Light Fluorescence Microscopy Research Articles

Relating Centromeric Topography in Fixed Human Chromosomes to α-Satellite DNA and CENP-B Distribution

Despite extensive analyses on the centromere and its associated proteins, detailed studies of centromeric DNA structure have provided limited information about its topography in condensed chromatin. We have developed a method with correlative fluorescence light microscopy and atomic force microscopy that investigates the physical and structural organization of α-satellite DNA sequences in the context of its associated protein, CENP-B, on human metaphase chromosome topography. Comparison of centromeric DNA and protein distribution patterns in fixed homologous chromosomes indicates that CENP-B and α-satellite DNA are distributed distinctly from one another and relative to observed centromeric ridge topography. Our approach facilitates correlated studies of multiple chromatin components comprising higher-order structures of human metaphase chromosomes.

In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17‐β‐estradiol analogue in breast adenocarcinoma cells

2-Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2-methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19), a non-commercially available 17-β-estradiol analogue, was tested on the breast adenocarcinoma MCF-7 cell line. The in vitro influence of 24 h exposure to 0.18 μM of C19 on MCF-7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization-optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19-treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19-treated samples. An increase in the number of cells present in the sub-G1 fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19-treated cells. C19 induced both apoptosis and autophagy in MCF-7 cells.

21. The emerging role of nanotechnology in anatomical pathology

Background Interpretation of subcellular localisation by immu-noelectron microscopy is often difficult due to low probe density on target structures. Correlation studies can provide more accurate context and spatial resolution but such approach has hindered by a lack of suitable probes, furthermore, conventional light microscopy will also limit the interpretation due to the resolution limit from diffraction. Nanoparticles such as quantum dots are recently being introduced in research applications. These nanocrystals offer the potential of universal markers and can be visualised by light and electron microscopy. Super-resolution light microscopy overcomes the limit of light diffraction hence it allows a higher image resolution than the conventional light microscopy. Aims To investigate quantum dot for use as immunocytochemi-cal probes for correlation between light and electron microscopy. Furthermore the image quality between super-resolution, confocal and fluorescence light microscopy were also assessed. Methods Quantum dot and colloidal gold probes have been assessed using anti-somatostatin labelling of secretory granules in human somatostatinoma tumour cells. Images of quantum dot labelled tissues were further examined under fluorescence light, confocal and super-resolution light microscopy. Results Antibody conjugated quantum dot immunolabelling was highly specific and can be visualised by light and electron microscopy. Super-resolution microscopy displayed superior details of the target structures compare to fluorescence light and confocal microscopy. Conclusions Quantum dot nanocrystals offer new possibilities for correlation studies incorporating light microscopy, electron microscopy and super resolution, providing a more sensitive and accurate assessment in diagnostic anatomical pathology. Interpretation of subcellular localisation by immu-noelectron microscopy is often difficult due to low probe density on target structures. Correlation studies can provide more accurate context and spatial resolution but such approach has hindered by a lack of suitable probes, furthermore, conventional light microscopy will also limit the interpretation due to the resolution limit from diffraction. Nanoparticles such as quantum dots are recently being introduced in research applications. These nanocrystals offer the potential of universal markers and can be visualised by light and electron microscopy. Super-resolution light microscopy overcomes the limit of light diffraction hence it allows a higher image resolution than the conventional light microscopy. To investigate quantum dot for use as immunocytochemi-cal probes for correlation between light and electron microscopy. Furthermore the image quality between super-resolution, confocal and fluorescence light microscopy were also assessed. Quantum dot and colloidal gold probes have been assessed using anti-somatostatin labelling of secretory granules in human somatostatinoma tumour cells. Images of quantum dot labelled tissues were further examined under fluorescence light, confocal and super-resolution light microscopy. Antibody conjugated quantum dot immunolabelling was highly specific and can be visualised by light and electron microscopy. Super-resolution microscopy displayed superior details of the target structures compare to fluorescence light and confocal microscopy. Quantum dot nanocrystals offer new possibilities for correlation studies incorporating light microscopy, electron microscopy and super resolution, providing a more sensitive and accurate assessment in diagnostic anatomical pathology.

