Resolution Fluorescence Microscopy Research Articles

Controlling microgel deformation via deposition method and surface functionalization of solid supports.

Soft matter at solid-liquid interfaces plays an important role in multiple scientific disciplines as well as in various technological fields. For microgels, representing highly interesting soft matter systems, we demonstrate that the preparation method, i.e. the way how the microgel is applied to the specific surface, plays a key role. Focusing on the three most common sample preparation methods (spin-coating, drop-casting and adsorption from solution), we performed a comparative study of the deformation behavior of microgels at the solid-liquid interface on three different surfaces with varying hydrophilicities. For in situ visualization of the deformation of pNIPMAM microgels, we conducted highly sensitive 3D super resolution fluorescence microscopy methods. We furthermore performed complementary molecular dynamics simulations to determine the driving force responsible for the deformation depending on the surface and the deposition method. The combination of experiments and simulations revealed that the simulated equilibrium structure obtained after simulation of the completely dry microgel after deposition is retained after rehydration and subsequent fluorescent imaging.

ℓ1-regularized maximum likelihood estimation with focused-spot illumination quadruples the diffraction-limited resolution in fluorescence microscopy.

Super-resolution fluorescence microscopy has proven to be a useful tool in biological studies. To achieve more than two-fold resolution improvement over the diffraction limit, existing methods require exploitation of the physical properties of the fluorophores. Recently, it has been demonstrated that achieving more than two-fold resolution improvement without such exploitation is possible using only a focused illumination spot and numerical post-processing. However, how the achievable resolution is affected by the processing step has not been thoroughly investigated. In this paper, we focus on the processing aspect of this emerging super-resolution microscopy technique. Based on a careful examination of the dominant noise source and the available prior information in the image, we find that if a processing scheme is appropriate for the dominant noise model in the image and can utilize the prior information in the form of sparsity, improved accuracy can be expected. Based on simulation results, we identify an improved processing scheme and apply it in a real-world experiment to super-resolve a known calibration sample. We show an improved super-resolution of 60nm, approximately four times beyond the conventional diffraction-limited resolution.

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment.

2072-P: Deletion of the Mitofusins 1 and 2 (Mfn1 and Mfn2) in the Pancreatic Beta Cell Disrupts Mitochondrial Structure and Function In Vitro and Strongly Impairs Glucose-Stimulated Insulin Secretion In Vivo

Background and Aims: Mitochondrial metabolism of glucose is vital for the initiation of insulin secretion in pancreatic beta cells. Whether mitochondrial ultra-structure, and the proteins controlling their fission and fusion, are essential for glucose recognition is less clear. In the present study, beta cell-selective adult mouse restricted deletion of Mfn1 and Mfn2 was used to investigate the role of mitochondrial fusion in insulin secretion and glucose homeostasis in vivo and in vitro. Methods: C57BL6 mice bearing Mfn1 and Mfn2 alleles with FloxP sites, were crossed to animals carrying an inducible Cre recombinase at the Pdx1 locus (PdxCreERT). After induction of recombination with tamoxifen, live beta cell fluorescence imaging of mitochondrial [Ca2+] and membrane potential were performed on isolated islets. Mitochondrial ultra-structure was measured using super resolution fluorescence and transmission electron microscopy. Intraperitoneal glucose tolerance, and insulin secretion in vivo and in vitro, were measured using standard techniques. Results: Mitochondrial length was strongly (to 77±0.9% of controls, p<0.0001) and selectively diminished in beta cells from Mfn1/2 KO mice. KO animals displayed higher fasting glycaemia than control animals at 11-12 weeks (8.6 vs. 6.4 mmol/L, p>0.05). An increase in circulating glucose levels was also observed (p<0.05 at 30 min and p<0.01 at 60 min) and was linked to a considerable (>five-fold) decrease in plasma insulin (5-15 min, p<0.0001) post-intraperitoneal glucose injection. Mitochondrial Ca2+ accumulation and membrane potential were significantly reduced (p<0.01) in response to high glucose in KO animals. Conclusions: Mitochondrial fusion and fission cycles are indispensable in the beta cell in order to preserve normal mitochondrial bioenergetics and glucose sensing, and may be disrupted in some forms of diabetes. Disclosure E. Georgiadou: None. P.L. Chabosseau: None. A. Tomas: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. Funding UK Wellcome Trust (WT098424AIA, 212625/Z/18/Z); UK Medical Researh Council (MR/R022259/1, MR/J0003042/1, MR/L020149/1, MR/L02036X/1, MR/N00275X/1); Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA16/0005485)

