Super Resolution Microscopy

Over the past decade, the different super-resolution microscopy methods have enabled cellular imaging with nanometer scale resolution; however, super-resolution microscopy is still not practical for many biologists due to limitations such as high illumination intensities, long recording times, fluorescence probe restrictions, and the high cost of super-resolution microscopes. Expansion Microscopy (Chen, Tillberg, Boyden; Science, 2015) circumvents these instrument-related issues by instead isotropically swelling the sample, bringing sub-diffraction limited structures into the size range viewable by conventional confocal and wide-field microscopes. We demonstrate the use of Expansion Microscopy to enable High Content Imaging in the super-resolution range with <110 nm lateral resolution using the CellInsight™ CX7 High Content Screening platform. This high content imaging of sub-diffraction limited structures in cells was done on multiplexed 4-color labeled cellular samples processed for Expansion Microscopy, and using a 20x/0.7 NA dry objective lens. The intracellular targets were fluorescently labeled using different multiplexed labeling strategies (immunofluorescence, targeted fluorescence probes, and expressed fluorescent proteins) that are compatible with Expansion Microscopy and are easily available. We also demonstrate the ability to image cyanine dyes, such as Alexa Fluor™ 647 dye, which was previously not possible by Expansion Microscopy. Images acquired with the CellInsight™ CX7 High Content Screening platform will be shown for different expanded, multiplexed, cellular targets. This will demonstrate the ease and accessibility in using the CellInsight™ CX7 High Content Screening platform to do quantitative imaging of multiplexed cellular structures in the super-resolution range.

Scope of the book:
This book focusses on the technical concepts of deep learning and its associated branch Neural Networks for the various dimensions of image processing applications. The proposed volume intends to bring together researchers to report the latest results or progress in the development of the above-mentioned areas. Since there is a deficit of books on this specific subject matter, the editors aim to provide a common platform for researchers working in this area to exhibit their novel findings.
Topics of Interest:
This book solicits contributions, which include the fundamentals in the field of Deep Artificial Neural Networks and Image Processing supported by case studies and practical examples. Each chapter is expected to be self-contained and to cover an in-depth analysis of real life applications of neural networks to image analysis.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.

Chromatin condensation plays an important role in the regulation of gene expression. Recently, it was shown that the transcriptional activation of Hoxd genes during vertebrate digit development involves modifications in 3D interactions within and around the HoxD gene cluster. This reorganization follows a global transition from one set of regulatory contacts to another, between two topologically associating domains (TADs) located on either side of
the HoxD locus. Here, we use 3D DNA FISH to assess the spatial
organization of chromatin at and around the HoxD gene cluster
and report that although the two TADs are tightly associated, they
appear as spatially distinct units. We measured the relative position of genes within the cluster and found that they segregate over long distances, suggesting that a physical elongation of the
HoxD cluster can occur. We analyzed this possibility by super-resolution imaging (STORM) and found that tissues with distinct transcriptional activity exhibit differing degrees of elongation. We also observed that the morphological change of the HoxD cluster in developing digits is associated with its position at the boundary between the two TADs. Such variations in the fine-scale architecture of the gene cluster suggest causal links among its spatial configuration, transcriptional activation, and the flanking chromatin context.

Graphical Abstract Highlights d Correlative cryo-imaging used to study biological processes in vitrified cells d Reovirus intracellular release mechanism is elucidated in 3D with combined cryo-imaging d Reovirus core particles start modifying carrier endosomes at 1 h after infection d Correlation of X-ray and fluorescence cryo-imaging requires no data deformation In Brief Kounatidis et al. describe a correlative microscopy platform that combines cryo-structured illumination fluorescence imaging with soft X-ray absorption imaging on the same sample allowing direct data correlation in 3D. The methods are used to track the intracellular progress of reovirus in human cells in the early stages of infection.

