleaching pathways in STED microscopy is through the triplet state [22]. ...
Article
Full-text available
With the growing popularity of cryogenic correlative light and electron microscopy, it is becoming increasingly important to bridge the resolution gap between these two modalities. At cryogenic temperatures, the photon yield of fluorophores is a few orders of magnitude higher than at room temperature, enabling localization precisions on the Ångström scale. The current challenge is to induce sparsity at cryogenic temperatures such that individual fluorescent molecules can be localized. In this paper, we demonstrate the progress of using polarized stimulated-emission depletion (STED) to induce sparsity at cryogenic temperatures and in vacuum. We generate linear polarization of arbitrary in-plane orientations to achieve polarized STED with a sparsity of 3.3:1. Furthermore, we have probed the dark-state lifetime of ATTO 647N at cryogenic temperatures and in vacuum at room temperature. This dark state in vacuum is long-lived (τ=38 ms) and could be the cause for reduced photostability of fluorophores under STED illumination in vacuum. The experiments were done on an in-house designed and built liquid nitrogen cryostat, enabling 30 hours of stable cryogenic fluorescence microscopy.
... However, conventional SR microscopy performed at room temperature is still not considered as a contestant in the arena of structural biology, where Angstrom-level information about the molecular architecture of proteins and protein complexes is sought after. To push the limit of fluorescence microscopy, one can perform measurements under cryogenic conditions (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). In addition to slowing down photochemistry, which allows each fluorophore to emit several orders of magnitude more photons than at room temperature (23)(24)(25), a key advantage of cryogenic temperatures is in offering superior sample preservation and high stability for Angstrom-scale structural studies. ...
... To push the limit of fluorescence microscopy, one can perform measurements under cryogenic conditions (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). In addition to slowing down photochemistry, which allows each fluorophore to emit several orders of magnitude more photons than at room temperature (23)(24)(25), a key advantage of cryogenic temperatures is in offering superior sample preservation and high stability for Angstrom-scale structural studies. In one implementation, cryogenic optical localization in 3D (COLD) was introduced, where the stochastic intensity blinking of organic dyes gave access to the positions of up to four labeling sites on a single protein (21). ...
Preprint
Full-text available
Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases, where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and the hexamer geometry of Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic, environmental and dynamic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology. Significance statement Fluorescence super-resolution microscopy has witnessed many clever innovations in the last two decades. Here, we advance the frontiers of this field of research by combining partial labeling and 2D image classification schemes with polarization-encoded single-molecule localization at liquid helium temperature to reach Angstrom resolution in three dimensions. We demonstrate the performance of the method by applying it to trimer and hexamer protein complexes. Our approach holds great promise for examining membrane protein structural assemblies and conformations in challenging native environments. The methodology closes the gap between electron and optical microscopy and offers an ideal ground for correlating the two modalities at the single-particle level. Indeed, correlative light and electron microscopy is an emerging technique that will provide new insight into cell biology.
... Collection of these amounts of photons is possible for uncaging dyes [17] which today have been used infrequently, in DNA PAINT approaches where an effectively endless reservoir of dyes is imaged [18] , or through imaging at cryogenic temperatures. [19][20][21] At cryogenic temperatures dyes have a very low rate of photobleaching and localization precisions below 1 nm are typical. For fluorescent proteins, however, freezing only offers a moderate increase in total photon count before bleaching. ...
... This matches with the relatively low NA of objective lenses that can be used in cryogenic setups (typically up to NA=0.7). [19,21] Hafi et al. already put forward the idea to use polarization as an effective way to introduce sparsity in combination with STED, similar to our setup in Figure 1. [27] Later Frahm and Keller showed however that their results were in fact not based on polarization control but due to post processing of the data by a sparsity enhancing deconvolution algorithm. ...
Preprint
Full-text available
Light microscopy allowing sub-diffraction limited resolution has been among the fastest developing techniques at the interface of biology, chemistry and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultra resolution structures, brings highly specific labeling of molecules in a large assemble to the table and inherently allows the detection of multiple colors, which enable the interrogation of multiple molecular species at the same time in the same sample. Here we discuss the problems to be solved in the coming years to aim for higher resolution and describe what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples like whole cells.
... Cryofixation provides superior sample fixation, which is less prone to artefacts, so adapting SR to vitrified cryosamples poses a valuable alternative to RT-SR 8 . Furthermore, the reduced photobleaching rate of fluorophores at low temperatures 5,[19][20][21] can increase the photon yield of single-molecules, leading to a sub-nanometre localisation precision 19,22 . Several workflows have previously been developed to combine SR microscopy and EM [23][24][25][26] . ...
... Cryofixation provides superior sample fixation, which is less prone to artefacts, so adapting SR to vitrified cryosamples poses a valuable alternative to RT-SR 8 . Furthermore, the reduced photobleaching rate of fluorophores at low temperatures 5,[19][20][21] can increase the photon yield of single-molecules, leading to a sub-nanometre localisation precision 19,22 . Several workflows have previously been developed to combine SR microscopy and EM [23][24][25][26] . ...
Article
Full-text available
Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.
... Collection of these amounts of photons is possible for uncaging dyes [17] which today have been used infrequently, in DNA PAINT approaches where an effectively endless reservoir of dyes is imaged [18] , or through imaging at cryogenic temperatures. [19][20][21] At cryogenic temperatures dyes have a very low rate of photobleaching and localization precisions below 1 nm are typical. For fluorescent proteins, however, freezing only offers a moderate increase in total photon count before bleaching. ...
