Structured Illumination Fluorescence Research Articles

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we have developed a cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 110 nm enough for the subsequent cryo-lamella fabrication. We have successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

P-257 Nanosphere-based assessment of zona pellucida permeability in humans and mammalian models

Abstract Study question What is the maximum particle size that can traverse through human zona pellucida (ZP) and that of other mammals? Summary answer Human oocytes ZP is impermeable to 40-100 nm nanospheres. ZP of other mammals is impermeable to 100 nm particles, and permeability changes along preimplantation development. What is known already ZP is a porous structure that protects oocytes and early embryos, being permeable to nourishing and signaling substances while blocking the entrance of immune cells and viruses into the perivitelline space. Size-dependent ZP permeability is poorly known and may differ between species, as in humans and rabbits ZP it is composed by 4 proteins (ZP1-4), while bovine and murine ZPs contain only 3 proteins (ZP2-4 in bovine, ZP1-3 in mice). Understanding permeability is crucial to determine embryo susceptibility to viral infections or large biological particles such as exosomes, and to assess possible damages inferred by artificial reproductive techniques. Study design, size, duration The objective has been to determine ZP permeability in mice, bovine, rabbit (wild-type or lacking ZP4) or human oocytes and/or embryos based on particle size. The assay was performed in 3 replicates of 3 samples per stage: mouse samples (oocytes, zygotes, 2-cell embryos and blastocysts) collected in vivo, bovine samples (oocytes, zygotes, 4-8 cell embryos and blastocysts) produced in vitro, rabbit samples (zygotes) collected in vivo and immature human oocytes discarded for clinical use. Participants/materials, setting, methods Permeability test consisted of incubating oocytes and embryos in 0.005 % solutions of carboxylate-modified polystyrene nanospheres (20, 40 and 100 nm) loaded with a fluorophore emitting at a 505/515 nm for 30 min. Following incubation and washing, samples were observed under a structured illumination fluorescence microscope to determine if particles are present inside ZP (i.e., ZP is permeable to that size of particle) or fluorescence cannot be detected (i.e., particles unable to traverse ZP). Main results and the role of chance ZPs of all species and stages blocked efficiently the pass of 100 nm-sized nanospheres. 40 mm nanospheres were blocked by the ZP of oocytes and zygotes in all species with the exception of rabbit zygotes lacking ZP4. ZP permeability changed during early development, as ZP from murine and bovine cleaved embryos and blastocyst were permeable to 40 nm-sized particles. Finally, with the exception or WT rabbit zygotes, ZP from all oocytes and embryos analyzed were permeable to 20 nm nanospheres. As a fluorescence control, murine and bovine oocytes without ZP were exposed to nanospheres of all sizes, which were clearly visualized attached to the oolema or into the ooplasm. Limitations, reasons for caution Experiments on cleavage and blastocysts stages were performed on animal models, so whether human ZP permeability changes following fertilization remains unknown. Wider implications of the findings Mammalian ZP acts as a selective barrier during early embryo development. Understanding particle size-dependent permeability is crucial to determine embryo susceptibility to viral infection or to signaling mediated by large particles such as extracellular vesicles and exosomes and to assess the impact of oocyte manipulation techniques. Trial registration number Not applicable

MmSIM: an open toolbox for accessible structured illumination microscopy.

Since the first practical super-resolution structured illumination fluorescence microscopes (SIM) were demonstrated more than two decades ago, the method has become increasingly popular for a wide range of bioimaging applications. The high cost and relative inflexibility of commercial systems, coupled with the conceptual simplicity of the approach and the desire to exploit and customize existing hardware, have led to the development of a large number of home-built systems. Several detailed hardware designs are available in the scientific literature, complemented by open-source software tools for SIM image validation and reconstruction. However, there remains a lack of simple open-source software to control these systems and manage the synchronization between hardware components, which is critical for effective SIM imaging. This article describes a new suite of software tools based on the popular Micro-Manager package, which enable the keen microscopist to develop and run a SIM system. We use the software to control two custom-built, high-speed, spatial light modulator-based SIM systems, evaluating their performance by imaging a range of fluorescent samples. By simplifying the process of SIM hardware development, we aim to support wider adoption of the technique. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Enhanced detection of oral dysplasia by structured illumination fluorescence lifetime imaging microscopy

We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).

