High throughput spectrally resolved super-resolution fluorescence microscopy with improved photon usage.

The natural characteristics of deoxyribonucleic acid (DNA) enable its advanced applications in nanotechnology as a special tool that can be detected by high-resolution imaging with precise localization. Super-resolution (SR) microscopy enables the examination of nanoscale molecules beyond the diffraction limit. With the development of SR microscopy methods, DNA nanostructures can now be optically assessed. Using the specific binding of fluorophores with their target molecules, advanced single-molecule localization microscopy (SMLM) has been expanded into different fields, allowing wide-range detection at the single-molecule level. This review discusses the recent progress in the SR imaging of DNA nano-objects using SMLM techniques, such as direct stochastic optical reconstruction microscopy, binding-activated localization microscopy, and point accumulation for imaging nanoscale topography. Furthermore, we discuss their advantages and limitations, present applications, and future perspectives.

The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules. An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed. Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and –lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates. In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60–1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed.

Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with “high-risk” human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access to the repair machinery. In conclusion, these data demonstrated a folate-dependent repair activity and chromatin re-organization on the SMLM nanoscale level. This offers new opportunities to further investigate folate-induced chromatin re-organization and the associated mechanisms.

The „Resolution Revolution in fluorescence microscopy over the last decade has given rise to a variety of techniques that allow imaging beyond the diffraction limit with a resolution power down into the nanometer range. With this, the field of so-called super-resolution microscopy was born. It allows to visualize cellular architecture at a molecular level and thereby achieve a resolution level that had been previously only accessible by electron microscopy approaches. One of these promising techniques is single molecule localization microscopy (SMLM) in its most varied forms such as direct stochastic optical reconstruction microscopy (dSTORM) which are based on the temporal separation of the emission of individual fluorophores. Localization analysis of the subsequently taken images of single emitters eventually allows to reconstruct an image containing super-resolution information down to typically 20 nm in a cellular setting. The key point here is the localization precision, which mainly depends on the image contrast generated the by the individual fluorophore’s emission. Thus, measures to enhance the signal intensity or reduce the signal background allow to increase the image resolution achieved by dSTORM. In my thesis, this is achieved by simply adding a reflective metal-dielectric nano-coating to the microscopy coverslip that serves as a tunable nano-mirror. I have demonstrated that such metal-dielectric coatings provide higher photon yield at lower background and thus substantially improve SMLM performance by a significantly increased localization precision, and thus ultimately higher image resolution. The strength of this approach is that ─ except for the coated cover glass ─ no specialized setup is required. The biocompatible metal-dielectric nano-coatings are fabricated directly on microscopy coverslips and have a simple three-ply design permitting straightforward implementation into a conventional fluorescence microscope. The introduced improved lateral resolution with such mirror-enhanced STORM (meSTORM) not only allows to exceed Widefield and Total Internal Reflection Fluorescence (TIRF) dSTORM performance, but also offers the possibility to measure in a simplified setup as it does not require a special TIRF objective lens. The resolution improvement achieved with meSTORM is both spectrally and spatially tunable and thus allows for dual-color approaches on the one hand, and selectively highlighting region above the cover glass on the other hand, as demonstrated here. Beyond lateral resolution enhancement, the clear-cut profile of the highlighted region provides additional access to the axial dimension. As shown in my thesis, this allows for example to assess the three-dimensional architecture of the intracellular microtubule network by translating the local localization uncertainty to a relative axial position. Even beyond meSTORM, a wide range of membrane or surface imaging applications may benefit from the selective highlighting and fluorescence enhancing provided by the metal-dielectric nano-coatings. This includes for example, among others, live-cell Fluorescence Correlation Spectroscopy and Fluorescence Resonance Energy Transfer studies as recently demonstrated.

To better understand the interactions between biological molecules, a high optical resolution in all three dimensions is crucial. The intrinsically lower axial resolution of microscopes however, is a limiting factor in fluorescence imaging, correspondingly in fluorescence based single molecule localization microscopy (SMLM). Here, we present a method to improve the axial localization precision in SMLM by combining point-spread-function engineering with total internal reflection fluorescence (TIRF) fields with decay lengths that vary within the on-time of a fluorophore. Such time-varying illumination field intensity allows one to extract additional axial location information from the emitted photons. With this time varying illumination approach, we show that axial localization is improved two-fold over TIRF-based SMLM using astigmatic PSFs. We calculate theoretical resolution gains for various imaging conditions via the Cramér Rao Lower Bound (CRLB), a commonly used metric to compute the best attainable localization precision in SMLM.

