Answering some questions about structured illumination microscopy.

Structured illumination microscopy (SIM) provides images of fluorescent objects at an enhanced resolution greater than that of conventional epifluorescence wide-field microscopy. Initially demonstrated in 1999 to enhance the lateral resolution twofold, it has since been extended to enhance axial resolution twofold (2008), applied to live-cell imaging (2009) and combined with myriad other techniques, including interferometric detection (2008), confocal microscopy (2010) and light sheet illumination (2012). Despite these impressive developments, SIM remains, perhaps, the most poorly understood 'super-resolution' method. In this article, we provide answers to the 13 questions regarding SIM proposed by Prakash et al. along with answers to a further three questions. After providing a general overview of the technique and its developments, we explain why SIM as normally used is still diffraction-limited. We then highlight the necessity for a non-polynomial, and not just nonlinear, response to the illuminating light in order to make SIM a true, diffraction-unlimited, super-resolution technique. In addition, we present a derivation of a real-space SIM reconstruction approach that can be used to process conventional SIM and image scanning microscopy (ISM) data and extended to process data with quasi-arbitrary illumination patterns. Finally, we provide a simple bibliometric analysis of SIM development over the past two decades and provide a short outlook on potential future work. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'

NBLAST: Rapid, Sensitive Comparison of Neuronal Structure and Construction of Neuron Family Databases.

Neural circuit mapping is generating datasets of tens of thousands of labeled neurons. New computational tools are needed to search and organize these data. We present NBLAST, a sensitive and rapid algorithm, for measuring pairwise neuronal similarity. NBLAST considers both position and local geometry, decomposing neurons into short segments; matched segments are scored using a probabilistic scoring matrix defined by statistics of matches and non-matches. We validated NBLAST on a published dataset of 16,129 single Drosophila neurons. NBLAST can distinguish neuronal types down to the finest level (single identified neurons) without a priori information. Cluster analysis of extensively studied neuronal classes identified new types and unreported topographical features. Fully automated clustering organized the validation dataset into 1,052 clusters, many of which map onto previously described neuronal types. NBLAST supports additional query types, including searching neurons against transgene expression patterns. Finally, we show that NBLAST is effective with data from other invertebrates and zebrafish. VIDEO ABSTRACT.This work was supported by the Medical Research Council [MRC file reference U105188491] and European Research Council Starting and Consolidator Grants to G.S.X.E.J., who is an EMBO Young Investigator.This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.neuron.2016.06.01

ELM: super-resolution analysis of wide-field images of fluorescent shell structures.

It is often necessary to precisely quantify the size of specimens in biological studies. When measuring feature size in fluorescence microscopy, significant biases can arise due to blurring of its edges if the feature is smaller than the diffraction limit of resolution. This problem is avoided if an equation describing the feature's entire image is fitted to its image data. In this paper we present open-source software, ELM, which uses this approach to measure the size of spheroidal or cylindrical fluorescent shells with a precision of around 10 nm. This has been used to measure coat protein locations in bacterial spores and cell wall diameter in vegetative bacilli, and may also be valuable in microbiological studies of algae, fungi and viruses. ELM is available for download at https://github.com/quantitativeimaging/ELM

Ellipsoid Localisation Microscopy

Multilayered protein coats are crucial to the dormancy, robustness, and germination of bacterial spores. In Bacillus subtilis spores, the coat contains over 70 distinct proteins. Identifying which proteins reside in each layer may provide insight into their distinct functions. We present image analysis methods that determine the order and geometry of concentric protein layers by fitting a model description for a spheroidal fluorescent shell image to optical micrographs of spores incorporating fluorescent fusion proteins. The radius of a spherical protein shell can be determined with <10 nm error by fitting an equation to widefield fluorescence micrographs. Ellipsoidal shell axes can be fitted with comparable precision. The layer orders inferred for B. subtilis and B. megaterium are consistent with measurements in the literature. The aspect ratio of elongated spores and the tendency of some proteins to localize near their poles can be quantified, enabling measurement of structural anisotropy.We gratefully acknowledge support from MedImmune through the Beacon collaboration, the EPSRC Centre for Doctoral Training in Sensor Technologies and Applications (EP/L015889/1), and thank Clemens Kaminski, Romain Laine and Jose Casas-Finet for inspiring discussions.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.bpj.2015.09.02

The natverse, a versatile toolbox for combining and analysing neuroanatomical data.

To analyse neuron data at scale, neuroscientists expend substantial effort reading documentation, installing dependencies and moving between analysis and visualisation environments. To facilitate this, we have developed a suite of interoperable open-source R packages called the natverse. The natverse allows users to read local and remote data, perform popular analyses including visualisation and clustering and graph-theoretic analysis of neuronal branching. Unlike most tools, the natverse enables comparison across many neurons of morphology and connectivity after imaging or co-registration within a common template space. The natverse also enables transformations between different template spaces and imaging modalities. We demonstrate tools that integrate the vast majority of Drosophila neuroanatomical light microscopy and electron microscopy connectomic datasets. The natverse is an easy-to-use environment for neuroscientists to solve complex, large-scale analysis challenges as well as an open platform to create new code and packages to share with the community

Proteins Encoded by the gerP Operon Are Localized to the Inner Coat in Bacillus cereus Spores and Are Dependent on GerPA and SafA for Assembly.

The germination of Bacillus spores is triggered by certain amino acids and sugar molecules which permeate the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here, we use the ellipsoid localization microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex-lytic enzymes within the inner coat. We also reveal that the GerPA protein alone can localize in the absence of all other GerP proteins and that it has an essential role for the localization of all other GerP proteins within the spore. Its essential role is also demonstrated to be dependent on SafA, but not CotE, for localization, which is consistent with an inner coat location. GerP-null spores are shown also to have reduced permeability to fluorescently labeled dextran molecules compared to wild-type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability.IMPORTANCE The bacterial spore coat comprises a multilayered proteinaceous structure that influences the distribution, survival, and germination properties of spores in the environment. The results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ellipsoid localization microscopy (ELM) image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, health care, and environmental sectors.1. Engineering and Physical Sciences Research Council Centre for Doctoral Training in Sensor Technologies and Applications (EP/L015889/1) 2. Cambridge Nehru Scholarship 3. Raymond and Beverly Sackler Foundation 4. MedImmun

A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication.

Herpesviruses are large and complex viruses that have a long history of coevolution with their host species. One important factor in the virus-host interaction is the alteration of intracellular morphology during viral replication with critical implications for viral assembly. However, the details of this remodeling event are not well understood, in part because insufficient tools are available to deconstruct this highly heterogeneous process. To provide an accurate and reliable method of investigating the spatiotemporal dynamics of virus-induced changes to cellular architecture, we constructed a dual-fluorescent reporter virus that enabled us to classify four distinct stages in the infection cycle of herpes simplex virus-1 at the single cell level. This timestamping method can accurately track the infection cycle across a wide range of multiplicities of infection. We used high-resolution fluorescence microscopy analysis of cellular structures in live and fixed cells in concert with our reporter virus to generate a detailed and chronological overview of the spatial and temporal reorganization during viral replication. The highly orchestrated and striking relocation of many organelles around the compartments of secondary envelopment during transition from early to late gene expression suggests that the reshaping of these compartments is essential for virus assembly. We furthermore find that accumulation of HSV-1 capsids in the cytoplasm is accompanied by fragmentation of the Golgi apparatus with potential impact on the late steps of viral assembly. We anticipate that in the future similar tools can be systematically applied for the systems-level analysis of intracellular morphology during replication of other viruses