Increased ROS-Dependent Fission of Mitochondria Causes Abnormal Morphology of the Cell Powerhouses in a Murine Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in humans and remains to have a fatal prognosis. Recent studies in animal models and human ALS patients indicate that increased reactive oxygen species (ROS) play an important role in the pathogenesis. Considering previous studies revealing the influence of ROS on mitochondrial physiology, our attention was focused on mitochondria in the murine ALS model, wobbler mouse. The aim of this study was to investigate morphological differences between wild-type and wobbler mitochondria with aid of superresolution structured illumination fluorescence microscopy, TEM, and TEM tomography. To get an insight into mitochondrial dynamics, expression studies of corresponding proteins were performed. Here, we found significantly smaller and degenerated mitochondria in wobbler motor neurons at a stable stage of the disease. Our data suggest a ROS-regulated, Ox-CaMKII-dependent Drp1 activation leading to disrupted fission-fusion balance, resulting in fragmented mitochondria. These changes are associated with numerous impairments, resulting in an overall self-reinforcing decline of motor neurons. In summary, our study provides common pathomechanisms with other ALS models and human ALS cases confirming mitochondria and related dysfunctions as a therapeutic target for the treatment of ALS.

Highly selective mitochondrial probes based on fluorinated pentamethinium salts: On two-photon properties and microscopic applications

Fluorescent dyes and probes have been of interest in the fields of chemistry and biology for a long time. However, with more recent applications in advanced techniques, such as two-photon and super-resolution fluorescence microscopy, higher demands are being placed on physico-chemical properties of such compounds. Another requirement is specific intracellular targeting which often remains a big challenge. Here, we present the synthesis of a series of far red-emitting fluorescent dyes based on a structure of fluorinated symmetrical ɣ-aryl substituted pentamethinium salts. We reveal the relationship between the structure and photophysical and biological properties of the compounds; a type of a side unit (indole, benzothiazole) and presence of fluorine atoms were investigated. It was found that substitution of benzothiazolium side units for indolium ones led to increased quantum yields and higher photostability of the dyes and that the presence of fluorine on the γ-aryl moiety did not exhibit significant influence on their photophysical properties. Regardless of the structural difference, all compounds localized in mitochondria of various cancerous and noncancerous cell lines and can be utilized in live-cell imaging using wide-field, confocal, two-photon and structured illumination fluorescence microscopy. Our findings suggest that the properties of symmetrical pentamethinium salts are affected to a higher extent by the type of a side unit rather than fluorine atoms on the γ-aryl moiety. Last but not least, we believe that such probes will further extend tools for mitochondrial research.

Microscope calibration using laser written fluorescence.

There is currently no widely adopted standard for the optical characterization of fluorescence microscopes. We used laser written fluorescence to generate two- and three-dimensional patterns to deliver a quick and quantitative measure of imaging performance. We report on the use of two laser written patterns to measure the lateral resolution, illumination uniformity, lens distortion and color plane alignment using confocal and structured illumination fluorescence microscopes.

A fixable probe for visualizing flagella and plasma membranes of the African trypanosome.

