Fluorescence Photoactivation Localization Microscopy Research Articles

Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry.

The organization and dynamics of plasma membrane receptors are a critical link in virus-receptor interactions, which finetune signaling efficiency and determine cellular responses during infection. Characterizing the mechanisms responsible for the active rearrangement and clustering of receptors may aid in developing novel strategies for the therapeutic treatment of viruses. Virus-receptor interactions are poorly understood at the nanoscale, yet they present an attractive target for the design of drugs and for the illumination of viral infection and pathogenesis. This study utilizes super-resolution microscopy and related techniques, which surpass traditional microscopy resolution limitations, to provide both a spatial and temporal assessment of the interactions of human JC polyomavirus (JCPyV) with 5-hydroxytrypamine 2 receptors (5-HT2Rs) subtypes during viral entry. JCPyV causes asymptomatic kidney infection in the majority of the population and can cause fatal brain disease, and progressive multifocal leukoencephalopathy (PML), in immunocompromised individuals. Using Fluorescence Photoactivation Localization Microscopy (FPALM), the colocalization of JCPyV with 5-HT2 receptor subtypes (5-HT2A, 5-HT2B, and 5-HT2C) during viral attachment and viral entry was analyzed. JCPyV was found to significantly enhance the clustering of 5-HT2 receptors during entry. Cluster analysis of infected cells reveals changes in 5-HT2 receptor cluster attributes, and radial distribution function (RDF) analyses suggest a significant increase in the aggregation of JCPyV particles colocalized with 5-HT2 receptor clusters in JCPyV-infected samples. These findings provide novel insights into receptor patterning during viral entry and highlight improved technologies for the future development of therapies for JCPyV infection as well as therapies for diseases involving 5-HT2 receptors.

Phosphatidylinositol 4,5-Bisphosphate Mediates the Co-Distribution of Influenza A Hemagglutinin and Matrix Protein M1 at the Plasma Membrane.

The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature-the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 μM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5–10 μM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

Measurement of Macromolecular Crowding in Rhodobacter sphaeroides under Different Growth Conditions.

ABSTRACTThe bacterial cytoplasm is a very crowded environment, and changes in crowding are thought to have an impact on cellular processes including protein folding, molecular diffusion and complex formation. Previous studies on the effects of crowding have generally compared cellular activity after imposition of stress. In response to different light intensities, in unstressed conditions, Rhodobacter sphaeroides changes the number of 50-nm intracytoplasmic membrane (ICM) vesicles, with the number varying from a few to over a thousand per cell. In this work, the effects of crowding induced by ICM vesicles in photoheterotrophic R. sphaeroides were investigated using a fluorescence resonance energy transfer (FRET) sensor and photoactivated localization microscopy (PALM). In low light grown cells where the cytoplasm has large numbers of ICM vesicles, the FRET probe adopts a more condensed conformation, resulting in higher FRET ratio readouts compared to high light cells with fewer ICM vesicles. The apparent diffusion coefficients of different sized proteins, PAmCherry, PAmCherry-CheY6, and L1-PAmCherry, measured via PALM showed that diffusion of protein molecules >27 kDa decreased as the number of ICM vesicles increased. In low light R. sphaeroides where the crowding level is high, protein molecules were found to diffuse more slowly than in aerobic and high light cells. This suggests that some physiological activities might show different kinetics in bacterial species whose intracellular membrane organization can change with growth conditions.

Intracellular Trafficking and Imaging Methods of Membrane-Bound Transcription Factors in Plants

Membrane-bound transcription factors (MTFs) differ from cytosolic transcription factors (TFs) and they innately bind to membranes. Under external stimuli, MTFs are released from various membranes, convert into the active form, and are transported into the nucleus for transcriptional regulation. Therefore, unlike most TFs, MTFs go through the unique process of transitioning from the membrane to intracellular regions. There are two typical mechanisms during this period: proteolytic processing and alternative splicing. However, other activation schemes have also recently emerged. To further understand these mechanisms, it is essential to study how MTFs transport within the cell and into the nucleus. Imaging techniques with high spatiotemporal resolution can partially resolve this process but new methods are required for future studies. In this review, we give an overview of the current knowledge of plant MTFs, including their identification, specific localization, and the difficulties in studying their cellular dynamics. We also discuss molecular mechanisms of MTF release and advanced methods, such as fluorescence correlation spectroscopy, single-particle tracking, and photoactivated localization microscopy, to further reveal their intracellular movement in living cells.