... doi: bioRxiv preprint first posted online Oct. 17, 2017; low NA of objective lenses that can be used in cryogenic setups (typically up to NA=0.7). [19,21] Hafi et al. already put forward the idea to use polarization as an effective way to introduce sparsity in combination with STED, similar to our setup in Figure 1. [27] Later Frahm and Keller showed however that their results were in fact not based on polarization control but due to post processing of the data by a sparsity enhancing deconvolution algorithm. ...
Article
Light microscopy, allowing sub‐diffraction‐limited resolution, has been among the fastest developing techniques at the interface of biology, chemistry, and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo‐electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultraresolution structures, brings highly specific labeling of molecules in a large assembly to the table and inherently allows the detection of multiple colors, which enables the interrogation of multiple molecular species at the same time in the same sample. Here, the problems to be solved in the coming years, with the aim of higher resolution, are discussed, and what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples, like whole cells, is described. Cryogenic super‐resolution fluorescence microscopy can be realized by utilizing fluorescence polarization control. Linearly polarized excitation and depletion lasers can be used to narrow the set of molecular dipole orientations that fluoresce. The enhanced cooling capacity and stability of the cryostat should enable resolutions down to the scale of individual emitters.
... 76,83,185 Synthetic fluorescent dyes represent a valuable alternative, as they avoid potential structural and functional artifacts sometimes found for FP-tagged proteins 186 and are often characterized by much brighter fluorescence. 99−101 Weisenburger et al. 187,188 recently demonstrated enhanced photostability of dye molecules at cryogenic temperatures and hence high-precision single-molecule localization. By monitoring fluorescence intensity time traces as molecules underwent blinking, the authors demonstrated cryogenic colocalization measurements for pairs of single Alexa Fluor 532 molecules that were labeled a few nanometers apart on in vitro DNA constructs. ...
... It is further worth noting that, at sufficiently high numerical aperture, the fixed dipole orientation of fluorophores in a vitrified sample may lead to significant (>10 nm) localization error for single molecules. 188,190 This issue is potentially addressable with evolving computational models 191 and new optical methods. 192 Correlating Cryo-Super-Resolution Microscopy with Cryo-Soft X-ray Tomography. ...
Article
Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.
... Alternatively, the molecule can be localized with scanning confocal arrangements (17), but also in this case the need for large photon numbers remains. Therefore, improving localization has so far concentrated on increasing molecular emission, particularly through anti-bleaching agents (18), special fluorophores (19), cryogenic conditions (20), transient (fluorogenic) labels (21,22), and fluorophore-metal interactions (23). However, apart from the fact that all these remedies entail restrictions when applied to (living) cells, none of them have addressed the problem of limited emission budget fundamentally. ...
... Two dimensional trajectories with 1200 localizations were generated for free isotropic Brownian motion with diffusion constants in the range of ∈ [0.01,1]µ 2 / and count rates of Γ ∈ [20,200] . 100 trajectories were generated for each parameter combination. ...
Article
Superresolution imaging in sharper focus An optical microscope cannot distinguish objects separated by less than half the wavelength of light. Superresolution techniques have broken this “diffraction limit” and provided exciting new insights into cell biology. Still, such techniques hit a limit at a resolution of about 10 nm. Balzarotti et al. describe another way of localizing single molecules called MINFLUX (see the Perspective by Xiao and Ha). As in photoactivated localization microscopy and stochastic optical reconstruction microscopy, fluorophores are stochastically switched on and off, but the emitter is located using an excitation beam that is doughnut-shaped, as in stimulated emission depletion. Finding the point where emission is minimal reduces the number of photons needed to localize an emitter. MINFLUX attained ∼1-nanometer precision, and, in single-particle tracking, achieved a 100-fold enhancement in temporal resolution. Science , this issue p. 606 ; see also p. 582
... Although the various super-resolution microscopy techniques 3-6,30-32 are now commonly used at room temperature, for example, in biological applications, their extension to low temperatures is in its infancy [33][34][35][36][37][38] , mainly because of the inherent experimental complications of cryogenics and unsuitable photo-physical properties of usual markers at these temperatures. At liquid helium temperatures, two approaches have been developed to localize individual aromatic molecules with a lateral precision well below the diffraction limit. ...
... Finally, in contrast with super-resolution microscopy methods based on single molecule localization 35,39 , we demonstrate that modulated-ESSat provides a unique capability to super-resolve non-blinking molecules with spectrally stable overlapping optical resonance. Two such molecules, which are not resolved with classical confocal microscopy in Fig. 4f, are clearly super-resolved in Fig. 4 (also Supplementary Fig. 6). ...
Article
Optical resolution of solid-state single quantum emitters at the nanometre scale is a challenging step towards the control of delocalized states formed by strongly and coherently interacting emitters. We have developed a simple super-resolution optical microscopy method operating at cryogenic temperatures, which is based on optical saturation of the excited state of single fluorescent molecules with a doughnut-shaped beam. Sub-10 nm resolution is achieved with extremely low excitation intensities, a million times lower than those used in room-temperature stimulated emission depletion microscopy. Compared with super-localization approaches, our technique offers a unique opportunity to super-resolve single molecules with overlapping optical resonance frequencies and paves the way to the study of coherent interactions between single emitters and to the manipulation of their degree of entanglement.
... But as generally photo-reactivity is hindered, also photoactivation, -conversion and -switching are a ected [60][61][62]. Mostly, photomodulation processes in a cryogenic environment are signi cantly slower which increases imaging time scales or are completely suppressed [30,[63][64][65][66]. ...
... Weisenburger et al. successfully photoswitched single synthetic Alexa Fluor 532 molecules at 4 K and showed similar photon counts to those at ambient temperatures but superior signal-to-noise ratios which led to a localization precision and colocalization of several uorophores at Ångstrom precision. Nevertheless, the uorescence "ontimes" ( uorescence signal until the uorophore is photoswitched into the dark "o -state") were long (up to hundreds of seconds) and similar to the time the uorophores spent in the dark o -states [66,67]. To date, only two publications report cryogenic SMLM imaging (Table 1 and [30,31]). ...