Integrated super resolution fluorescence microscopy and transmission electron microscopy

In correlative light and electron microscopy (CLEM), the capabilities of fluorescence microscopy (FM) and electron microscopy (EM) are united. FM combines a large field of view with high sensitivity for detecting fluorescence, which makes it an excellent tool for identifying regions of interest. EM has a much smaller field of view but offers superb resolution that allows studying cellular ultrastructure. In CLEM, the potentials of both techniques are combined but a limiting factor is the large difference in resolution between the two imaging modalities. Adding super resolution FM to CLEM reduces the resolution gap between FM and EM; it offers the possibility of identifying multiple targets within the diffraction limit and can increase correlation accuracy.CLEM is usually carried out in two separate setups, which requires transfer of the sample. This may result in distortion and damage of the specimen, which can complicate finding back regions of interest. By integrating the two imaging modalities, such problems can be avoided.Here, an integrated super resolution correlative microscopy approach is presented based on a wide-field super resolution FM integrated in a Transmission Electron Microscope (TEM). Switching imaging modalities is accomplished by rotation of the TEM sample holder. First imaging experiments are presented on sections of Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin both with Protein A-Gold, and Alexa Fluor®647. TEM and FM images were overlaid using fiducial markers visible in both imaging modalities with an overlay accuracy of 28 ± 11 nm. This is close to the optical resolution of ~50 nm.

Micropatterned Adhesion Sites for Spheroid Cultivation Under Flow

The focus of cell based assays in recent years switched from 2D to 3D applications using spheroids and organoid cell assemblies due to the higher validity of the experimental outcome concerning in vivo relevance. However, spheroid generation, continues media exchange for good nutrients supply and high quality imaging over a long period of time is often very laborious. We use a perfusable channel system with micropatterned adhesion ligands on a highly passivated coverslip, which enables high resolution fluorescence microscopy, to generate numerous homogenously distributed spheroids in parallel within one channel. In this system spheroids can either be directly generated by simple injection of a large number of cells and a self-sorting process or by addition of preformed spheroids, which will bind to the patterned adhesion sites. Due to the immobilization of spheroids to the adhesion spots, long-term observation of the same spheroids can be performed with many spheroids in parallel. Perfusion of the spheroid chip at low flow rates not only enables the homogenous supply of all spheroids with nutrients, but also the application of a defined shear stress, metabolite analysis together with toxicological screenings or even co-culture of multiple spheroid types. With our micropatterned channel system we were able to show that by applying a flow the overall spheroid growth increases over time and spheroid size is very homogenous. By applying very high flow rates spheroids can be harvested from the surfaces, collected and further analyzed. The flow also influences the drug response of spheroids towards a more in vivo like reaction, showing that 3D spheroid cultivation under flow is the method of choice for generating relevant experimental results.

Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation.