The protozoan Trypanosoma brucei sp. cause diseases in humans and animals. Studies of T. brucei cell biology have revealed unique features, such as major endocytic events being limited to a single region, and mitochondrial genome segregation mediated via basal bodies. Further understanding of trypanosome cell biology can be facilitated with super-resolution fluorescence microscopy. Lack of a plasma membrane probe for fixed trypanosomes remains a persistent problem in need of a working solution. Herein, we report protocols developed using mCLING in super-resolution structured illumination fluorescence microscopy (SR-SIM). mCLING comprehensively labels flagellar membranes, including nascent intracellular stages. To extend its usefulness for trypanosome biology we optimized mCLING in combination with organelle-specific antibodies for immunofluorescence of basal bodies or mitochondria. Then in work with live trypanosomes, we demonstrated internalization of mCLING into endocytic stations that overlap with LysoTracker in acidic organelles. Greater detail of the intracellular location of mCLING was obtained with SR-SIM after pulsing trypanosomes with the probe, and allowing continuous uptake of fluorescent concanavalin A (ConA) destined for lysosomes. In most cases, ConA and mCLING vesicles were juxtaposed but not coincident. A video of the complete image stack at the 15 min time point shows zones of mCLING staining surrounding patches of ConA, consistent with persistence of mCLING in membranes of compartments that contain luminal ConA. In summary, these studies establish mCLING as a versatile trypanosome membrane probe compatible with super-resolution microscopy that can be used for detailed analysis of flagellar membrane biogenesis. In addition, mCLING can be used for immunofluorescence in fixed, permeabilized trypanosomes. Its robust staining of the plasma membrane eliminates a need to overlay transmitted light images on fluorescence pictures obtained from widefield, confocal, or super-resolution microscopy.

Enhancing in vivo tumor boundary delineation with structured illumination fluorescence molecular imaging and spatial gradient mapping.

Fluorescence imaging, in combination with tumor-avid near-infrared (NIR) fluorescent molecular probes, provides high specificity and sensitivity for cancer detection in preclinical animal models, and more recently, assistance during oncologic surgery. However, conventional camera-based fluorescence imaging techniques are heavily surface-weighted such that surface reflection from skin or other nontumor tissue and nonspecific fluorescence signals dominate, obscuring true cancer-specific signals and blurring tumor boundaries. To address this challenge, we applied structured illumination fluorescence molecular imaging (SIFMI) in live animals for automated subtraction of nonspecific surface signals to better delineate accumulation of an NIR fluorescent probe targeting α4β1 integrin in mice bearing subcutaneous plasma cell xenografts. SIFMI demonstrated a fivefold improvement in tumor-to-background contrast when compared with other full-field fluorescence imaging methods and required significantly reduced scanning time compared with diffuse optical spectroscopy imaging. Furthermore, the spatial gradient mapping enhanced highlighting of tumor boundaries. Through the relatively simple hardware and software modifications described, SIFMI can be integrated with clinical fluorescence imaging systems, enhancing intraoperative tumor boundary delineation from the uninvolved tissue.

Structured illumination fluorescence Fourier ptychographic microscopy

We apply a Fourier ptychographic algorithm for fluorescent samples using structured illumination. The samples are illuminated with structured light patterns and the raw imaging data using traditional structured illumination fluorescence microscopy (SIM) are acquired. We then extract equivalent oblique illuminated images of fluorescent samples from the SIM images. An optimized Fourier ptychography algorithm is proposed, which ensures the fidelity of the reconstructed the super-resolution results. This method can break the diffraction limit to a resolution of λ/4, and has a better signal-to-noise ratio (SNR) than SIM, especially when the background noise is high.

Super-resolution structured illumination fluorescence microscopy of the lateral wall of the cochlea: the Connexin26/30 proteins are separately expressed in man.

Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This "battery" is located "off-site" in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.