Protein Bodies in Cotyledon Cells Exhibit Differential Patterns of Legumin-Like Proteins Mobilization during Seedling Germinating States

Olive (Olea europaea L.) tree is one of the most extensive and important agricultural crop in Mediterranean countries due to its beneficial health and nutritional properties and its high economic value. Currently, olive tree constitutes the sixth most important cultivated plant in the world, spreading from the Mediterranean region of origin to new production areas such as Australia, South and North America and South Africa. However, the mobilization processes of storage materials i.e. reserve proteins during seed germination, which are largely involved in essential physiological process including plant growth and development, remain poorly understood. Morphometric and immunohistochemistry analyses of protein bodies contained in olive seed storage tissues, cotyledon and endosperm, were performed by using different microscopy techniques, including light (bright-field and fluorescence) microscopy and transmission electron microscopy. Furthermore, we used legumin-like proteins (11S-type globulins) as a molecular marker to study the mobilization of reserve proteins from PBs of cotyledons at germinating seedling stages by using immunofluorescence assays. Results demonstrated that cotyledon and endosperm are characterized by distinct PBs populations containing legumin-like proteins, distinctly discriminated by the number of PBs per cell and tissue, size, immunofluorescence and histochemical staining. These features reflect differential PBs biogenesis during development and maturation processes in olive seed tissues endosperm and cotyledon, in relation to proteins (polypeptides) final composition, SSPs processing and/or packaging during seed maturation. Three different mobilization patterns of legumin-like proteins were identified for the first time in cotyledon PBs during seedling germinating process. Mature proteins composition and/or processing, cell types and enzyme composition and/or differential activation have been discussed as key features determining how proteins mobilize from PBs for further degradation in the cotyledon.

Molecular crosstalk between apoptosis and autophagy induced by a novel 2-methoxyestradiol analogue in cervical adenocarcinoma cells

Background2-Methoxyestradiol has been shown to induce both autophagy and apoptosis in various carcinogenic cell lines. Although a promising anti-cancer agent, it has poor bioavailability and rapid in vivo metabolism which decreases its efficiency. In order to improve 2-methoxyestradiol’s anti-proliferative properties, a novel 2-methoxyestradiol analogue, 2-ethyl-3-O-sulphamoyl-estra-1,3,5 (10)16-tetraene (ESE-16), was previously in silico-designed in our laboratory. This study investigated ESE-16 for its anti-proliferative potential on a cervical adenocarcinoma cell (HeLa) cell line. Additionally, the possible intracellular crosstalk mechanisms between the two types of cell death were investigated.Methods and resultsHeLa cells exposed to 0.5 μM ESE-16 for 24 hours showed morphological evidence of both apoptotic and autophagic death pathways as assessed by polarization-optical transmitted light differential interference contrast microscopy, fluorescent microscopy and transmission electron microscopy. Flow cytometric cyclin B1 quantification revealed induction of programmed cell death after halting cell cycle progression in metaphase. Confocal microscopy demonstrated that ESE-16 caused microtubule fragmentation. Flow cytometric analysis of cell cycle progression and phosphatidylserine flip determination confirmed induction of apoptosis. Moreover, an increase in aggresome formation and microtubule-associated protein light chain, LC3, was demonstrated indicative of autophagy. Both caspase 8 and 3 were upregulated in a spectrophotometric analysis, indicating the involvement of the extrinsic pathway of apoptotic induction.ConclusionsWe conclude that the novel in silico-designed compound, ESE-16, exerts its anti-proliferative effect on the tumorigenic human epithelial cervical (HeLa) cells by sequentially targeting microtubule integrity, resulting in a metaphase block, causing induction of both autophagic and apoptotic cell death via a crosstalk mechanism that involves the extrinsic pathway. Future investigations will expand on signal transduction pathways involved in both apoptosis and autophagy for assessment of ESE-16 effects on microtubule dynamic instability parameters.

Kerion due to microsporum gypseum in a 1-month-old infant

Microsporum gypseum is a rare cause of kerion in infancy. Light microscopy, fluorescent microscopy and fungal culture of the infected hair aids in early and correct diagnosis. Griseofulvin is the drug of choice for ectothrix fungi. We report a case of neonatal kerion caused by M. gypseum occurring at the age of 1 month, successfully treated with griseofulvin.