Article
Full-text available
During the last few decades, correlative fluorescence light and electron microscopy (FLM-EM) has gained increased interest in the life sciences concomitant with the advent of fluorescence light microscopy. It has become, accompanied by numerous developments in both techniques, an important tool to study bio-cellular structure and function as it combines the specificity of fluorescence labeling with the high structural resolution and cellular context information given by the EM images. Having the recently introduced single-molecule localization microscopy techniques (SMLM) at hand, FLM-EM can now make use of improved fluorescence light microscopy resolution, single-molecule sensitivity and quantification strategies. Here, currently used methods for correlative SMLM and EM including the special requirements in sample preparation and imaging routines are summarized and an outlook on remaining challenges concerning methods and instrumentation is provided.
... However, the resolution gap between conventional fluorescence and electron microscopy presents a challenge, and many attempts have been made to apply super-resolution techniques such as stimulated emission depletion or SMLM at liquid-nitrogen or liquid-helium temperatures. At these temperatures, most photophysical transitions are either greatly slowed down or completely frozen, which has so far impeded the achievement of super-resolution fluorescence imaging under cryogenic conditions (with very few exceptions [72][73][74][75][76]. SOFI could offer an interesting alternative as it does not require controlled photoswitching with long on and off times but can use any intensity fluctuations. ...
... Consequently, fast freezing techniques are applicable for preserving a wide range of biological structures, from individual proteins to complex tissues. Apart from fast freezing techniques, studies have shown that hydrophilic polymers can be also used to preserve the structures of some proteins or protein complexes at low temperatures (14)(15)(16)(17). ...
... In addition, those polarization tailored beams have also paved the way towards versatile applications in recent nanophotonic experiments by enabling selective excitation of nanoparticle eigen-modes [8,9], or controllable directional emission and waveguidecoupling of single plasmonic nanoantennas [10]. In this work, we combine several aspects of both research fields to present a novel approach towards high-precision position sensing, a discipline, which is of paramount importance in modern nanometrology [11][12][13][14][15][16][17][18], because of its special role in super-resolution microscopy [19][20][21][22]. Our all-optical technique for localization of a single nanoantenna is thereby based on encoding the position of the antenna in its laterally directional scattering pattern. ...
Preprint
Controlling the propagation and coupling of light to sub-wavelength antennas is a crucial prerequisite for many nanoscale optical devices. Recently, the main focus of attention has been directed towards high-refractive-index materials such as silicon as an integral part of the antenna design. This development is motivated by the rich spectral properties of individual high-refractive-index nanoparticles. Here, we take advantage of the interference of their magnetic and electric resonances, to achieve remarkably strong lateral directionality. For controlled excitation of a spherical silicon nanoantenna we use tightly focused radially polarized light. The resultant directional emission depends on the antenna's position relative to the focus. This approach finds application as a novel position sensing technique, which might be implemented in modern nanometrology and super-resolution microscopy setups. We demonstrate in a proof-of-concept experiment, that a lateral resolution in the Angstrom regime can be achieved.
... Introduction.-Dipole emitters such as molecules, quantum dots and nano-antennas represent fundamental building blocks in various nano-optical experiments [1][2][3][4]. In general, the emission characteristics of such dipoles depend on the relative phases and amplitudes of the three electric and/or magnetic dipolar components. ...
Preprint
We investigate points of circular polarization in the far field of elliptically polarized dipoles and establish a relation between the angular position and helicity of these C points and the dipole moment. In the case of highly eccentric dipoles, the C points of opposite handedness exhibit only a small angular separation and occur in the low intensity region of the emission pattern. In this regard, we introduce an optical weak measurement approach that utilizes the transverse electric (azimuthal) and transverse magnetic (radial) far-field polarization basis. Projecting the far field onto a spatially varying post-selected polarization state reveals the angular separation and the helicity of the C points. We demonstrate the applicability of this approach and determine the elliptical dipole moment of a particle sitting on an interface by measuring the C points in its far field.
... The early work on spCryo-LM demonstrated co-localization of two organic fluorophores on a DNA backbone, reaching sub-nanometer accuracy [132]. The method was then extended to resolving two fluorophores bound to the C termini of the cytosolic GtCitA PAS protein domain and four fluorophores bound to the four biotin sites of single streptavidin molecules [60]. ...
Article
Full-text available
Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light micros-copy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.
... The first of these factors has been the main focus of efforts for improving image resolution, via hardware, e.g., by combining improved structured illumination conditions with single-molecule detection (Kalisvaart et al., 2022;Zhan et al., 2022), DNA-PAINT with STORM (Cnossen et al., 2020), using cryo-SMLM and ad hoc software corrections (Schneider et al., 2020;Hinterer et al., 2022), or via computational methods that enhance localization precision through engineering of the PSF or other approaches as applied to a single (Li M. et al., 2022) or multiple fluorophores (colocalization precision) (Koyama-Honda et al., 2005;Donnert et al., 2007;Lemmer et al., 2009; Frontiers in Molecular Biosciences frontiersin.org Curthoys et al., 2013;Weisenburger et al., 2014;Georgieva et al., 2016;Willems and MacGillavry, 2022). Data post-processing via deep-learning approaches (Möckl et al., 2019) and high-throuput parallel computing with highperformance clusters (Munro et al., 2019) are increasingly being employed to enhance localization precision in SMLM. ...
Article
Full-text available
Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200 nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4 nm–10 nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.