Mitochondrial sirtuins (Sirts) control important cellular processes related to stress. Despite their regulatory importance, however, the dynamics and subcellular distributions of Sirts remain debatable. Here, we investigate the subcellular localization of sirtuin 4 (Sirt4), a sirtuin variant with a mitochondrial targeting sequence (MTS), by expressing Sirt4 fused to the superfolder green fluorescent protein (Sirt4-sfGFP) in HeLa and pancreatic β-cells. Super resolution fluorescence microscopy revealed the trapping of Sirt4-sfGFP to the outer mitochondrial membrane (OMM), possibly due to slow mitochondrial import kinetics. In many cells, Sirt4-sfGFP was also present within the cytosol and nucleus. Moreover, the expression of Sirt4-sfGFP induced mitochondrial swelling in HeLa cells. In order to bypass these effects, we applied the self-complementing split fluorescent protein (FP) technology and developed mito-STAR (mitochondrial sirtuin 4 tripartite abundance reporter), a tripartite probe for the visualization of Sirt4 distribution between mitochondria and the nucleus in single cells. The application of mito-STAR proved the importation of Sirt4 into the mitochondrial matrix and demonstrated its localization in the nucleus under mitochondrial stress conditions. Moreover, our findings highlight that the self-complementation of split FP is a powerful technique to study protein import efficiency in distinct cellular organelles.

High-resolution computed tomography reveals dynamics of desiccation and rehydration in fern petioles of a desiccation-tolerant fern.

Desiccation-tolerant (DT) plants can dry past -100MPa and subsequently recover function upon rehydration. Vascular DT plants face the unique challenges of desiccating and rehydrating complex tissues without causing structural damage. However, these dynamics have not been studied in intact DT plants. We used high resolution micro-computed tomography (microCT), light microscopy, and fluorescence microscopy to characterize the dynamics of tissue desiccation and rehydration in petioles (stipes) of intact DT ferns. During desiccation, xylem conduits in stipes embolized before cellular dehydration of living tissues within the vascular cylinder. During resurrection, the chlorenchyma and phloem within the stipe vascular cylinder rehydrated before xylem refilling. We identified unique stipe traits that may facilitate desiccation and resurrection of the vascular system, including xylem conduits containing pectin (which may confer flexibility and wettability); chloroplasts within the vascular cylinder; and an endodermal layer impregnated with hydrophobic substances that impede apoplastic leakage while facilitating the upward flow of water within the vascular cylinder. Resurrection ferns are a novel system for studying extreme dehydration recovery and embolism repair in the petioles of intact plants. The unique anatomical traits identified here may contribute to the spatial and temporal dynamics of water movement observed during desiccation and resurrection.

Höchstmögliche räumlich-zeitliche Auflösung in der Lichtmikroskopie

Super resolution (SR) fluorescence microscopy techniques STED, SIM and PALM/STORM have revolutionized light microscopy and provide resolution of imaging way below the general limit of resolution as defined by the Abbe law. Single molecule tracking (SMT) allows the pursuit of movement of single proteins or lipids in real time, revealing in vivo dwell times and binding kinetics, a further milestone in live cell imaging. A combination of SR and SMT is a logical next evolution step in the goal to study dynamic processes in live cells at a maximal spatial and temporal resolution.

Brief update on endocytosis of nanomedicines.

The complexity of nanoscale interactions between biomaterials and cells has limited the realization of the ultimate vision of nanotechnology in diagnostics and therapeutics. As such, significant effort has been devoted to advancing our understanding of the biophysical interactions of the myriad nanoparticles. Endocytosis of nanomedicine has drawn tremendous interest in the last decade. Here, we highlight the ever-present barriers to efficient intracellular delivery of nanoparticles as well as the current advances and strategies deployed to breach these barriers. We also introduce new barriers that have been largely overlooked such as the glycocalyx and macromolecular crowding. Additionally, we draw attention to the potential complications arising from the disruption of the newly discovered functions of the lysosomes. Novel strategies of exploiting the inherent intracellular defects in disease states to enhance delivery and the use of exosomes for bioanalytics and drug delivery are explored. Furthermore, we discuss the advances in imaging techniques like electron microscopy, super resolution fluorescence microscopy, and single particle tracking which have been instrumental in our growing understanding of intracellular pathways and nanoparticle trafficking. Finally, we advocate for the push towards more intravital analysis of nanoparticle transport phenomena using the multitude of techniques available to us. Unraveling the underlying mechanisms governing the cellular barriers to delivery and biological interactions of nanoparticles will guide the innovations capable of breaching these barriers.