Bacterial replication and chromosome segregation are highly organized both in space and in time. However, spatial analysis is hampered by the resolution limit of conventional fluorescence microscopy. In this study, we incubate rapidly-growing Escherichia coli with 5-ethynyl-2 0 -deoxyuridine (EdU), label the resulting EdU-DNA with photoswitchable fluorophores, and image incorporated molecules with an average experimental precision of 13 nm. During the segregation process, nucleoids develop highlydefined and cell-cycle dependent hetero-structures, which contain discrete DNA fibers with diameters far below the diffraction limit. Strikingly, these structures appear temporally shifted between sister chromosomes, an asymmetry which accumulates for ongoing replication rounds. Moreover, nucleoid positioning and expansion along the bacterial length axis fit into an elongation-mediated segregation model in fast growing E. coli cultures. This is supported by close proximity of the nucleoids to the bacterial plasma membrane, the nature of the observed hetero-structures and recently found interactions of membrane-associated proteins with DNA.

The mammalian nucleus is filled with self-organizing, nanometer-scale nucleoprotein machines that carry out DNA replication, RNA biogenesis, and DNA repair. We discuss, as a model, the nonhomologous end-joining (NHEJ) machine, which repairs DNA double-strand breaks. The NHEJ machine consists of six core polypeptides and 10-20 ancillary polypeptides. A full understanding of its design principles will require measuring the behavior of single NHEJ complexes in living cells, using a Nano Toolbox that includes bright, stable, biocompatible fluorophores, efficient protein and nucleic acid tagging strategies, and sensitive, high-resolution imaging methods. Taking inspiration from natural examples, it may be possible to adapt and redesign the NHEJ machine to precisely correct mutations responsible for common human diseases, such as K-ras in lung cancer, or human papillomavirus E6 and E7 genes in cervical and oral cancers. the DNA damage . NHEJ also occurs within domains of modified chromatin containing roughly 2 million base pairs of DNA [3] and must be able to join ends of virtually any nucleotide sequence. The uniqueness of individual repair complexes exemplifies why it is essential to study the behavior of single nucleoprotein complexes, rather than population averages, in order to obtain insights into their design principles.

techniques based on localizing individual fluorophores, such as photoactivated localization microscopy (PALM) 6 , fluorescence PALM (FPALM) 7 and stochastic optical reconstruction microscopy (STORM) 8 . Key to these techniques is the consecutive onand off-switching of fluorophores within a spectral detection window 9 . The imaging procedure starts with the fluorophores in an off (dark) state, either by using fluorescent proteins that are natively expressed in an off state 6,7 or by using fluorophores that are converted into an off state by a suitable illumination procedure 8 . Next, a random but sparse subset of fluorophores is activated through illumination of the sample with light of suitable wavelength and intensity. This subset is then imaged by light of an npg

The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the ttubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

Photoluminescence (PL) of organometal halide perovskite materials reflects the charge dynamics inside of the material and thus contains important information for understanding the electro-optical properties of the material. Interpretation of PL blinking of methylammonium lead iodide (MAPbI 3) nanostructures observed on polycrystalline samples remains puzzling owing to their intrinsic disordered nature. Here, we report a novel method for the synthesis of high-quality single-crystal MAPbI 3 nanorods and demonstrate a single-crystal study on MAPbI 3 PL blinking. At low excitation power densities, two-state blinking was found on individual nanorods with dimensions of several hundred nanometers. A super-resolution localization study on the blinking of individual nanorods showed that single crystals of several hundred nanometers emit and blink as a whole, without showing changes in the localization center over the crystal. Moreover, both the blinking ON and OFF times showed power-law distributions, indicating trapping−detrapping processes. This is further supported by the PL decay times of the individual nanorods, which were found to correlate with the ON/OFF states. Furthermore, a strong environmental dependence of the nanorod PL blinking was revealed by comparing the measurements in vacuum, nitrogen, and air, implying that traps locate close to crystal surfaces. We explain our observations by proposing surface charge traps that are likely related to under-coordinated lead ions and methylammonium vacancies to result in the PL blinking observed here.