Caveolae are plasma membrane invaginations whose formation requires caveolin-1 (Cav1), the adaptor protein polymerase I, and the transcript release factor (PTRF or CAVIN1). Caveolae have an important role in cell functioning, signaling, and disease. In the absence of CAVIN1/PTRF, Cav1 forms non-caveolar membrane domains called scaffolds. In this work, we train machine learning models to automatically distinguish between caveolae and scaffolds from single molecule localization microscopy (SMLM) data. We apply machine and deep learning algorithms to discriminate biological structures from SMLM data. Our work is the first that is leveraging machine and deep learning approaches to automatically identifying biological structures from SMLM data. In particular, we develop and compare three binary classification methods to identify whether or not a given 3D cluster of Cav1 proteins is a caveolae. The first uses a random forest classifier applied to 28 hand-picked features, the second uses a convolutional neural net (CNN) applied to a projection of the point clouds onto three planes, and the third uses a PointNet model, a recent development that can directly take point clouds as its input. We validate our methods on a dataset of super-resolution microscopy images of PC3 prostate cancer cells labeled for Cav1. Specifically, we have images from two cell populations: 10 PC3 and 10 CAVIN1/PTRF-transfected PC3 cells (PC3-PTRF cells) that form caveolae. We obtained a balanced set of 1714 different cellular structures. Our results show that both the random forest on hand selected features and the deep learning approach achieve high accuracy in distinguishing the intrinsic features of the caveolae and non-caveolae biological structures. More specifically, both random forest and deep CNN classifiers achieve classification accuracy reaching 94% on our test set, while the PointNet model only reached 83% accuracy. We also discuss the pros and cons of the different approaches. Additional information about individual files is in the accompanying CSV file (item_metadata.csv). This dataset was originally deposited in the Simon Fraser University institutional repository.

The advent of single molecule localization microscopy (SMLM) has transformed our capacity to investigate biological structures at the nanoscale. While the research focus has long been on improving localization precision, systematic errors caused by optical aberrations are often overlooked. In the case of 3D SMLM, such errors have the potential to significantly impair the quality of the resulting images. In this paper, we present an imaging and data processing approach that jointly estimates both, molecule positions and optical aberrations in SMLM. Therefore, the method minimizes systematic errors in SMLM reconstructions without the necessity of additional experimental calibration steps, such as the recording of fluorescent bead z-stacks. We investigate the reliability of this approach, especially in situations where the joint retrieval can be expected to be ill-posed, i.e., whenever the sample is "flat" and provides little diversity among the captured single molecule images. To enhance the reliability of the inverse problem solution, we suggest utilizing small SMLM data sets acquired at one or more slightly defocused "auxiliary" planes. We investigate the effectiveness of our approach through numerical simulations and imaging experiments of a calibration probe and nuclear pore complexes. Our method is simple and integrates seamlessly into existing SMLM setups without necessitating modifications or added complexity to the system.

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.

Precisely pinpointing the positions of emitters within the diffraction limit is crucial for quantitative analysis or molecular mechanism investigation in biomedical research but has remained challenging unless exploiting single molecule localization microscopy (SMLM). Via integrating experimental spot dataset with deep learning, we develop a new approach, Digital-SMLM, to accurately predict emitter numbers and positions for sub-diffraction-limit spots with an accuracy of up to 98 % and a root mean square error as low as 14 nm. Digital-SMLM can accurately resolve two emitters at a close distance, e.g. 30 nm. Digital-SMLM outperforms Deep-STORM in predicting emitter numbers and positions for sub-diffraction-limited spots and recovering the ground truth distribution of molecules of interest. We have validated the generalization capability of Digital-SMLM using independent experimental data. Furthermore, Digital-SMLM complements SMLM by providing more accurate event number and precise emitter positions, enabling SMLM to closely approximate the natural state of high-density cellular structures.

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 ⋅ 106 to 11 ⋅ 106 photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.