... [3,4] Nevertheless, the fluorescence intermittency originating from the existence of triplet dark states can be exploited in cryogenic superresolution localization schemes based on stochastic blinking of single emitters. [5][6][7][8][9] One of the applications of single molecules as bright emitters is as very sensitive probes of their local environment by means of perturbations on their optical transitions. The ideal case of an uninterrupted stream of photons, caused by a very low triplet yield and/or a very short triplet lifetime, would act as a stable information source for probing local effects. ...
Article
Full-text available
The front cover artwork is provided by Prof. Michel Orrit's group at the University of Leiden, The Netherlands. The image shows the structures of the dibenzothiophene host molecule and perylene guest molecule with its fluorescence emission spectrum on the bottom. The symbols and arrows refer to the reverse intersystem crossing (rISC) observed for single perylene molecules in dibenzothiophene host crystals, which typically have a needle shape and are shown in the background. Read the full text of the Article at 10.1002/cphc.202100679.
... [3,4] Nevertheless, the fluorescence intermittency originating from the existence of triplet dark states can be exploited in cryogenic superresolution localization schemes based on stochastic blinking of single emitters. [5][6][7][8][9] One of the applications of single molecules as bright emitters is as very sensitive probes of their local environment by means of perturbations on their optical transitions. The ideal case of an uninterrupted stream of photons, caused by a very low triplet yield and/or a very short triplet lifetime, would act as a stable information source for probing local effects. ...
Article
Full-text available
Intersystem crossing to the long‐lived metastable triplet state is often a strong limitation on fluorescence brightness of single molecules, particularly for perylene in various matrices. In this paper, we report on a strong excitation‐induced reverse intersystem crossing (rISC), a process where single perylene molecules in a dibenzothiophene matrix recover faster from the triplet state, turning into bright emitters at saturated excitation powers. With a detailed study of single‐molecule fluorescence autocorrelations, we quantify the effect of rISC. The intrinsic lifetimes found for the two effective triplet states (8.5±0.4 ms and 64±12 ms) become significantly shorter, into the sub‐millisecond range, as the excitation power increases and fluorescence brightness is ultimately enhanced at least fourfold. Our results are relevant for the understanding of triplet state manipulation of single‐molecule quantum emitters and for markedly improving their brightness.
... The mean photon yield of ATTO 647N (uncorrected for spectra) of 5 · 10 6 photons at 240 W/cm 2 and 3·10 6 photons at 360 W/cm 2 agrees well with the result found by Li et al. (3.5 · 10 6 photons at 300 W/cm 2 ) [7]. The maximal photon yield at liquid nitrogen temperatures is lower than at liquid helium temperatures, with only ∼ 5% emitting 10 7 photons or more compared to > 20% at liquid helium temperatures as found by Weisenburger et al. [16]. Although the photon yield is lower, liquid nitrogen is more suitable for fluorescence microscopy as phonons are frozen out at liquid helium temperatures leading to a significant narrowing of the excitation and emission lines [17], making efficient excitation of a fluorophore difficult. ...
Preprint
Full-text available
Single Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super-resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen temperatures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 · 10 ⁶ to 11 · 10 ⁶ photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.
... 12,13 In that case, dipole orientation effects can no longer be neglected, and they have become even more important in light of the extraordinary Ångström-size localization accuracy that can, in principle, be achieved at cryogenic temperatures due to the dramatically enhanced photostability of dyes at these temperatures. [14][15][16] Recently, Lew and co-worker presented an elegant solution to this problem: By using a custom-build polarizer (broadband metasurface mask) that transmits only the azimuthally polarized component of the fluorescence light, they could demonstrate that the resulting images of single molecules do no longer show orientation-dependent lateral shifts with respect to their actual positions. 17 A disadvantage of this method was that it required the fabrication of a rather complex metasurface structure, resulting in about ∼50% loss of the emission. ...
Article
Full-text available
Single-Molecule Localization Microscopy (SMLM) has become one of the most important methods of super-resolution fluorescence microscopy. It is based on the precise localization of single molecules in wide-field microscopy images. It is well known that the localization accuracy can show a significant bias if the imaged molecules have a fixed orientation and are located either close to an interface or not exactly within the focal plane of the microscope. In this Letter, we propose a simple solution to this problem, which is based on polarization-resolved imaging. This method can be easily implemented into any existing SMLM setup, and we demonstrate its performance by imaging single dye molecules embedded into a polymer film, which fixes their orientation in space.
... The spatial resolution of optical microscopy is restricted by the Abbe diffraction limit to about half the wavelength of emitted light. While this can be improved by single-molecule localization techniques, [7] especially at cryogenic temperatures, [8,9] PL alone can never resolve the nanoscale physical structure of individual emitters in far-field microscopy due to its detection by electric-dipole radiation. Cryo-ET is ac omplementary technique to single-molecule PL spectroscopy as it provides excellent spatial resolution, but no information about the corresponding electronic structure.The combination of these two powerful approaches allows for the correlation of physical and electronic structure of individual emitters and can lead to insight inaccessible by either technique independently. ...
Article
Full-text available
Cryogenic single‐particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single‐particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in‐plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in‐plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.
... Introduction.-Dipole emitters such as molecules, quantum dots and nano-antennas represent fundamental building blocks in various nano-optical experiments [1][2][3][4]. In general, the emission characteristics of such dipoles depend on the relative phases and amplitudes of the three electric and/or magnetic dipolar components. ...
Article
Full-text available
We investigate points of circular polarization in the far field of elliptically polarized dipoles and establish a relation between the angular position and helicity of these C points and the dipole moment. In the case of highly eccentric dipoles, the C points of opposite handedness exhibit only a small angular separation and occur in the low intensity region of the emission pattern. In this regard, we introduce an optical weak measurement approach that utilizes the transverse electric (azimuthal) and transverse magnetic (radial) far-field polarization basis. Projecting the far field onto a spatially varying postselected polarization state reveals the angular separation and the helicity of the C points. We demonstrate the applicability of this approach and determine the elliptical dipole moment of a particle sitting on an interface by measuring the C points in its far field.