Frustrated Phagocytic Spreading Dynamics End in Distinct Non-Muscle Myosin II Dependent Contraction

Phagocytosis is the process by which individual cells engulf large (up to 10 μm) particles. It is the hallmark behavior of macrophages and neutrophils, being the process through which those cells ingest and degrade pathogens and debris from the body. The biochemical cascade involved with triggering Fc-receptor mediated phagocytosis (i.e. antibody dependent phagocytosis) has been well-studied; however, questions regarding how molecular scale signals coordinate such a large-scale process remain unanswered. To help address those questions, we developed a high-throughput method for quantitatively characterizing single-cell phagocytosis dynamics using Frustrated Phagocytosis (phagocytosis of a large flat surface). Our work reveals that FP response depends on opsonin density in a binary manner: above a minimum ligand density cells undergo a period of rapid spreading, below that threshold they do not. Surprisingly, the dynamic behavior (e.g. spreading-rate, and final contact-area) of cells undergoing FP is independent of opsonin density. We also find that FP includes a late-stage myosin II dependent contractile period, during which the cell exerts significant forces on the underlying substrate. Based on these findings we posit that phagocytosis dynamics are determined by two major factors. First, after Fc-receptor stimulation, phagocytic spreading is driven by the recruitment and polymerization of F-actin at the leading edge of the cell. This polymerization force drives the cell to spread along the target particle with dynamics determined by the viscoelastic properties of the cell, not signal diffusion or chemical kinetics. Second, we argue that late-stage contraction is driven by the formation of an F-acin “contractile belt” that runs parallel to the cell perimeter. Structured illumination fluorescence reveals F-actin bundles are formed during contraction and we hypothesize that late-stage myosin II activity is responsible for their formation. Funding: NSF CAREER-DMR #0848797 & NSF-PoLS #0848797.

Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells.

Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500 μM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability.

Chemo-spectroscopic sensor for carboxyl terminus overexpressed in carcinoma cell membrane

Certain carboxyl groups of the plasma membrane are involved in tumorgenesis processes. A gold core-hydroxyapatite shell (AuHA) nanocomposite is introduced as chemo-spectroscopic sensor to monitor these carboxyl groups of the cell membrane. Hydroxyapatite (HA) plays the role both of a chemical detector and of a biocompatible Raman marker. The principle of detection is based on chemical interaction between the hydroxyl groups of the HA and the carboxyl terminus of the proteins. The AuHA exhibits a surface enhanced Raman scattering (SERS) signal at 954cm−1 which can be used for its localization. The bio-sensing capacity of AuHA towards human skin epidermoid carcinoma (A431) and Chinese hamster ovary (CHO) cell lines is investigated using Raman microspectroscopic imaging. The localization of AuHA on cells is correlated with scanning electron microscopy, transmission electron microscopy and structured illumination fluorescence microscopy. This qualitative approach is a step towards a quantitative study of the proteins terminus. From the Clinical EditorThis method would enable further studies on the molecular profiling of the plasma membrane, in an attempt to provide accurate cell identification. Using a gold core-hydroxyapatite shell (AuHA) nanocomposite, the authors in this paper showed the feasibility of detecting and differentiating cell surface molecules by surface enhanced Raman scattering.

Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator.

We describe a two-beam interference structured illumination fluorescence microscope. The novelty of the presented system lies in its simplicity. A programmable spatial light modulator (ferroelectric LCoS) in an intermediate image plane enables precise and rapid control of the excitation pattern in the specimen. The contrast of the projected light pattern is strongly influenced by the polarization state of the light entering the high NA objective. To achieve high contrast, we use a segmented polarizer. Furthermore, a mask with six holes blocks unwanted components in the spatial frequency spectrum of the illumination grating. Both these passive components serve their purpose in a simpler and almost as efficient way as active components. We demonstrate a lateral resolution of 114.2 ± 9.5 nm at a frame rate of 7.6 fps per reconstructed 2D slice.