www.erasmusmc.nl/globe-consortium or [email protected] I n tr od uct io n The combination of genome sequence and structure, its annotation and experimental data in an accessible and comprehensible way is a major challenge. Increasingly there is a large number of extremely divergent data sets: the sequence itself, genes, regulatory regions, various forms of reoccurring sequence features and clone sets etc. Currently, one possibility to represent this information in a visual form -and thus to reveal its scientific meaning -is to use genome browsers such as "Ensembl" or "The UCSC Genome Browser". These browsers have been beneficial in the understanding of the complex organization of genomes. However, there are also limitations concerning their focus on linear presentation, standardized input and data bank accessibility. Also customizability by a remote user with special requirements is difficult. The GLOBE-Consortium is developing ways to visualize multi-dimensional data sets from various sources in an easily accessible way. This allows the integration of these data sets into a single holistic display system giving a biological oriented view of genomes and advancing basic research, diagnostics and new treatments.

Supper-resolution (SR) methods are classified into two different methods: image registration (IR)-based
methods and example-based methods. The proposed joint SR method is focused on estimating highresolution
(HR) video sequences from low-resolution (LR) ones by combining the two different methods.
The IR-based SR method collects information from adjacent frames to reconstruct HR images in the video
sequence. Example-based SR methods give good textures and strong edges in the result HR video. In this
paper, IR-based and example-based SR methods are fused based on the gradient features. The proposed
joint SR method gives smaller peak signal to noise ratio than the example-based method, however it shows
better reconstruction results on high-level features such as characters in images. Experimental result of the
proposed joint SR method shows less noise and higher contrast than the example-based method

Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED) microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides ultrastructural information in biological samples as an alternative to immuno-electron microscopy.

Highlights d Correlative cryo-imaging used to study biological processes in vitrified cells d Reovirus intracellular release mechanism is elucidated in 3D with combined cryo-imaging d Reovirus core particles start modifying carrier endosomes at 1 h after infection d Correlation of X-ray and fluorescence cryo-imaging requires no data deformation

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diff...

Over the past decade, the different super-resolution microscopy methods have enabled cellular imaging with nanometer scale resolution; however, super-resolution microscopy is still not practical for many biologists due to limitations such as high illumination intensities, long recording times, and the high cost of super-resolution microscopes. Expansion Microscopy (Chen, Tillberg, Boyden; Science, 2015) circumvents these instrument-related issues by instead isotropically swelling the sample, bringing sub-diffraction limited structures into the size range viewable by conventional confocal and wide-field microscopes. This presentation will demonstrate additional advances and applications of Expansion Microscopy including (1) multiplexed 4-color fluorescence imaging of cells, including the ability to image cyanine dyes, such as Alexa Fluor™ 647 dye, which was previously not possible; (2) use of an inexpensive bench top microscope (Evos™ FL Cell imaging System) with a 60x/1.42 NA oil immersion objective lens to get an effective lateral resolution of <74 nm; and (3) the ability to do High Content Imaging in the super-resolution range with <104 nm lateral resolution using the CellInsight™ CX7 High Content Analysis platform, despite using a lower resolution 20x/0.7 NA dry objective lens. Labeling intracellular targets for these applications used different multiplexed fluorescent labeling strategies (immunofluorescence, targeted fluorescence probes, and expressed fluorescent proteins) that are compatible with Expansion Microscopy and are easily available. Images acquired with the different imaging platforms and labeling methods will be shown for different expanded, multiplexed, cellular targets. This will demonstrate Expansion Microscopy's ease and accessibility in imaging multiplexed sub-diffraction limited cellular structures, as well as it's potential for enabling automated quantitative imaging (High Content Imaging) in the super-resolution range.

It is shown that super-resolution microscopy can be performed by using dielectric microcylinders. We used silica (n ~ 1.46) optical fibers, chemically etched down to ~ 7 µm diameter, placed on top of a commercial Blu-ray ® disk. We clearly resolved one-dimensional arrays of 200 nm width stripes separated by 100 nm width grooves. Potential applications of this imaging technique include microfluidics and nanophotonics applications.