New sources of resistance to Meloidogyne incognita race 3 in wild cotton accessions and histological characterization of the defence mechanisms

Accessions of Gossypium spp., some of them never previously tested, were evaluated for resistance to a local isolate of Meloidogyne incognita race 3 in greenhouse experiments. Nematode infection was characterized based on the galling and egg mass indexes and the reproduction factors (RF). Root‐knot nematode reproduction among the newly tested accessions varied from highly susceptible (AS0188, AS0189) to moderately resistant (MT123 no. 3), and some accessions showed highly reduced nematode reproduction (CIR1343, CIR1348, Fai Mui). Histological observations of two resistant accessions (G. barbadense CIR1348 and G. hirsutum TX‐25, respectively) showed that resistance occurs through a two‐stage mechanism in the first accession and through a single‐stage mechanism in the second. Parasitism is blocked early after second‐stage juvenile (J2) penetration or during its initial tissue migration (CIR1348) and the development of later‐stage juveniles into female adults is suppressed at a later stage (TX‐25 and CIR1348). Fluorescence and bright light microscopy showed that root cells surrounding nematodes exhibit a hypersensitivity‐like reaction, with the accumulation of presumably phenolic compounds and the presence of necrotic cells that limit the development of nematodes and the formation of giant cells. Underdeveloped giant cells with degenerated cytoplasmic content were found in small numbers in CIR1348 and in large numbers in TX‐25, along with deformed nematodes. The full characterization of the defence mechanisms of novel sources of resistance to the root‐knot nematode in cotton constitutes a first step towards their use in crop improvement.

Three-Dimensional Architecture of the Rod Sensory Cilium and Its Disruption in Retinal Neurodegeneration

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Superresolution microscopy for microbiology

This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of superresolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate superresolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments.

Superhydrophobic and Adhesive Properties of Surfaces: Testing the Quality by an Elaborated Scanning Electron Microscopy Method

In contrast to advancements in the fabrication of new superhydrophobic materials, the characterization of their water repellency and quality is often coarse and unsatisfactory. In view of the problems and inaccuracies, particularly in the measurement of very high contact angles, we developed alternative methods for the characterization of superhydrophobic surfaces. It was found that adhering water remnants after immersion are a useful criterion in determining the repellency quality. In this study, we introduce microscopy methods to detect traces of water-resembling test liquids on superhydrophobic surfaces by scanning electron microscopy (SEM) or fluorescence light microscopy (FLM). Diverse plant surfaces and some artificial superhydrophobic samples were examined. Instead of pure water, we used aqueous solutions containing a detectable stain and glycerol in order to prevent immediate evaporation of the microdroplets. For the SEM examinations, aqueous solutions of lead acetate were used, which could be detected in a frozen state at -90 °C with high sensitivity using a backscattered electron detector. For fluorescence microscopy, aqueous solutions of auramine were used. On different species of superhydrophobic plants, varying patterns of remaining microdroplets were found on their leaves. On some species, drop remnants occurred only on surface defects such as damaged epicuticular waxes. On others, microdroplets regularly decorated the locations of increased adhesion, particularly on hierarchically structured surfaces. Furthermore, it is demonstrated that the method is suitable for testing the limits of repellency under harsh conditions, such as drop impact or long-enduring contact. The supplementation of the visualization method by the measurement of the pull-off force between a water drop and the sample allowed us to determine the adhesive properties of superhydrophobic surfaces quantitatively. The results were in good agreement with former studies of the water repellency and contact angles. In contrast to contact angle measurements, the acqusition of SEM images with high resolution and wide depth of sharpness gives better insight into the wetting behavior and susceptibility of the structural elements of the superhydrophobic surfaces.

Microdistribution of tetrodotoxin in two species of blue-ringed octopuses (Hapalochlaena lunulata and Hapalochlaena fasciata) detected by fluorescent immunolabeling

Blue-ringed octopuses (genus Hapalochlaena) possess the potent neurotoxin tetrodotoxin (TTX). We examined the microdistribution of TTX in ten tissues of Hapalochlaena lunulata and Hapalochlaena fasciata by immunolabeling for fluorescent light microscopy (FLM). We visualized TTX throughout the posterior salivary gland, but the toxin was concentrated in cells lining the secretory tubules within the gland. Tetrodotoxin was present just beneath the epidermis of the integument (mantle and arms) and also concentrated in channels running through the dermis. This was suggestive of a TTX transport mechanism in the blood of the octopus, which would also explain the presence of the toxin in the blood-rich brachial hearts, gills, nephridia, and highly vascularized Needham's sac (testes contents). We also present the first report of TTX in any cephalopod outside of the genus Hapalochlaena. A specimen of Octopus bocki from French Polynesia contained a small amount of TTX in the digestive gland.