Dynamic lateral organization of opioid receptors (kappa, muwt and muN40D ) in the plasma membrane at the nanoscale level.

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOPwt), and its naturally occurring isoform (MOPN40D) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble‐averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside‐enriched domains and partial association with cholesterol‐enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor‐specific. KOP‐containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOPN40D. Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOPwt, whereas this effect was not observed for MOPN40D.

Receptor-Receptor Interactions of G Protein-Coupled Receptors in the Carotid Body: A Working Hypothesis.

In the carotid body (CB), a wide series of neurotransmitters and neuromodulators have been identified. They are mainly produced and released by type I cells and act on many different ionotropic and metabotropic receptors located in afferent nerve fibers, type I and II cells. Most metabotropic receptors are G protein-coupled receptors (GPCRs). In other transfected or native cells, GPCRs have been demonstrated to establish physical receptor–receptor interactions (RRIs) with formation of homo/hetero-complexes (dimers or receptor mosaics) in a dynamic monomer/oligomer equilibrium. RRIs modulate ligand binding, signaling, and internalization of GPCR protomers and they are considered of relevance for physiology, pharmacology, and pathology of the nervous system. We hypothesize that RRI may also occur in the different structural elements of the CB (type I cells, type II cells, and afferent fibers), with potential implications in chemoreception, neuromodulation, and tissue plasticity. This ‘working hypothesis’ is supported by literature data reporting the contemporary expression, in type I cells, type II cells, or afferent terminals, of GPCRs which are able to physically interact with each other to form homo/hetero-complexes. Functional data about cross-talks in the CB between different neurotransmitters/neuromodulators also support the hypothesis. On the basis of the above findings, the most significant homo/hetero-complexes which could be postulated in the CB include receptors for dopamine, adenosine, ATP, opioids, histamine, serotonin, endothelin, galanin, GABA, cannabinoids, angiotensin, neurotensin, and melatonin. From a methodological point of view, future studies should demonstrate the colocalization in close proximity (less than 10 nm) of the above receptors, through biophysical (i.e., bioluminescence/fluorescence resonance energy transfer, protein-fragment complementation assay, total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy and photoactivated localization microscopy, X-ray crystallography) or biochemical (co-immunoprecipitation, in situ proximity ligation assay) methods. Moreover, functional approaches will be able to show if ligand binding to one receptor produces changes in the biochemical characteristics (ligand recognition, decoding, and trafficking processes) of the other(s). Plasticity aspects would be also of interest, as development and environmental stimuli (chronic continuous or intermittent hypoxia) produce changes in the expression of certain receptors which could potentially invest the dynamic monomer/oligomer equilibrium of homo/hetero-complexes and the correlated functional implications.

Antimicrobial agent triclosan disrupts mitochondrial structure, revealed by super-resolution microscopy, and inhibits mast cell signaling via calcium modulation

The antimicrobial agent triclosan (TCS) is used in products such as toothpaste and surgical soaps and is readily absorbed into oral mucosa and human skin. These and many other tissues contain mast cells, which are involved in numerous physiologies and diseases. Mast cells release chemical mediators through a process termed degranulation, which is inhibited by TCS. Investigation into the underlying mechanisms led to the finding that TCS is a mitochondrial uncoupler at non-cytotoxic, low-micromolar doses in several cell types and live zebrafish. Our aim was to determine the mechanisms underlying TCS disruption of mitochondrial function and of mast cell signaling. We combined super-resolution (fluorescence photoactivation localization) microscopy and multiple fluorescence-based assays to detail triclosan's effects in living mast cells, fibroblasts, and primary human keratinocytes. TCS disrupts mitochondrial nanostructure, causing mitochondria to undergo fission and to form a toroidal, “donut” shape. TCS increases reactive oxygen species production, decreases mitochondrial membrane potential, and disrupts ER and mitochondrial Ca2+ levels, processes that cause mitochondrial fission. TCS is 60 × more potent than the banned uncoupler 2,4-dinitrophenol. TCS inhibits mast cell degranulation by decreasing mitochondrial membrane potential, disrupting microtubule polymerization, and inhibiting mitochondrial translocation, which reduces Ca2+ influx into the cell. Our findings provide mechanisms for both triclosan's inhibition of mast cell signaling and its universal disruption of mitochondria. These mechanisms provide partial explanations for triclosan's adverse effects on human reproduction, immunology, and development. This study is the first to utilize super-resolution microscopy in the field of toxicology.