The sigma-F dependent gene sspA affects septum positioning development of spore chains in the filamentous bacterium, Streptomyces coelicolor. Septation was visualised using cell wall staining with fluorescent wheat germ agglutinin (green). Each pre-spore

Molecular MicrobiologyVolume 91, Issue 2 p. i-i Front CoverFree Access The sigma-F dependent gene sspA affects septum positioning development of spore chains in the filamentous bacterium, Streptomyces coelicolor. Septation was visualised using cell wall staining with fluorescent wheat germ agglutinin (green). Each pre-spore compartment contains a single chromosome stained by propidium iodide (red). The main image and the insert were taken using laser-scanning confocal microscopy and super-resolution, three-dimensional structured illumination fluorescence microscopy, respectively. For further details readers are referred to the article by Tzanis et al. on pp. 363–380 of this issue. First published: 10 January 2014 https://doi.org/10.1111/mmi.12497AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume91, Issue2January 2014Pages i-i RelatedInformation

Structured illumination fluorescence microscopy with distorted excitations using a filtered blind-SIM algorithm

Structured illumination microscopy (SIM) is a powerful technique for obtaining super-resolved fluorescence maps of samples, but it is very sensitive to aberrations or misalignments affecting the excitation patterns. Here, we present a reconstruction algorithm that is able to process SIM data even if the illuminations are strongly distorted. The approach is an extension of the recent blind-SIM technique, which reconstructs simultaneously the sample and the excitation patterns without a priori information on the latter. Our algorithm was checked on synthetic and experimental data using distorted and nondistorted illuminations. The reconstructions were similar to that obtained by up-to-date SIM methods when the illuminations were periodic and remained artifact-free when the illuminations were strongly distorted.

Structured illumination-based super-resolution optical microscopy for hemato- and cyto-pathology applications.

Structured illumination fluorescence microscopy utilizes interfering light and the moiré effect to enhance spatial resolution to about a half of that of conventional light microscopy, i.e. approximately 90 nm. In addition to the enhancement in the x and y directions, it also allows enhancement of resolution in the z- direction by the same factor of two (to approximately 220 nm), making it a powerful tool for 3-D morphology studies of fluorescently labeled cells or thin tissue sections. In this report, we applied this technique to several types of blood cells that are commonly seen in hematopathology. Compared with standard brightfield and ordinary fluorescence microscopy images, the 3-D morphology results clearly reveal the morphological features of different types of normal blood cells. We have also used this technique to evaluate morphologies of abnormal erythrocytes and compare them with those recorded on normal cells. The results give a very intuitive presentation of morphological structures of erythrocytes with great details. This research illustrates the potential of this technique to be used in hematology and cyto-pathology studies aimed at identifying nanometer-sized features that cannot be distinguished otherwise with conventional optical microscopy.

Structured Illumination-Based Super-Resolution Optical Microscopy for Hemato- and Cyto-Pathology Applications

Structured illumination fluorescence microscopy utilizes interfering light and the moiré effect to enhance spatial resolution to about a half of that of conventional light microscopy, i.e. approximately 90 nm. In addition to the enhancement in thexandydirections, it also allows enhancement of resolution in thez- direction by the same factor of two (to approximately 220 nm), making it a powerful tool for 3-D morphology studies of fluorescently labeled cells or thin tissue sections. In this report, we applied this technique to several types of blood cells that are commonly seen in hematopathology. Compared with standard brightfield and ordinary fluorescence microscopy images, the 3-D morphology results clearly reveal the morphological features of different types of normal blood cells. We have also used this technique to evaluate morphologies of abnormal erythrocytes and compare them with those recorded on normal cells. The results give a very intuitive presentation of morphological structures of erythrocytes with great details. This research illustrates the potential of this technique to be used in hematology and cyto-pathology studies aimed at identifying nanometer-sized features that cannot be distinguished otherwise with conventional optical microscopy.

Dual-modality fluorescence and full-field optical coherence microscopy for biomedical imaging applications.

Full-field optical coherence microscopy (FFOCM) is a high-resolution interferometric technique that is particularly attractive for biomedical imaging. Here we show that combining it with structured illumination fluorescence microscopy on one platform can increase its versatility since it enables co-localized registration of optically sectioned reflectance and fluorescence images. To demonstrate the potential of this dual modality, a fixed and labeled mouse retina was imaged. Results showed that both techniques can provide complementary information and therefore the system could potentially be useful for biomedical imaging applications.