... Nevertheless, several approaches to increase the resolution of microscopes at cryogenic temperatures could be achieved. Kaufmann et al.[17]utilize the effect of photoswitching[18,19]of fluorescent marker proteins by using a 0.75 NA MO for super-resolution fluorescence cryogenic-microscopy. Shibata et al. developed a microscope with NA of 0.9, where the MO is mounted inside of the cryogenic vacuum space[20]. One method exceeding an NA of unity, was achieved by using solid immersion lenses[21]exhibiting an NA of 1.23[22]with the disadvantage of a very small field of view. ...
Article
Full-text available
Here we report a simple way to enhance the resolution of a confocal scanning microscope under cryogenic conditions. Using a microscope objective (MO) with high numerical aperture (NA = 1.25) and 1-propanol as an immersion fluid with low freezing temperature we were able to reach an imaging resolution at 160 K comparable to ambient conditions. The MO and the sample were both placed inside the inner chamber of the cryostat to reduce distortions induced by temperature gradients. The image quality of our commercially available MO was further enhanced by scanning the sample (sample scanning) in contrast to beam scanning. The ease of the whole procedure marks an essential step towards the development of cryo high-resolution microscopy and correlative light and electron cryo microscopy (cryoCLEM).
... Although Moor and Mühlethaler (4) and Dubochet and McDowall (5) followed different postprocessing and imaging routines (freeze-etching versus direct imaging, respectively), the basic principle of both preparation procedures is the same: the physical arresting of the sample in a frozen-hydrated state, which is achieved by the vitrification of the sample within milliseconds, resulting in a sample embedded in amorphous ice, thereby avoiding structural changes due to ice crystal growth. The convincing structural preservation achieved in the case of isolated cell organelles (6,7), viruses (8), bacteria (9,10), eukaryotic cells (11) and other softmatter applications (12), and the technical improvements in sample preparation and handling (13)(14)(15)(16)(17)(18)(19), caused this approach to spread out into other fields of microscopy like soft x-ray (20) and light microscopy (21,22), and enabled new hybrid approaches like correlative cryo-light and electron microscopy (23). ...
Article
Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented.
... As localization precision improves with newly designed fluorescent labels and other methodological improvements, the need to also guarantee localization accuracy over the FOV becomes increasingly urgent. Single-molecule experiments that reach subnanometer precision in two dimensions require careful estimation of detector nonuniformity and dipole mislocalization errors [25,53], and we expect that the extension of these ultraprecise experiments to three dimensions will also require that fielddependent aberrations be carefully corrected. Another relevant trend is the increased use of sCMOS rather than EMCCD detectors for single-molecule localization [54]. ...
Article
Full-text available
The localization of single fluorescent molecules enables the imaging of molecular structure and dynamics with subdiffraction precision and can be extended to three dimensions using point spread function (PSF) engineering. However, the nanoscale accuracy of localization throughout a 3D single-molecule microscope’s field of view has not yet been rigorously examined. By using regularly spaced subdiffraction apertures filled with fluorescent dyes, we reveal field-dependent aberrations as large as 50–100 nm and show that they can be corrected to less than 25 nm over an extended 3D focal volume. We demonstrate the applicability of this technique for two engineered PSFs, the double-helix PSF and the astigmatic PSF. We expect these results to be broadly applicable to 3D single-molecule tracking and superresolution methods demanding high accuracy.
... In addition, those polarization tailored beams have also paved the way towards versatile applications in recent nanophotonic experiments by enabling selective excitation of nanoparticle eigen-modes [8,9], or controllable directional emission and waveguidecoupling of single plasmonic nanoantennas [10]. In this work, we combine several aspects of both research fields to present a novel approach towards high-precision position sensing, a discipline, which is of paramount importance in modern nanometrology [11][12][13][14][15][16][17][18], because of its special role in super-resolution microscopy [19][20][21][22]. Our all-optical technique for localization of a single nanoantenna is thereby based on encoding the position of the antenna in its laterally directional scattering pattern. ...
Article
Full-text available
Controlling the propagation and coupling of light to sub-wavelength antennas is a crucial prerequisite for many nanoscale optical devices. Recently, the main focus of attention has been directed towards high-refractive index materials such as silicon as an integral part of the antenna design. The development is motivated by the rich spectral properties of individual high-refractive index nanoparticles, featuring magnetic and electric resonances in the visible regime, whose interference may yield remarkably strong directivity. Here, we use tightly focused radially polarized light for controlled excitation of a spherical silicon nanoantenna. The resultant emission can be highly directional, depending on the antenna's position relative to the focus. This approach finds application as a novel position sensing technique, a discipline, which is of paramount importance in modern nanometrology, because of its special role in super-resolution microscopy. We yield a lateral resolution in the Angstrom regime.
Article
Single-molecule localization microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass-transition temperature hampers efficient cryo-photoswitching. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent proteins at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at ∼110 K. At this cryogenic temperature, on-off photoswitching involves the formation of two off-states in cis conformation with blue-shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these off-states can be switched back to the fluorescent on-state by 405 nm light, while both of them are sensitive to UV light at 355 nm. Superior recovery to the fluorescent on-state by 355 nm light was confirmed at the single-molecule level. This suggests, as also shown by simulations, that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 and possibly other FPs could improve the effective labeling efficiency achievable with this technique. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins.