Apoptosis induced neurotoxicity of Di-n-butyl-di-(4-chlorobenzohydroxamato) Tin (IV) via mitochondria-mediated pathway in PC12 cells

The severe toxicity of antitumor organotin (IV) compounds limits their application in clinic, however, the toxic mechanism is still unclear. Di-n-butyl-di-(4-chlorobenzohydroxamato) Tin (IV) (DBDCT), an antitumor agent with high activity and obvious neurotoxicity was chosen as a typical diorganotin (IV) compound to investigate its neurotoxic mechanism using PC12 cells and comprehensive methods. Treatment with DBDCT resulted in a dose- and time-dependent growth inhibition of PC12 cells. The changes in cell morphology were observed using light microscopy, fluorescence microscopy and transmission electron microscopy. PC12 cell apoptosis induced by DBDCT was confirmed by annexin V/propidium iodide staining, and characterized by cleavage of caspase-9 and caspase-3 proteins. DBDCT induced the release of cytochrome c from the mitochondria to the cytosol and the generation of reactive oxygen species. DBDCT up-regulated the expression of Bax, down-regulated the expression of Bcl-2, and significantly increased the ratio of Bax/Bcl-2. DBDCT also caused the phosphorylation of JNK and p38MAPK. In rats exposed to DBDCT, apoptosis was also observed in brain, as shown by the detection of cleaved caspase-9 and caspase-3 proteins and increased TUNEL positive staining. In conclusion, the results demonstrated that DBDCT caused the neurotoxicity by inducing apoptosis via mitochondria-mediated pathway.

Construction of retrovirus vector taking MDR1/ACBC1 and its transfection into human placenta derived mesenchymal stem cells

In the study, we used both the methods of perfusion and density gradient centrifugation to isolate and purify mesenchymal stem cells (MSCS) from placenta tissue, and constructed a retroviral vector with multiple drug resistant genes, and the green fluorescent protein (GFP) has been used as an indicative mark. The 293T cell was transfected by the retroviral vector PMX-flag-MDR1-GFP together with its peripheral membrane protein gene. After the infective and replication–defective retrovirus were acquired, we transfected them into human placenta-derived mesenchymal stem cells (HPMSCs). We successfully observed the expression of the reporter gene-GFP by using the green light fluorescence microscope and the p-glycoprotein (P-gp) expressed by exogenous gene MDR1 by Western Blotting. All these facts indicated that the retroviral vector PMX-flag-MDR1-GFP had successfully been transfected into HPMSCs and the exogenous gene multidrug resistance (MDR)1 was detected as normally expressed. The daunorubicin (DNR) pump experiment proved that P-gp of HPMSCs transfected with PMX-flag-MDR1-GFP was of biological activity. The result indicates that MDR1 retroviral vector can transfect the HPMSCs. Not only can the exogenous gene be expressed, but also the expression protein had the biological activity. The conclusion lays a solid foundation of the clinical application of MDR1 genetic therapy. Keywords: Transfect, human placenta-derived mesenchymal stem cells, multidrug resistance (MDR)1 gene.

Encapsulation of protein microfiber networks supporting pancreatic islets

Networks of discrete, genipin-crosslinked gelatin microfibers enveloping pancreatic islets were incorporated within barium alginate microcapsules. This novel technique enabled encapsulation of cellular aggregates in a spherical fibrous matrix <300 μm in diameter. Microfibers were produced by vortex-drawn extrusion within an alginate support matrix. Optimization culminated in a hydrated fiber diameter of 22.3 ± 0.4 μm, a significant reduction relative to that available through current gelatin microfiber spinning techniques, while making the process more reliable and less labor intensive. Microfibers were encapsulated at 40 vol % within 294 ± 4 μm 1.6% barium alginate microparticles by electrostatic-mediated dropwise extrusion. Pancreatic islets extracted from Sprague Dawley rats were encapsulated within the microparticles and analyzed over 21 days. Acridine orange and propidium iodide fluorescent viability staining and light microscopy indicated a significant increase in viability for islets within the fiber-embedded particles relative to fiber-free controls at days 7, 14, and 21. The fiber-embedded system also promoted cellular aggregate cohesion, reducing the incidence of dispersed islet morphologies within the capsules from 31 to 8% at day 21. Further enquiry into benefits of islet encapsulation within a protein fiber network will be the subject of future investigation.