Dynamical artifacts, such as mechanical drift, advection, and hydrodynamic flow, can adversely affect multi-probe dynamic imaging and passive particle-tracking microrheology experiments. Alternatively, active driving by molecular motors can cause interesting non-Brownian motion of probes in local regions. Existing drift-correction techniques, which require large ensembles of probes or fast temporal sampling, are inadequate for handling complex spatio-temporal drifts and non-Brownian motion of localized domains containing relatively few probes. Here, we report an analytical method based on local collective motion (LCM) analysis of as few as two probes for detecting the presence of non-Brownian motion and for accurately eliminating it to reveal the underlying Brownian motion. By calculating an ensemble-average, time-dependent, LCM mean square displacement (MSD) of two or more localized probes and comparing this MSD to constituent single-probe MSDs, we can identify temporal regimes during which either thermal or athermal motion dominates. Single-probe motion, when referenced relative to the moving frame attached to the multi-probe LCM trajectory, provides a true Brownian MSD after scaling by an appropriate correction factor that depends on the number of probes used in LCM analysis. We show that LCM analysis can be used to correct many different dynamical artifacts, including spatially varying drifts, gradient flows, cell motion, time-dependent drift, and temporally varying oscillatory advection, thereby offering a significant improvement over existing approaches.

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diff...

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated ...

Calculated and measured resolution figures are presented for confocal microscopes with different pinhole sizes and for nonlinear (2-photon and second harmonic) microscopes. A modest degree of super-resolution is predicted for a confocal microscope but in practice this is not achievable and confocal fluorescence gives little resolution improvement over widefield. However, practical non-linear microscopes do approach their theoretical resolution and therefore show no resolution disadvantage relative to confocal microscopes in spite of the longer excitation wavelength.

Multi-molecular protein complexes are critical to many cellular functions, including signaling, DNA transcription and enzymatic reactions. In spite of their importance, current research techniques such as biochemistry and diffraction-limited microscopy cannot resolve the heterogeneity and nanoscale organization of protein complexes in intact cells. Here we describe a technique that enables the study of multi-molecular protein complexes at the single molecule level in intact cells. The technique uses photoactivated localization microscopy (PALM) to resolve individual proteins with a resolution down to 20nm in intact cells, and second-order statistics to study the spatial interactions of the proteins. We demonstrate the feasibility of this technique by studying signaling complexes that form in activated T cells. We first use single color PALM imaging and univariate second-order statistics to resolve the clustering of Linker for Activation of T cells (LAT) at the plasma membrane (PM) of the cells. We then use two color PALM and bivariate second-order statistics to resolve the interaction of LAT with key interacting proteins. We discuss potential caveats in studying molecular clustering and the robustness of the technique to study bimolecular interactions. Our proposed technique, combined with older techniques, could help shed new light on the nature of multimolecular protein complexes and their significance to cell function.

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actinlike protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

Stochastic optical reconstruction microscopy (STORM) is a promising method for the visualization of ultra-fine mitochondrial structures. However, this approach is limited to monitoring dynamic intracellular events owing to its low temporal resolution. We developed a new strategy to capture mitochondrial dynamics using a compressed sensing STORM algorithm following raw data pre-treatments by a noise-corrected principal component analysis and K-factor image factorization. Using STORM microscopy with a vicinal-dithiol-proteins targeting probe, visualizing mitochondrial dynamics was attainable with spatial and temporal resolutions of 45 nm and 0.8 s, notably, dynamic mitochondrial tubulation retraction of~746 nm in 1.2 s was monitored. The labeled conjugate was observed as clusters (radii,~90 nm) distributed on the outer mitochondrial membranes, not yet reported as far as we know. This strategy is promising for the quantitative analysis of intracellular behaviors below the optical diffraction limit.