Article
Super-resolved cryogenic correlative light and electron tomography is an emerging method that provides both the single-molecule sensitivity and specificity of fluorescence imaging, and the molecular scale resolution and detailed cellular context of tomography, all in vitrified cells preserved in their native hydrated state. Technical hurdles that limit these correlative experiments need to be overcome for the full potential of this approach to be realized. Chief among these is sample heating due to optical excitation which leads to devitrification, a phase transition from amorphous to crystalline ice. Here we show that much of this heating is due to the material properties of the support film of the electron microscopy grid, specifically the absorptivity and thermal conductivity. We demonstrate through experiment and simulation that the properties of the standard holey carbon electron microscopy grid lead to substantial heating under optical excitation. In order to avoid devitrification, optical excitation intensities must be kept orders of magnitude lower than the intensities commonly employed in room temperature super-resolution experiments. We further show that the use of metallic films, either holey gold grids, or custom made holey silver grids, alleviate much of this heating. For example, the holey silver grids permit 20× the optical intensities used on the standard holey carbon grids. Super-resolution correlative experiments conducted on holey silver grids under these increased optical excitation intensities have a corresponding increase in the rate of single-molecule fluorescence localizations. This results in an increased density of localizations and improved correlative imaging without deleterious effects from sample heating.
Chapter
Oligomerization of biomolecules on the plasma membrane drives vital cellular functions. Despite the importance of detecting and characterizing these molecular interactions, there is an apparent lack of proper techniques capable of unraveling the exact composition of multimolecular complexes directly on the (live) cell membrane, particularly when present at high surface densities. In this chapter, commonly applied single-molecule fluorescence microscopy approaches are reviewed in terms of their suitability for detecting molecular aggregates down to the smallest biologically relevant unit: a molecular dimer. Amongst these techniques, Thinning Out Clusters while Conserving Stoichiometry of Labeling (TOCCSL) – a modality based on single-molecule brightness and co-localization analysis – is discussed in detail. The chapter lists basic principles of TOCCSL, its implementation on single-molecule fluorescence microscopes, analysis of brightness and co-localization-based microscopy data, and guidelines on how to choose parameters and labels to determine the number of labeled subunits within a multimolecular assembly. At the end, a tabular list of TOCCSL applications conducted so far is provided.KeywordsBrightness analysisCo-localization analysisMultimolecular complexesPlasma membrane structureSingle-molecule microscopyThinning out clusters while conserving stoichiometry of labeling (TOCCSL)
Article
The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72 , 253–278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.
Article
We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Thesis
The trapping and manipulation of small objects are desirable for a number of reasons such as force measurements at the nanoscale, imaging, and controlled diffusion studies. Therefore, many techniques have been developed over the years such as optical tweezers, Paul traps, anti-Brownian electrokinetic traps and geometry-induced electrostatic traps. In this work, we use the electrostatic forces generated by either an electrical double layer at the solid-liquid interface or an externally applied electric field to influence the motion of charged particles. In the first part, we report on the nanopipette geometry-induced electrostatic trapping and manipulation of gold nanoparticles in an aqueous solution. A trapping potential is created in the junction of a glass nanopipette and a substrate to trap and deliver a gold nanosphere in the vicinity of a single colloidal quantum dot which is embedded at the polymer-liquid interface. By scanning the trapped nanoparticle across the emitter's near field, we demonstrate around 8-fold plasmonic enhancement of fluorescence and a lateral resolution of about 45 nm in imaging the emitter. Furthermore, we also employ supported lipid bilayer to act as conveyor belts for either a single emitter or a plasmonic nanoparticle to bring one into the vicinity of the other. The second part of this thesis addresses some of the fundamental questions regarding the diffusion of gold nanoparticles in non-parabolic potentials. We first investigate the diffusion of gold nanoparticles moving in a nanochannel under the influence of an externally applied electrostatic force and experimentally demonstrate that anomalous diffusion can also be observed in an uncrowded environment such as a Brownian particle in pure water. This leads us to the first experimental observation of subdiffusion with a tunable exponent and an observation of the transition from free diffusion to trapping. Importantly, we have discovered that this transition is not sharp, but rather occurs via an intermediate regime, where the particles are subdiffusive at the ensemble level, yet individual trajectories are qualitatively different. This type of behaviour is known as weak ergodicity breaking. In nonergodic systems, ensemble quantities like the time-averaged displacement along a trajectory fluctuate from particle to particle. Therefore, single particle tracking measurements cannot infer the average diffusive dynamics of the system. In the later parts of this dissertation, we show how we circumvent this problem by characterizing the excursion times, which is found to be in direct correspondence with the transient diffusion exponent and the magnitude of the potential. Weak ergodicity breaking and anomalous diffusion are known to arise in logarithmic potentials even at infinite times, thus, they lie outside the framework of ordinary statistical mechanics. In the last part of this work, we explain the peculiar properties of diffusion dynamics in logarithmic potentials and present a numerical study of the nanoparticles' diffusion in such potentials. Finally, we propose several experimental schemes for the realisation of logarithmic potentials and mapping nonergodicity by real-time imaging of individual particles.
Article
A closer look: Correlative single‐particle cryogenic photoluminescence spectroscopy and cryogenic electron tomography analysis of CdSSe/ZnS core/shell quantum dots reveals spontaneous splitting of the photoluminescence spectrum of single quantum dots into two distinct emission bands ≈80 meV apart. These surprising photoluminescence dynamics are directly correlated with tomographic structures. Abstract Cryogenic single‐particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single‐particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in‐plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in‐plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.
Article
Full-text available
We introduce an interferometric single-molecule localization method for super-resolution fluorescence microscopy. Fluorescence molecules are located by the intensities of multiple excitation patterns of an interference fringe, providing around a twofold improvement in the localization precision compared with the conventional imaging with the same photon budget. We demonstrate this technique by resolving nanostructures down to 5 nm in size over a large 25 × 25 μm² field of view. © 2019, The Author(s), under exclusive licence to Springer Nature America, Inc.