As one of the super-resolved optical imaging techniques, single molecule localization microscopy (SMLM) received considerable attention due to its impressive spatial resolution. Compared with other fluorescence imaging techniques, SMLM has one particular request for the fluorophores, that is, continuous ‘on’ and ‘off’ behaviors of their signals (referred to as ‘blinking’). Hence, we present here a kind of super blinking and biocompatible nanoprobes (denoted as SBNs) for SMLM. The SBNs have two main advantages, first, they possess an outstanding fluorescence blinking. Second, they are biocompatible since they are based on bovine serum albumin (BSA). The SBNs are fabricated by doping organic dyes into BSA nanoparticles. We fabricated two kinds of SBNs, one was doped with Alexa Fluor 647 (A647) and the other was doped with Alexa Fluor 594 (A594). Especially for A594 doped SBNs, the improved blinking of A594 doped SBNs induced a better localization precision as compared with A594 alone. Moreover, SMLM imaging of breast cancer cells and exosomes using the SBNs was successfully realized with high spatial resolutions. The work demonstrated here provides a new strategy to prepare novel kinds of super blinking fluorescent agents for SMLM, which broadens the selection of suitable fluorophores for SMLM.

Single-molecule localization microscopy (SMLM) has revolutionized the study of biological phenomena by providing exquisite nanoscale spatial resolution. However, optical aberrations induced by sample and system imperfections distort the single-molecule emission patterns (i.e. PSFs), leading to reduced precision and resolution of SMLM, particularly in three-dimensional (3D) applications. While various methods, both analytical and instrumental, have been employed to mitigate these aberrations, a comprehensive analysis of how different types of commonly encountered aberrations affect single-molecule experiments and their image formation remains missing. In this study, we addressed this gap by conducting a quantitative study of the theoretical precision limit for position and wavefront distortion measurements in the presence of aberrations. Leveraging Fisher information and Cramér-Rao lower bound (CRLB), we quantitively analyzed and compared the effects of different aberration types, including index mismatch aberrations, on localization precision in both biplane and astigmatism 3D modalities as well as 2D SMLM imaging. Furthermore, we studied the achievable wavefront estimation precision from aberrated single-molecule emission patterns, a pivot step for successful adaptive optics in SMLM through thick specimens. This analysis lays a quantitative foundation for the development and application of SMLM in whole-cells, tissues and with a large field of view, providing in-depth insights into the behavior of different aberration types in single-molecule imaging and thus generating theoretical guidelines for developing highly efficient aberration correction strategies and enhancing the precision and reliability of 3D SMLM.

Single molecule localization microscopy (SMLM) is a powerful tool for imaging biological targets at the nanoscale. In this report, we present SMLM imaging of telomeres and centromeres using fluorescence in situ hybridization (FISH). The FISH probes were fabricated by decorating CdSSe/ZnS quantum dots (QDs) with telomere or centromere complementary DNA strands. SMLM imaging experiments using commercially available peptide nucleic acid (PNA) probes labeled with organic fluorophores were also conducted to demonstrate the advantages of using QDs FISH probes. Compared with the PNA probes, the QDs probes have the following merits. First, the fluorescence blinking of QDs can be realized in aqueous solution or PBS buffer without thiol, which is a key buffer component for organic fluorophores’ blinking. Second, fluorescence blinking of the QDs probe needs only one excitation light (i.e. 405 nm). While fluorescence blinking of the organic fluorophores usually requires two illumination lights, that is, the activation light (i.e. 405 nm) and the imaging light. Third, the high quantum yield, multiple switching times and a good optical stability make the QDs more suitable for long-term imaging. The localization precision achieved in telomeres and centromeres imaging experiments is about 30 nm, which is far beyond the diffraction limit. SMLM has enabled new insights into telomeres or centromeres on the molecular level, and it is even possible to determine the length of telomere and become a potential technique for telomere-related investigation.

Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells. The protocol makes use of a strategy to preserve the fluorescence and photo-switching capabilities of standard fluorescent proteins, such as GFP and YFP, to enable single-molecule localization microscopy (SMLM) in-resin sections followed by transmission electron microscopy (TEM) imaging. This results in a fivefold improvement in resolution in the fluorescence image and a more precise correlation of the distribution of fluorescently labeled molecules with EM ultrastructure compared with conventional CLEM.

Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. The charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and ∼10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision >3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40 s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative dequenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of super-resolution fluorescence microscopy of live cells.