Fibre-based spectral ratio endomicroscopy for contrast enhancement of bacterial imaging and pulmonary autofluorescence.

Fibre-based optical endomicroscopy (OEM) permits high resolution fluorescence microscopy in endoscopically accessible tissues. Fibred OEM has the potential to visualise pathologies targeted with fluorescent imaging probes and provide an in vivo in situ molecular pathology platform to augment disease understanding, diagnosis and stratification. Here we present an inexpensive widefield ratiometric fibred OEM system capable of enhancing the contrast between similar spectra of pathologically relevant fluorescent signals without the burden of complex spectral unmixing. As an exemplar, we demonstrate the potential of the platform to detect fluorescently labelled Gram-negative bacteria in the challenging environment of highly autofluorescent lung tissue in whole ex vivo human lungs.

3,3'-thiodipropanol as a versatile refractive index-matching mounting medium for fluorescence microscopy.

High resolution fluorescence microscopy requires optimization of the protocols for biological sample preparation. The optical and chemical characteristics of mounting media are among the things that could be modified to achieve optimal image formation. In our search for chemical substances that could perform as mounting media, 3,3'-thiodipropanol (TDP) emerged as a sulfide with potentially interesting characteristics. In this work, several tests of its performance as a mounting medium for fluorescence microscopy of biological samples were performed, including the labeling of filamentous actin with fluorescent phalloidins. The refractive index dispersion curve of pH-adjusted TDP was experimentally obtained in the visible range and compared to the dispersion curves of commercial and lab-made mounting media. The effects on the fluorescence of commonly used dyes were tested by using TDP as a solvent and measuring the relative fluorescence quantum yield of the dyes. By being able to mix TDP in any concentration with water and 2,2'-thiodiethanol (TDE), it was possible not only to fine-tune the refractive index of the resulting solution, but also to preserve the compatibility of TDP with the most popular and efficient fluorescent actin staining used in biological microscopy.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles.

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of <em>Chlamydomonas</em> Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Improving the spectral resolution in fluorescence microscopy through shaped-excitation imaging

The visualization of distinct molecular species represents an important challenge of bio-imaging research. In past decades, the development of multicolor fluorescent (FL) labels has greatly improved our ability to track biological analytes, paving the way for important advances in understanding the cell dynamics. It remains challenging, however, to visualize a large number of different fluorephores simultaneously. Owing to a spectrally broad absorption of fluorescent dyes, only up to five color categories can be resolved at once. Here, we demonstrate a general strategy for distinguishing between multiple fluorescent targets in acquired microscopy images with improved accuracy. The present strategy is enabled through spectral shaping of the excitation light with an optical filter that uniquely attenuates the light absorption of each fluorophore in the investigated sample. The resulting emission changes, induced by such excitation modulation, are therefore target-specific and can be used for identifying various fluorescent species. The technique is demonstrated through an accurate identification of 8 different CdSe dyes with absorption maxima spanning the 520–620 spectral range. It is subsequently applied for accurate measurements of the pH balance in buffers emulating a metabolism of tumor cells.