Article
Full-text available
Super‐resolution light microscopy (SRM) enables imaging of biomolecules within cells with nanometer precision. Cryo‐fixation by vitrification offers optimal structure preservation of biological specimens and permits sequential cryo electron microscopy (cryoEM) on the same sample, but is rarely used for SRM due to various technical challenges and the lack of fluorophores developed for vitrified conditions. Here, a protocol to perform correlated cryoSRM and cryoEM on intact mammalian cells using fluorescent proteins and commercially available equipment is described. After cell culture and sample preparation by plunge‐freezing, cryoSRM is performed using the reversibly photoswitchable fluorescent protein rsEGFP2. Next, a super‐resolved image is reconstructed to guide cryoEM imaging to the feature of interest. Finally, the cryoSRM and cryoEM images are correlated to combine information from both imaging modalities. Using this protocol, a localization precision of 30 nm for cryoSRM is routinely achieved. No impediments to successive cryoEM imaging are detected, and the protocol is compatible with a variety of cryoEM techniques. When the optical set‐up and analysis pipeline is established, the total duration of the protocol for experienced cryoEM users is 3 days, not including cell culture.
Conference Paper
We discuss a nanolocalization technique with sub-nanometer localization resolution based on position dependent transverse Kerker scattering, obtained via interference of tailored electric and magnetic dipole moments.
Article
Single-molecule super-resolution fluorescence microscopy con-ducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labelling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of four, attributable to reduced photobleaching.
Article
Single Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super‐resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen tempe‐ ratures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 to 11 million photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.
Article
Among imaging techniques, fluorescence microscopy is a unique method to noninvasively image individual molecules in whole cells. If the three-dimensional spatial precision is improved to the angstrom level, various molecular arrangements in the cell can be visualized on an individual basis. We have developed a cryogenic reflecting microscope with a numerical aperture of 0.99 and an imaging stability of 0.05 nm in standard deviation at a temperature of 1.8 K. The key optics to realize the cryogenic performances is the reflecting objective developed by our laboratory. With this cryogenic microscope, an individual fluorescent molecule (ATTO647N) at 1.8 K was localized with standard errors of 0.53 nm (x), 0.31 nm (y), and 0.90 nm (z) when 10(6) fluorescence photons from the molecule were accumulated in 5 min.
Article
Protein function can be modulated or dictated by the amplitude and timescale of biomolecular motion, therefore it is imperative to study protein dynamics. Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique capable of studying timescales of motion that range from those faster than molecular reorientation on the picosecond timescale to those that occur in real-time. Across this entire regime, NMR observables can report on the amplitude of atomic motion, and the kinetics of atomic motion can be ascertained with a wide variety of experimental techniques from real-time to milliseconds and several nanoseconds to picoseconds. Still a four orders of magnitude window between several nanoseconds and tens of microseconds has remained elusive. Here, we highlight new relaxation dispersion NMR techniques that serve to cover this “hidden-time” window up to hundreds of nanoseconds that achieve atomic resolution while studying the molecule under physiological conditions.
Article
We introduce Cryogenic Optical Localization in 3D (COLD), a method to localize multiple fluorescent sites within a single small protein with Angstrom resolution. We demonstrate COLD by determining the conformational state of the cytosolic Per-ARNT-Sim domain from the histidine kinase CitA of Geobacillus thermodenitrificans and resolving the four biotin sites of streptavidin. COLD provides quantitative 3D information about small- to medium-sized biomolecules on the Angstrom scale and complements other techniques in structural biology.
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Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.
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We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.
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Nature has evolved many molecular machines such as kinesin, myosin, and the rotary flagellar motor powered by an ion current from the mitochondria. Direct observation of the step-like motion of these machines with time series from novel experimental assays has recently become possible. These time series are corrupted by molecular and experimental noise that requires removal, but classical signal processing is of limited use for recovering such step-like dynamics. This paper reports simple, novel Bayesian filters that are robust to step-like dynamics in noise, and introduce an L1-regularized, global filter whose sparse solution can be rapidly obtained by standard convex optimization methods. We show these techniques outperforming classical filters on simulated time series in terms of their ability to accurately recover the underlying step dynamics. To show the techniques in action, we extract step-like speed transitions from Rhodobacter sphaeroides flagellar motor time series.
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Brighter dyes in heavy water: A simple and cost-effective method increases the brightness of a whole class of commonly used red-emitting fluorophores, including ATTO655, ATTO680, and ATTO700. Replacing water (H2 O) by heavy water (D2 O) in the imaging buffer doubles the fluorescence quantum yield of these dyes and significantly improves the localization precision in super-resolution imaging.
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The plethora of available scientific cameras of different types challenges the biologically oriented experimenter when picking the appropriate camera for his experiment. In this study, we chose to investigate camera performances in a typical nonsingle molecule situation in life sciences, that is, quantitative measurements of fluorescence intensity changes from video data with typically skewed intensity distributions. Here, intensity profile dynamics of pH-sensors upon triggered changes of pH-environments in living cells served as a model system. The following camera types were tested: sCMOS, CCD (scientific and nonscientific) and EM-CCD (back- and front-illuminated). We found that although the EM-CCD cameras achieved the best absolute spatial SNR (signal-to-noise ratio) values, the sCMOS was at least of equal performance when the spatial SNR was related to the effective dynamic range, and it was superior in terms of temporal SNR. In the measurements of triggered intensity changes, the sCMOS camera had the advantage that it used the smallest fraction of its dynamic range when depicting intensity changes, and thus featured the best SNR at full usage of its dynamic range. Microsc. Res. Tech., 2013. © 2013 Wiley Periodicals, Inc.
