Resolution Fluorescence Microscopy Research Articles

High resolution fluorescence microscopy evidence on the transport of immunoglobulins. Differences between mammalian IgG, F(abʹ)2 and avian IgY

We describe the subcellular localization of horse F(abʹ)2 and IgG, and ostrich IgY labeled with fluorescein isothiocyanate (FITC) administered IV to mice. We used wide field high sensitivity fluorescence microscopy deblurred by 3-dimensional blind deconvolution of kidney, liver, lungs and brain sections. Sections were obtained from mice sacrificed 15 min, 1 or 5 h after receiving FITC-immunoproteins, counter-stained with DAPI (4′,6′-diamidino-2-phenylindole) and Evans blue. FITC-IgG and its fractions are rapidly taken up and extravasated by vascular endothelium. FITC-IgG and FITC-F(abʹ)2 appear to be quickly secreted by glomeruli endothelium and to be reabsorbed along all nephron segments. FITC-IgG and FITC-F(abʹ)2 appeared 15 min after IV injection within bronchial, alveolar and bile duct epithelium. Hepatocytes were loaded with fluorescence after 15 min of administration. Fluorescence was absent from brain slices, except for the endothelium of some vessels in brain ventricles which appeared intensely fluorescent. Fluorescence appeared in intracellular vesicles which conferred the tissues a glowing foamy aspect for up to 5 h after inoculation. Arterial elastic layers were intensely green after horse FITC-Ig inoculation. Ostrich FITC-IgY behaved completely differently to horse Ig's; only 1 h after injection it was possible to observe small brightly green scarce vesicles in vascular endothelium of arteries, interstitial kidney capillaries between nephron tubules and were also scarce in glomeruli endothelium; FITC-IgY appeared only in hepatic sinusoids in the liver. No IgY was seen in bronchial and alveolar endothelium, in bile ducts or in hepatocytes.

Preparation strategy and illumination of three-dimensional cell cultures in light sheet–based fluorescence microscopy

A device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions, is described. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. Layers of the sample, of about 10 μm or less in diameter, are, thus, illuminated selectively and imaged by high resolution fluorescence microscopy. SPIM is operated at low light exposure even if a larger number of layers is imaged and is easily combined with laser scanning microscopy. Chinese hamster ovary cells expressing a membrane-associated green fluorescent protein are used for preliminary tests, and the uptake of the fluorescent marker, acridine orange via a microfluidic system, is visualized to demonstrate its potential in cancer research such as for the detection of cellular responses to anticancer drugs.

Horse IgG- and ostrich IgY-F(ab′)2 groups have different affinities for mice erythrocytes and lymphocytes. Implications for avian immunoglobulin therapeutic usefulness

We used high sensitivity and resolution fluorescence microscopy to study the interaction of ostrich IgY, horse F(ab′)2 and horse IgG with mice lymphocyte and erythrocyte plasma membrane. The immunoglobulins were labeled with fluorescein isotiocyanate (FITC). Our results show an interaction of IgY with lymphocyte plasma membrane which does not result in endocytosis of the labeled protein. Less IgG and its F(ab′)2 fraction bind to lymphocytes, and this binding seems to be followed by endocytosis producing a diffuse cytoplasmic fluorescence in most lymphocytes exposed to FITC-IgG or FITC-F(ab′)2. Cytoplasmic fluorescence resembling FITC was not observed in lymphocytes exposed to FITC-IgY. Receptors in the erythrocyte membrane also differentiate between avian and horse Ig; while erythrocytes exposed to horse Igs became intensely fluorescent for at least 5 h, no erythrocyte labeling occurred when FITC-IgY was used. Our results suggest that IgY may be a stronger activator of adaptive immunity than horse IgG in mammals. Adaptive immunity against IgY is detrimental to its IV therapeutic use in humans and other mammals.

Improved resolution in fluorescence microscopy using quantum correlations

Breaking the diffraction limit in microscopy by utilizing the quantum properties of light has been the goal of intense research in recent years. We propose a super-resolution technique based on nonclassical emission statistics of fluorescent markers, routinely used as contrast labels for bioimaging. The technique can be readily implemented with current technology.