The Role of Probe Photophysics in Localization-Based Superresolution Microscopy

Fluorescent proteins are used extensively for biological imaging applications; photoactivatable and photoconvertible fluorescent proteins (PAFPs) are used widely in superresolution localization microscopy methods such as fluorescence photoactivation localization microscopy and photoactivated localization microscopy. However, their optimal use depends on knowledge of not only their bulk fluorescence properties, but also their photophysical properties at the single molecule level. We have used fluorescence correlation spectroscopy and cross-correlation spectroscopy to quantify the diffusion, photobleaching, fluorescence intermittency, and photoconversion dynamics of Dendra2, a well-known PAFP used in localization microscopy. Numerous dark states of Dendra2 are observed both in inactive (green fluorescent) and active (orange fluorescent) forms; the interconversion rates are both light- and pH-dependent, as observed for other PAFPs. The dark states limit the detected count rate per molecule, which is a crucial parameter for localization microscopy. We then developed, to our knowledge, a new mathematical estimate for the resolution in localization microscopy as a function of the measured photophysical parameters of the probe such as photobleaching quantum yield, count rate per molecule, and intensity of saturation. The model was used to predict the dependence of resolution on acquisition parameters such as illumination intensity and time per frame, demonstrating an optimal set of acquisition parameters for a given probe for a variety of measures of resolution. The best possible resolution was then compared for Dendra2 and other widely used probes, including Alexa dyes and quantum dots. This work establishes a framework for determination of the best possible resolution using a localization microscope to image a particular fluorophore, and suggests that development of probes for use in superresolution localization microscopy must consider the count rate per molecule, the saturation intensity, the photobleaching yield, and, crucially, management of bright/dark state transitions, to optimize image resolution.

Blind sparse inpainting reveals cytoskeletal filaments with sub-Nyquist localization.

Single-molecule localization microscopy (SMLM), such as stochastic optical reconstruction microscopy and (fluorescence) photoactivated localization microscopy, has enabled superresolution microscopy beyond the diffraction limit. However, the temporal resolution of SMLM is limited by the time needed to acquire sufficient sparse single-molecule activation events to successfully construct a superresolution image. Here, a novel fast SMLM technique is developed to achieve superresolution imaging within a much shortened duration. This technique does not require a faster switching rate or a higher activation density, which may cause signal degradation or photodamage/bleaching, but relies on computational algorithms to reconstruct a high-density superresolution image from a low-density one using the concept of blind image inpainting. Our results demonstrate that the technique reduces the acquisition time by up to two orders of magnitude compared to the conventional method while achieving the same high resolution. We anticipate our technique to enable future real-time live cell imaging with even higher resolution.

Cytokinesis: Going Super-Resolution in Live Cells

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying molecular organization, but mostly in fixed cells. New work using high-speed fluorescence photoactivation localization microscopy now reveals the organization of cytokinesis nodes and contractile rings in live fission yeast cells.

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was ∼125 nm wide and ∼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring.

Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations.

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample.

Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Ježek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

Division of Mitochondrial Nucleoids Visualized by Biplane FPALM/dSTORM

Mitochondrial DNA (mtDNA) is contained in nucleoids with accessory proteins and gene expression machinery proteins. To visualize them and screen sizes, 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with Eos conjugated to the mitochondrial (mt) transcription factor-A (TFAM) [1], was employed in HepG2 cells. Also, 3D-immunocytochemistry of TFAM, mt-single-stranded-DNA-binding protein (mtSSB), or DNA, was used with the direct stochastic optical reconstruction microscopy (dSTORM). The localized points of FPALM/dSTORM data were remodeled using Delaunay tetrahedron modeling or by principal component analysis (PCA). Alternatively, PCA ellipsoid nucleoid images were normalized by Delaunay volume to obtain symmetrical ellipsoid (DVSE) models. For TFAM FPALM average DVSE of 80×80×159 nm dimensions were yielded, confirmed by TFAM dSTORM giving 78×78×170 nm; whereas mtDNA- and mtSSB-contoured ellipsoids were on average 65×65×152 nm and 44×44×133 nm, reflecting the nucleoid core and a space of unfolded mtDNA (or third-strand structures, such as D-loop), respectively. Due to the 2-fold lower axial vs. lateral resolution, only bulky DVSE models with a high aspect ratio and tilted towards the xy-plane were considered as two proximal nucleoids and suspicious to reflect a state immediately after the nucleoid division following mtDNA replication. Such nucleoid dividing pairs were visualized. The existence of twin nucleoids in mtDNA-dSTORM 3D images, representing snapshot of mtDNA “doubling”, i.e., predicted outcome of replication, supports our hypothetical assignments of similar nucleoids twins, when visualized via TFAM. The visualized nucleoid pairs (bulky, exhibiting high aspect ratios along the mt tubular axis) might represent direct observations of mt nucleoid division after mtDNA replication. Supported by grant 13-02033 (GACR).[1] M.J. Mlodzianoski et al. “Sample drift correction in 3D fluorescence photoactivation localization microscopy”. Opt. Express19, 15009-15019 (2011).