Recent advances in superresolution fluorescence microscopy have been limited by a belief that surpassing twofold resolution enhancement of the Rayleigh resolution limit requires stimulated emission or the fluorophore to undergo state transitions. Here we demonstrate a new superresolution method that requires only image acquisitions with a focused illumination spot and computational post-processing. The proposed method utilizes the focused illumination spot to effectively reduce the object size and enhance the object sparsity and consequently increases the resolution and accuracy through nonlinear image post-processing. This method clearly resolves 70nm resolution test objects emitting ~530nm light with a 1.4 numerical aperture (NA) objective, and, when imaging through a 0.5NA objective, exhibits high spatial frequencies comparable to a 1.4NA widefield image, both demonstrating a resolution enhancement above twofold of the Rayleigh resolution limit. More importantly, we examine how the resolution increases with photon numbers, and show that the more-than-twofold enhancement is achievable with realistic photon budgets.

AbstractThe group 1 metabotropic glutamate receptors 1 and 5 have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout-controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well-described Homer proteins. We confirmed the presence of mGluR1/5 complex by (1) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (2) co-expression in HEK293 cells followed by co-immunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (3) super-resolution microscopy imaging of hippocampal primary neurons to show co-localization of the mGluR1/5 in the synapse.This article is protected by copyright. All rights reserved

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diff...

In this paper we present a technique aimed for simultaneous detection of multiple types of gold nanoparticles (GNPs) within a biological sample, using lock-in detection. We image the sample using a number of modulated laser beams that correspond to the number of GNP species that label a given sample. The final image where the GNPs are spatially separated is obtained computationally. The proposed method enables the simultaneous superresolved imaging of different areas of interest within biological sample and also the spatial separation of GNPs at sub-diffraction distances, making it a useful tool in the study of intracellular trafficking pathways in living cells. Molecular imaging of cells is an important tool for the investigation of biological systems. The ability to trace and track different cells or different components within a cell has an important role in understanding cellular trafficking pathways, identifying receptor expression and facilitating the understanding of cellular signaling pathways which can lead to the design of effective therapies for medical applications 1–7. Therefore, a method for identifying the binding of labeling markers to a cell and the ability to trace it is of great importance. One common method for labeling is to use fluorescent dyes and fluorescent proteins (FPs) as biomarkers 8–11. However, these markers are photo toxic to living cells and their photochemical activity is destroyed after multiple cycles of switching on and off and in addition, their use is limited to visible wavelengths 12–16. GNPs provide an alternative choice for labeling as they are nontoxic, have a long lasting activity, are inexpensive and easy to produce and have spectral absorption/reflectance peak suitable for a variety of wavelengths 17–19. GNPs exhibit localized surface plasmon resonance (SPR), which is manifested by enhanced absorption and scattering at a specific optical frequency when are under optical illumination that matches this resonant wavelength 20. The peak resonance wavelength of the GNPs is governed by their shape, size and the refractive index of the environment 21–24. The most basic GNP shapes include spheres, which have a peak resonance around 532 nm (depending on their exact dimensions), and rods which usually have two resonance peaks; one due to the transverse oscillation of the electrons (which may be around 520 nm) and the other due to the longitudinal plasmon resonance at longer wavelengths that depends on the aspect ratio of the nanorod. The larger the aspect ratio is, the wavelength of the second resonance will become longer. Recently, the use of gold nanoparticles (GNPs) as biomarkers has been demonstrated 25–28 , however, the ability to simultaneously trace different types of GNPs that are site-specifically labeling tissues, cells or areas within a cell is a difficult and more complicated task. One reported study uses a hyperspectral darkfield microspectroscopy system that was developed in order to record the scattering spectra of cells labeled with molecular tags that scatter strongly in distinct spectral windows. This system operated using specific types of GNPs and requires a custom designed system 29. Another limitation stems from the diffraction limit. When imaged, each GNP becomes a point spread function (PSF) due to the diffraction limit set by the Rayleigh criterion 30. As a consequence, areas with high GNPs concentration will appear as large spots. This can be a limitation for applications that require

Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where superresolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of~300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.