Quantifying Actin mRNA Expression with Super Resolution Fluorescence Microscopy

The copy number of a specific mRNA and its subcellular localization in cells is a mode by which cells can regulate protein synthesis. Beta-actin mRNA is transported to filopodia upon serum stimulation, regions of cell growth and movement where actin polymerization is essential (1). The copy number of mRNA molecules directly translates to the amount of actin monomers that are synthesized. Quantifying the actin mRNA copy numbers provides a measure of how polymerization of actin is regulated.To this extent, we apply stochastic super-resolution microscopy with photoswitchable fluorescent probes combined with single-molecule localization (2,3) for quantitative studies of mRNA concentration and localization in eukaryotic cells. For this purpose, we apply various labelling approaches for mRNA, including well-established methods such as fluorescence in-situ hybridization (FISH) (4). Focussing on actin mRNA, we derive quantitative information on copy numbers, and we apply coordinate-based algorithms to analyze our single-molecule super-resolution data.References(1) Ben-Ari et al. (2010) J Cell Sci, 123, 1761-1774.(2) Betzig et al. (2006) Science, 313, 1642-1645.(3) Heilemann et al. (2008) Angew. Chemie, 47, 6172-6176.(4) Levskey, J. M.; Singer, R. (2003) Journal of Cell Science, 116, 2833-2838.

A Novel Assay That Integrates the Effects of Multiple Agonists At the Single-Platelet Level,

Abstract 3258 Background:At sites of vascular injury, platelets are exposed to multiple agonists that lead to overall activation and platelet plug formation. Signaling pathways induced by these agonists are known to interact with each other. For example, collagen binding to the platelet collagen receptors, α2β1 integrin, CD36, and glycoprotein VI, induces inside-out signaling that ultimately leads to the activation of the glycoprotein IIb/IIIa receptor for fibrinogen on the platelet surface (Nakamura et al, JCB, 1999).An assay capable of tracking the biological effects of multiple different agonists simultaneously within a single platelet would enhance our understanding of how platelets integrate these different signals at the single-platelet level. This assay would have advantages over current clinical assays in that platelet function tests such as platelet aggregometry or the PFA-100 assess only collective behavior of platelet populations without single platelet resolution. Flow cytometry achieves single platelet resolution but cannot monitor the dynamic changes induced by agonists over time. Platelet adhesion assays have the capability to track individual platelets over time via microscopy, but currently cannot simultaneously monitor the different effects of multiple agonists and their potential interactions.To that end, we developed a modified platelet adhesion assay by using microcontact printing to “stamp” distinct micro-to-nanoscale patterns of different platelet agonists and ligands on glass coverslips. Different parts of each platelet are then exposed to different agonists/ligands with our assay and how the different downstream biological signals interact, synergize, and potentially compete can be monitored overall time at the single platelet level via epifluorescence microscopy. Results:Using microfabrication techniques, patterned polydimethylsiloxane (PDMS) stamps were” inked” with two different known platelet agonists/ligands: Collagen type 1 conjugated with FITC (green) and fibrinogen conjugated to Alexa Fluor 594 (red). Using those stamps, protein micropatterns were then “microstamped” and transferred onto the glass surface, creating spatially distinct microprints with two different proteins (Fig. 1). The protein surface was then blocked with 1% BSA. [Display omitted] Platelets were isolated and dyed with a fluorescent membrane marker. Platelets were suspended in Tyrode's buffer (20 million platelets per mL) and incubated with the double micro-patterned surface. After incubation, the surface was washed and imaged with epifluorescence microscopy using a 40x objective.For these initial proof-of-concept experiments, we observed that when individual platelets are simultaneously exposed to separate areas of fibrinogen as well as collagen, they exhibit a strong “preference” for the fibrinogen over collagen (Fig. 2). Indeed, 79.1% of platelets were observed to almost completely (>75% of the surface area) migrate to the fibrinogen micropattern from the collagen micropattern (Fig. 3). Few platelets were spread equally between the two protein micropatterns, which suggest that platelets eventually “choose” which agonist to settle before it get fully spread and immobilized. Interestingly, platelets with more than 75 % surface area on collagen (8.96 % of total platelets measured) tended to be smaller in size, have filopodia, and have intense granule staining compared to platelets on fibrinogen, which were more fully spread with broad lamellipodia. These data suggest that the platelets that migrated to the fibrinogen micropattern compared to those that preferred collagen may be physiologically distinct. [Display omitted] [Display omitted] Conclusions and Ongoing Efforts: These data establish the viability of our system to investigate the integrative effects of different agonists at the single platelet level. This assay will enable the further understanding of how different agonist-induced signaling pathways interact. Ongoing experiments will include other agonists/ligands such as von Willebrand factor and thrombin. Although the current data assesses morphologic changes of platelets, we are focusing our efforts on using high resolution fluorescence microscopy to measure and monitor the spatial dynamics of calcium signaling, protein phosphorylation of relevant signaling pathways, and cytoskeletal rearrangement when different parts of single platelets are exposed to different agonists. Disclosures:No relevant conflicts of interest to declare.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Effects of aberrations and specimen structure in conventional, confocal and two‐photon fluorescence microscopy

Specimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point-like, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.