High-Speed Super-Resolution Imaging of Live Fission Yeast Cells.

We describe a step-by-step method for high-speed fluorescence photoactivation localization microscopy (FPALM) of live fission yeast cells. The resolution with this method is tenfold better than spinning disk confocal microscopy.

Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging.

Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents--inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non-invasive microscopy in animals and humans using ultrasound. We anticipate that ultrafast ultrasound localization microscopy may become an invaluable tool for the fundamental understanding and diagnostics of various disease processes that modify the microvascular blood flow, such as cancer, stroke and arteriosclerosis.

3D localization of high particle density images using sparse recovery.

If particles are too close in space, their images may be overlapped when they are observed with microscopes because of diffraction limitation, which makes them difficult to be distinguished or localized. This limitation also affects the efficiency of localization of those single-particle-localization microcopies, such as stochastic optical reconstruction microscopy (STORM) and (fluorescence) photoactivated localization microscopy [(F)PALM]. In this work, we developed a 3D sparse recovery (3D-SR) method, with the aim of localizing particles with high density in three dimensions, which cannot be resolved using original STROM or (F)PALM. A cylindrical lens was introduced to a traditional wide-field microscope in order to form the 3D point spread function for 3D-SR. The performance of the 3D-SR method was evaluated using simulation. Simulated results demonstrated that, even for particle densities as high as 4 μm-2 on a transversal projection, particles could still be localized with high accuracy. The standard deviations were found to be 25.59nm along the transverse direction and 50.42nm along the axial direction. Compared with the existing 3D localization methods used in high particle density cases, such as 3D-DAOSTORM, 3D-SR allows a higher activated fluorophore density per frame.

Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids.

Oligomer aggregation of green-to-red photoconvertible fluorescent protein Eos (EosFP) is a natural feature of the wild‑type variant. The aim of the present study was to follow up mitochondrial nucleoid behavior under natural conditions of living cells transfected with mitochondrial single‑strand DNA‑binding protein (mtSSB) conjugated with EosFP. HEPG2 and SH‑SY5Y cells were subjected to lentiviral transfection and subsequently immunostained with anti‑DNA, anti‑transcription factor A, mitochondrial (TFAM) or anti‑translocase of the inner membrane 23 antibodies. Fluorescent microscopy, conventional confocal microscopy, superresolution biplane fluorescence photo-activation localization microscopy and direct stochastic optical reconstruction microscopy were used for imaging. In the two cell types, apparent couples of equally‑sized mtSSB‑EosFP‑visualized dots were observed. During the time course of the ongoing transfection procedure, however, a small limited number of large aggregates of mtSSB‑EosFP‑tagged protein started to form in the cells, which exhibited a great co‑localization with the noted coupled positions. Antibody staining and 3D immunocytochemistry confirmed that nucleoid components such as TFAM and DNA were co‑localized with these aggregates. Furthermore, the observed reduction of the mtDNA copy number in mtSSB‑EosFP‑transfected cells suggested a possible impairment of nucleoid function. In conclusion, the present study demonstrated that coupled nucleoids are synchronized by mtSSB‑EosFP overexpression and visualized through their equal binding capacity to mtSSB‑EosFP‑tagged protein. This observation suggested parallel replication and transcription activity of nucleoid couples native from a parental one. Preserved coupling in late stages of artificial EosFP‑mediated aggregation of tagged proteins suggested a rational manner of mitochondrial branching that may be cell-type specifically dependent on hierarchical nucleoid replication.

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.