Negative-Stain Electron Microscopy of Inside-Out FtsZ Rings Reconstituted on Artificial Membrane Tubules Show Ribbons of Protofilaments

FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.

Stretching the rules: monocentric chromosomes with multiple centromere domains.

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3–5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel “meta-polycentric” functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.

Histochemical and Anatomical Studies of Phloem and Xylem Cells of Jackfruit (&lt;i&gt;Artocarpus heterophyllus&lt;/i&gt; Lam.) Tree

The localization of starch, lipid and protein, nuclei in the phloem and xylem cells of stem of Jackfruit trees (Artocarpus heterophyllus) has been studied. The optical digital images of anatomical features and localization of nuclei and reserve materials were obtained by light microscopy and fluorescence microscopy. Starch granules in the ray parenchyma cells were more abundant in the outer xylem close to the cambium than in the inner xylem and phloem. Radial localization of starch granules provided more clear data than transverse and tangential observations. Lipid and protein appeared as droplets and were uniformly distributed in the outer xylem. The parenchyma cells of phloem have large amount of lipid bodies but those were almost absent in cambium and xylem ray parenchyma cells. The results on the localization of storage starch, lipid droplets and proteins in phloem, cambium and xylem cells indicating that reserve materials might be important for wood formation in jackfruit trees. This data would be helpful for further study in tree breeding program and clarification of the mechanism of utilization of such reserve materials and their distribution pattern within the cells. DOI: http://dx.doi.org/10.3329/ijns.v2i1.10876 International Journal of Natural Sciences (2012), 2(1): 01- 07

Cassette series designed for live‐cell imaging of proteins and high‐resolution techniques in yeast

During the past decade, it has become clear that protein function and regulation are highly dependent upon intracellular localization. Although fluorescent protein variants are ubiquitously used to monitor protein dynamics, localization and abundance; fluorescent light microscopy techniques often lack the resolution to explore protein heterogeneity and cellular ultrastructure. Several approaches have been developed to identify, characterize and monitor the spatial localization of proteins and complexes at the suborganelle level, yet many of these techniques have not been applied to yeast. Thus, we have constructed a series of cassettes containing codon-optimized epitope tags, fluorescent protein variants that cover the full spectrum of visible light, a TetCys motif used for fluorescein arsenical hairpin (FlAsH)-based localization, and the first evaluation in yeast of a photoswitchable variant, mEos2, to monitor discrete subpopulations of proteins via confocal microscopy. This series of modules, complete with six different selection markers, provides the optimal flexibility during live-cell imaging and multicolour labelling in vivo. Furthermore, high-resolution imaging techniques include the yeast-enhanced TetCys motif, which is compatible with diaminobenzidine photo-oxidation used for protein localization by electron microscopy, and mEos2, which is ideal for super-resolution microscopy. We have examined the utility of our cassettes by analysing all probes fused to the C-terminus of Sec61, a polytopic membrane protein of the endoplasmic reticulum of moderate protein concentration, in order to directly compare fluorescent probes, their utility and technical applications. Our series of cassettes expand the repertoire of molecular tools available to advance targeted spatiotemporal investigations using multiple live-cell, super-resolution or electron microscopy imaging techniques.

Type VI secretion requires a dynamic contractile phage tail-like structure

Type VI secretion systems are bacterial virulence-associated nanomachines composed of proteins that are evolutionarily related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.

Scattered light fluorescence microscopy in three dimensions

Recently, we have proposed a method to image fluorescent structures behind turbid layers at diffraction limited resolution using wave-front shaping and the memory effect. However, this was limited to a raster scanning of the wave-front shaped focus to a two dimensional plane. In applications, it can however be of great importance to be able to scan a three dimensional volume. Here we show that this can be implemented in the same setup. This is achieved by the addition of a parabolic phase shift to the shaped wave-front. Via the memory effect, this phase shift leads to a shift of the interference based focus in the z-direction, thus opening the possibility of three dimensional imaging using scattered light fluorescence microscopy. Here, we show an example of such a three dimensional image of fluorescent nano-beads taken behind a turbid layer more than 10 mean free paths thick. Finally, we discuss the differences of the scanning in the z-direction with that in the x-y plane and the corresponding possibilities and limitations of the technique.