Localization Of Individual Molecules Research Articles

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Quantitative Analysis of Anchoring Proteins of the Inhibitory Synapse through Single Molecule Localization Techniques

The post-synaptic area of the inhibitory synapse is characterized by sub-resolved clusters of the scaffold protein Gephyrin [1] that anchors GABAA receptor (GABAAR). Super-resolution techniques [2] based on Individual Molecule Localization (IML) [3] provide the capability to quantitatively study these subcellular structures, giving access to its protein distribution at the molecular level.In this work the highly heterogeneous spatial distribution of Gephyrin cluster along the 3D neuronal network has been investigated through dual color STORM (Stochastic Optical Reconstruction Microscopy) to characterize the post-synaptic area, for example reveling the interaction between Gephyrin and GABAARs.To monitor the response of Gephyrin cluster to Long-Term Potentiation of inhibition (iLTP) their functional link with the synaptic terminals has been highlighted. The insight provided at the molecular level makes IML techniques the suitable tool for a quantitative study of Gephyrin distribution at synaptic level. Quantitative approach based on clustering analysis [4] provided access to a more comprehensive set of parameters able to define the response to iLTP, such as the area and the density of the scaffold protein. At the fluorescent tag level, to fulfill the requests of the quantitative analysis, an irreversibly photoactivatable fluorescent protein has been used (mEos), knowing its photophysical properties [5].[1] Petrini E. M. et al, Nat. Comm., 2014.[2] Deschout H. et al., Nat. Methods, 2014.[3] Specht C.G. et al. Neuron, 2013.[4] Pavan M. et al. IEEE Trans. Pattern Analysis and Machine Intelligence, 2007.[5] Durisic N. et al., Nat. Methods, 2014.

Taylor series expansion based multidimensional image reconstruction for confocal and 4pi microscopy

We propose and experimentally demonstrate a three-dimensional (3D) image reconstruction methodology based on Taylor series approximation (TSA) in a Bayesian image reconstruction formulation. TSA incorporates the requirement of analyticity in the image domain, and acts as a finite impulse response filter. This technique is validated on images obtained from widefield, confocal laser scanning fluorescence microscopy and two-photon excited 4pi (2PE-4pi) fluorescence microscopy. Studies on simulated 3D objects, mitochondria-tagged yeast cells (labeled with Mitotracker Orange) and mitochondrial networks (tagged with Green fluorescent protein) show a signal-to-background improvement of 40% and resolution enhancement from 360 to 240 nm. This technique can easily be extended to other imaging modalities (single plane illumination microscopy (SPIM), individual molecule localization SPIM, stimulated emission depletion microscopy and its variants).

Light-sheet confined super-resolution using two-photon photoactivation.

Light-sheet microscopy is a useful tool for performing biological investigations of thick samples and it has recently been demonstrated that it can also act as a suitable architecture for super-resolution imaging of thick biological samples by means of individual molecule localization. However, imaging in depth is still limited since it suffers from a reduction in image quality caused by scattering effects. This paper sets out to investigate the advantages of non-linear photoactivation implemented in a selective plane illumination configuration when imaging scattering samples. In particular, two-photon excitation is proven to improve imaging capabilities in terms of imaging depth and is expected to reduce light-sample interactions and sample photo-damage. Here, two-photon photoactivation is coupled to individual molecule localization methods based on light-sheet illumination (IML-SPIM), allowing super-resolution imaging of nuclear pH2AX in NB4 cells.

Super-resolution measurements related to uncertainty relations in optical and biological fluorescence systems

Super-resolution effects in optical and fluorescence biological systems are analyzed, and their relations with uncertainty relations are discussed. Super-resolutions obtained in the optical systems, including especially NSOM, SIM, hyperlens and Pendry lens, are related to an increase of the spatial frequencies in the object plane, leading to very small effective space uncertainties, and thus the resolution is increased far beyond the Abbe limit. Super-resolution measurements obtained in the fluorescent biological systems STED, FPALM and RESOLFT are related to space-momentum uncertainty relation. An example of a four-level STED system is analyzed, in analogy to a four-level laser system, but the special space dependence of the STED light is taken into account, restricting the fluorescence from extremely small volume, and thus extremely high resolution is obtained. Localization of individual molecules is described by the FPALM method where, by illuminating the fluorescent molecule by a ‘read out’ laser, many fluorescent photons are emitted by the same molecule before it decays or transmitted to non-fluorescent states. The relation between this effect and the space-momentum uncertainty relation is discussed. While in the STED method very high laser intensities are needed, in its variant method known as RESOLFT, the super-resolution measurements can be obtained by much weaker light intensities. This new method is analyzed, and the relation between this method and space-momentum uncertainty relation is explained.

Ultrahigh accuracy imaging modality for super-localization microscopy.

Super-localization microscopy encompasses techniques that depend on the accurate localization of individual molecules from generally low light images. The obtainable localization accuracies, however, are ultimately limited by the image detector's pixelation and noise. We present the Ultrahigh Accuracy Imaging Modality (UAIM), which enables the attainment of accuracies approaching what is only achievable without detector pixelation and noise, and is experimentally observed to provide a >200% accuracy improvement over conventional imaging.