Integration of Soft X-ray Tomography and High Resolution Fluorescence Microscopy

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Targeted delivery of fluorogenic peptide aptamers into live microalgae by femtosecond laser photoporation at single-cell resolution

Microalgae-based metabolic engineering has been proven effective for producing valuable substances such as food supplements, pharmaceutical drugs, biodegradable plastics, and biofuels in the past decade. The ability to accurately visualize and quantify intracellular metabolites in live microalgae is essential for efficient metabolic engineering, but remains a major challenge due to the lack of characterization methods. Here we demonstrate it by synthesizing fluorogenic peptide aptamers with specific binding affinity to a target metabolite and delivering them into live microalgae by femtosecond laser photoporation at single-cell resolution. As a proof-of-principle demonstration of our method, we use it to characterize Euglena gracilis, a photosynthetic unicellular motile microalgal species, which is capable of producing paramylon (a carbohydrate granule similar to starch). Specifically, we synthesize a peptide aptamer containing a paramylon-binding fluorescent probe, 7-nitrobenzofurazan, and introduce it into E. gracilis cells one-by-one by suppressing their mobility with mannitol and transiently perforating them with femtosecond laser pulses at 800 nm for photoporation. To demonstrate the method’s practical utility in metabolic engineering, we perform spatially and temporally resolved fluorescence microscopy of single live photoporated E. gracilis cells under different culture conditions. Our method holds great promise for highly efficient microalgae-based metabolic engineering.

Spectral Resolution in Fluorescence Microscopy in Strongly Scattering Media

Spectral phasors were introduced as alternative to spectral demixing, for the determination of FRET and for the measurement of dipolar relaxation in membranes. The basic concept in the spectral phasor approach is that the entire spectrum is not needed for some analysis, but only some of the elements of the spectral distribution are sufficient. In the case in which the spectrum is dominated by few spectral bands, this approach is very efficient and provides fast and valid alternative to the analysis of the full spectrum. One example of a system that could benefit from the phasor analysis is in tissue spectroscopy in which the light to be collected has undergone multiple scattering so that it cannot be easily focused on the entrance slit of a dispersive element or transformed in a parallel beam for use with hyperspectral cameras. In a typical application of thick tissue spectroscopy, the light, after being transmitted through the tissue, is emitted over a very large cone angle. All the schemes designed to capture this transmitted (or fluorescence) light use a relatively narrow collection angle with severe reduction of the signal. In the microscopy field, we introduced in 2011 a microscope design (DIVER) that can capture a very large solid emission angle (close to 2pi) and we have shown that this microscope, depending on the tissue thickness, can capture several orders of magnitude more light than conventional microscopes using epi detection. A limitation of the DIVER design is that interference filters cannot be used in the emission path since the light is impinging on the surface of the filter in front of the detector with a very large angular distribution. Here we propose a simple device to overcome this problem (Supported by NIH P41-GM103540 and NIH P50-GM076516).

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells.

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.e. depression, anxiety, and addictions), neuroendocrinological alterations, and neurodegenerative diseases. Here, we present a strategy to label GPCRs with a small fluorescent antagonist that permits the observation of the receptor in live cells through stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. The marker, an aza-BODIPY derivative, was designed based on computational docking studies, then synthesized, and finally tested in biological cells. Experiments on hippocampal neurons demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395. A quantitative analysis of two color STORM images enabled the determination of the binding affinity of the new marker in the cellular environment.

Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission.

The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance the fluorescent signal that is ascribed to the selected feature. The image acquisition is facilitated by using considerable illumination, bright probes at a relatively high concentration in order to make the fluorescent signal significantly more intense than the background signal. Here, we present two methods for completely removing the background signal in spectrally resolved fluorescence microscopy. The methodology is applicable for all probes with narrow and well-defined emission bands (Full width half-maximum < 20 nm). Here, we use two lanthanide based probes exploiting the narrow emission lines of europium(III) and terbium(III) ions. We used a model system with zeolites doped with lanthanides immobilized in a polymer stained with several fluorescent dyes regularly used in bioimaging. After smoothing the spectral data recorded in each pixel, they are differentiated. Method I is based on the direct sum of the gradient, while method II resolves the fluorescent signal by subtracting a background calculated via the gradient. Both methods improve signal-to-background ratio significantly and we suggest that spectral imaging of lanthanide-centered emission can be used as a tool to obtain absolute contrast in bioimaging.