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Members of the transmembrane AMPA receptor-regulatory protein (TARP) family modulate AMPA receptor (AMPA-R) trafficking and function. AMPA-Rs consist of four pore-forming subunits. Previous studies show that TARPs are an integral part of the AMPA-R complex, acting as accessory subunits for mature receptors in vivo. The TARP/AMPA-R stoichiometry was previously measured indirectly and found to be variable and dependent on TARP expression level, with at most four TARPs associated with each AMPA-R complex. Here, we use a single-molecule technique in live cells that selectively images proteins located in the plasma membrane to directly count the number of TARPs associated with each AMPA-R complex. Although individual GFP-tagged TARP subunits are observed as freely diffusing fluorescent spots on the surface of Xenopus laevis oocytes when expressed alone, coexpression with AMPA-R-mCherry immobilizes the stargazin-GFP spots at sites of AMPA-R-mCherry, consistent with complex formation. We determined the number of TARP molecules associated with each AMPA-R by counting bleaching steps for three different TARP family members: γ-2, γ-3, and γ-4. We confirm that the TARP/AMPA-R stoichiometry depends on TARP expression level and discover that the maximum number of TARPs per AMPA-R complex falls into two categories: up to four γ-2 or γ-3 subunits, but rarely above two for γ-4 subunit. This unexpected AMPA-R/TARP stoichiometry difference has important implications for the assembly and function of TARP/AMPA-R complexes.
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The asymmetric nature of single-molecule (SM) dipole emission patterns limits the accuracy of position determination in localization-based super-resolution fluorescence microscopy. The degree of mislocalization depends highly on the rotational mobility of SMs; only for SMs rotating within a cone half angle α > 60° can mislocalization errors be bounded to ≤ 10 nm. Simulations demonstrate how low or high rotational mobility can cause resolution degradation or distortion in super-resolution reconstructions.
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The possibility to visualize and image the arrangement of proteins within the cell at the molecular level has always been an attraction for scientists in biological research. In particular, for signalling molecules such as GPCRs (G-protein-coupled receptors), the existence of protein aggregates such as oligomers or clusters has been the topic of extensive debate. One of the reasons for this lively argument is that the molecular size is below the diffraction-limited resolution of the conventional microscopy, precluding the direct visualization of protein super-structures. On the other hand, new super-resolution microscopy techniques, such as the PALM (photoactivated localization microscopy), allow the limit of the resolution power of conventional optics to be broken and the localization of single molecules to be determined with a precision of 10-20 nm, close to their molecular size. The application of super-resolution microscopy to study the spatial and temporal organization of GPCRs has brought new insights into receptor arrangement on the plasma membrane. Furthermore, the use of this powerful microscopy technique as a quantitative tool opens up the possibility for investigating and quantifying the number of molecules in biological assemblies and determining the protein stoichiometry in signalling complexes.
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Single-molecule spectroscopy has developed into an important method for probing protein structure and dynamics, especially in structurally heterogeneous systems. A broad range of questions in the diversifying field of protein folding have been addressed with single-molecule Förster resonance energy transfer (FRET) and photo-induced electron transfer (PET). Building on more than a decade of rapid method development, these techniques can now be used to investigate a wide span of timescales, an aspect that we focus on in this review. Important current topics range from the structure and dynamics of unfolded and intrinsically disordered proteins, including the coupling of folding and binding, to transition path times, the folding and misfolding of larger proteins, and their interactions with molecular chaperones.
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Sub-diffraction-limit imaging can be achieved by sequential localization of photoactivatable fluorophores, for which the image resolution depends on the number of photons detected per localization. We report a strategy for fluorophore caging that creates photoactivatable probes with high photon yields. Upon photoactivation, these probes can provide 10(4)-10(6) photons per localization and allow imaging of fixed samples with resolutions of several nanometers. This strategy can be applied to many fluorophores across the visible spectrum.
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Providing previously obscured positional and orientational information, a novel wide-field optical microscope capable of visualizing three-dimensional orientational dynamics of individual room temperature molecules has been developed. Utilizing a single detector, this facile method enables simultaneous observation of all molecular orientations without polarization optics. Such methods not only provide conclusive, nondestructive evidence of single molecule observation, but will also make single molecule orientational studies accessible to a wide range of researchers. Analysis of observed molecular emission patterns not only directly and noninvasively reveals true 3-D orientational dynamics of individual molecules, but also demonstrates that perceived molecular position is strongly dependent on orientation.
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In pursuit of a better understanding of how electronic excitation migrates within complex structures, the concept of resonance energy transfer is being extended and deployed in a wide range of applications. Utilizing knowledge of the quantum interactions that operate in natural photosynthetic systems, wide-ranging molecular and solid-state materials are explored in the cause of more efficient solar energy harvesting, while advances in theory are paving the way for the development and application of fundamentally new mechanisms. In this review, an introduction to the underlying processes that cause singlet-singlet and triplet-triplet energy transfer leads into a discussion of how a new conception of these fundamental processes has emerged over recent years. Illustrative examples relevant to laser science and photonics are described, including photosynthetic light-harvesting, light-activated sensors, processes of cooperative and accretive energy pooling and quantum cutting in rare earth-doped crystals, and incoherent triplet-triplet energy upconversion in molecular solutions.
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We summarize experimental observations of fluorescence intermittency of single semiconductor nanocrystals and single molecules. We review the main models proposed earlier to explain the broad power-law distributions of on- and off-blinking times. We argue that a self-trapping model with a distribution of trapping distances can account for most, if not all, observations to date. We propose possible scenarios for photo-ionization, the switching to states with long on-times and the influence of disorder and surfaces on the trapping dynamics.
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