Super Resolution Imaging of Thick Samples by Means of Selective Plane Illumination Microscopy

Recently, super-resolution techniques based on single molecule localization became a popular tool to understand biological processes and image structures below the diffraction limit. In the last few years a fast development in the field allowed multicolor and 3D super-resolution imaging of biological samples. However, super-resolution techniques are currently well established at the cellular level but their application to more complicate samples, such as tissues and embryos, still remain a challenging task. within this context, recently, the coupling of localization based techniques and selective plane illumination microscopy [1], allowed to extend the application range to thicker tissues ( 50 μm), by coupling far-field individual molecule localization (IML) and selective plane illumination microscopy (SPIM) both in the linear and non linear regime. Particular attention is addressed to the advantages provided by two photon photo-activation in scattering environments.IML-SPIM allows to image cellular spheroids with sub-diffraction resolution in depth and can be applied to image cellular substructures in Zebrafish early developmental stages as well.(1) Cella Zanacchi F. et al., “Live-cell 3D super-resolution imaging in thick biological samples.,” Nat Methods, 8, no. 12, pp. 1047-1049, (2011)(2) Truong T. V. et al. “Deep and fast live imaging with two-photon scanned light-sheet microscopy.,” Nat Methods, 8, no. 9, pp. 757-760, (2011). Huisken, J., et al. Science 305, 1007-1009 (2004).

2D/3D Super-Resolution Microscopy by Means of Individual Molecule Localization (IML) and Selective Plane Illumination Microscopy

In the last few years localization based techniques, which exploit photoactivation, photoconversion or ground state depletion of fluorescent molecules, became a popular tool for super-resolution imaging of biological samples. Recently, approaches based on two photon excitation have been implemented in order to get axial confinement of the photoactivation process allowing for 3D super-resolution imaging of biological samples [1]. However, a topic of interest is still represented by the widening of super-resolution applications to thick samples (>15 μm). Within this scenario, light sheet based fluorescence microscopy techniques provide optical sectioning since illumination is confined to a thin planar region perpendicular to the detection axis and represent a suitable tool to confine the photoactivation process along the optical axis. In particular, single plane illumination microscopy (SPIM), has been proved to be a useful tool for biological investigations of thick living samples [2].Here we demonstrate three-dimensional super-resolution live cell imaging through thick biological specimen (>50 μm), by coupling far-field individual molecule localization (IML) and selective plane illumination microscopy (SPIM).The confined excitation provided by SPIM and the improved signal-to-noise ratio allows for nanometric localization of single molecules in thick scattering samples. A PALM approach [3] and elliptical stretching of the point spread function allow to perform 3D super-resolution imaging of biological live samples in depth (up to 100 μm). IML-SPIM allowed to image cellular spheroids with < 35 nm lateral precision and sub-diffraction resolution in depth [4].(1) York, A.G. et al. Nat Methods, 8, 327–333 (2011).(2) Huisken, J., et al. Science305, 1007–1009 (2004).(3) Hess, S.T., Girirajan,T.P.K. and Mason, M.D. Biophys J., 91(11), 4258–4272 (2006).(4) Cella Zanacchi et al. Nature Methods (accepted).

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

Single molecule image deconvolution. I. Standard deviation analysis of immobile fluorescent molecules

Single molecule fluorescence imaging has been a powerful technique in studying individual processes not accessible by bulk, ensemble-averaged measurements [1]. Improvements in image analysis are required for high temporal and spatial precision in the localization of single fluorescent molecules. We present the first thorough standard deviation analysis for point spread functions (PSFs) of single immobile fluorescent molecules. Using this new single molecule image deconvolution (SMID) method, we show that 3D localization of individual molecules with sub-nanometer precision can be achieved. We have derived an expression estimating the standard error of the PSF's standard deviation, incorporating experimental effects of the number of collected photons, finite pixel size, and background noise. The localization precision obtained via this expression is approximately 1.5 times better than the current available methods. The use of SMID to extract subexposure dynamics of mobile molecules will also be discussed.[1]. Wang, Y. M, R. H, Austin, & Cox, E. C. 2006 Physical Review Letters 97, 048302(1-4).

Syndecan and tenascin: Induction during early tooth morphogenesis and possible interactions

Cell-matrix interactions in morphogenesis The important role of the extraceUular matrix (ECM) in regulation of organ morphogenesis has been recognized for several decades (Grobstein, 1967; Bernfield et al., 1972; Lash and Vasan, 1978; Hay, 1981; Toole, 1981). When the main categories of matrix constituents, the collagens, other glycoproteins and proteoglycans with their different glycosaminoglycan side chains had been identified, their roles were studied in the development of different embryonic organs. Experiments where their accumulation was perturbed indicated developmental significance for molecules from all different categories, and metabolic labelling studies suggested that they were developmentally regulated (KaUman and Grobstein, 1966; Bernfield et al., 1972; Hetem et al., 1985). Subsequently, the localization of individual molecules by specific antibodies and immunohistology indicated that the distribution patterns of different matrix molecules correlated with morphogenetic changes, suggesting again developmental roles for the ECM