High accuracy FIONA–AFM hybrid imaging

Multi-protein complexes are ubiquitous and play essential roles in many biological mechanisms. Single molecule imaging techniques such as electron microscopy (EM) and atomic force microscopy (AFM) are powerful methods for characterizing the structural properties of multi-protein and multi-protein–DNA complexes. However, a significant limitation to these techniques is the ability to distinguish different proteins from one another. Here, we combine high resolution fluorescence microscopy and AFM (FIONA–AFM) to allow the identification of different proteins in such complexes. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, we are able to align fluorescence and AFM information to ≥8 nm accuracy. This accuracy is sufficient to identify individual fluorescently labeled proteins in most multi-protein complexes. We investigate the limitations of localization precision and accuracy in fluorescence and AFM images separately and their effects on the overall registration accuracy of FIONA–AFM hybrid images. This combination of the two orthogonal techniques (FIONA and AFM) opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5–10 nm) information about the conformational properties of multi-protein complexes and the fluorescence can indicate spatial relationships of the proteins in the complexes.

Steady state and time-resolved studies of pyrene in solution and in single microcrystals

Single microcrystals of pyrene have been studied by steady state and time resolved fluorescence microscopy. The fluorescence spectra of microcrystals exhibit vibrational structure unlike the broad spectrum observed in pyrene excimer. A risetime is observed in the decay curves of the concentrated solutions, indicating the excimer formation. In contrast, the fluorescence decay profiles of the single microcrystals are nonexponential in nature and the decay times vary with their size and the wavelength of emission. This behaviour has been explained mainly by considering the pyrene dimer stabilized in the ground state.

An ASIC1 and ENaC hybrid channel is the amiloride sensitive channel in human glioma.

High grade gliomas express multiple members of the Epithelial Sodium Channel (ENaC)/Degenerin (Deg) family, displaying a basally active amiloride sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC) and Acid Sensing Ion Channel (ASIC) subunits. Using immunohistochemisty we show a higher expression level of ASIC1, αENaC, and γENaC in glioblastoma biopsy tissue compared with non‐neoplastic brain tissue. To test our hypothesis we made dominant negative (DN) cDNAs for ASIC1, αENaC, and γENaC. D54‐MG cells transfected with the DN constructs for ASIC1, αENaC or γENaC showed reduced protein expression and a significant reduction in the amiloride sensitive whole cell current as compared with untransfected D54‐MG cells. Using co‐immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC at the plasma membrane of D54‐MG cells, consistent with these subunits interacting with each other to form an ion channel. Data using Total Internal Resolution Fluorescent Microscopy (TIRFM) also suggest that ASIC1 and ASIC2 interact with γENaC in CHO‐K1 cells. These data suggest that different ENaC/Deg subunits combine to form a hybrid channel that likely underlies the amiloride‐sensitive current in human gliomas. This study was supported by NIH Grants CA101952 and DK037206.

Light Dose is a Limiting Factor to Maintain Cell Viability in Fluorescence Microscopy and Single Molecule Detection

A test system for cell viability based on colony formation has been established and applied to high resolution fluorescence microscopy and single molecule detection. Living cells were irradiated either by epi-illumination or by total internal reflection (TIR) of a laser beam, and light doses where at least 90% of irradiated cells survived were determined. These light doses were in the range of a few J/cm2 up to about 200 J/cm2 depending on the wavelength of illumination as well as on the presence or absence of a fluorescent dye (e.g., the membrane marker laurdan). In general, cells were less sensitive to TIR than to epi-illumination. However, comparably high light doses needed for repetitive excitation of single molecules limit the application of super-resolution microscopy to living cells.

Photoswitching of monomeric and dimeric DNA-intercalating cyanine dyes for super-resolution microscopy applications

A growing trend in far-field super resolution fluorescence microscopy based on single molecule photoswitching involves the replacement of photoactivatable fluorophores by common organic dyes in which photoswitching reactions or blinking can be induced. This alternative strategy can greatly simplify the sample preparation and imaging scheme in some cases, and enables its application to a wider range of biological systems. This methodology has been applied successfully to unveil the nanoscale organisation of proteins, but little progress has been seen to date in DNA super-resolution imaging. Previous results have shown that blinking can be induced in the DNA-intercalating dimeric dye YOYO-1 in combination with a reducing buffer, and in turn super-resolution images of DNA can be reconstructed. However, monomeric intercalating dyes like YO-PRO-1 are more advantageous for biological applications. This paper shows that both YO-PRO-1 and YOYO-1 can be used in super-resolution imaging, and different sample preparation strategies are compared in terms of spatial resolution and homogeneity of the reconstructed super-resolution images. Moreover, ensemble and single-molecule experiments provide insight into the switching mechanism. The dyes YOYO-1 and YO-PRO-1 hold great potential for their use in nanoscale imaging of DNA topology in biology and nanoscience.

Structured Illumination and Image Inversion Interferometry

An overview of recent advances in high resolution fluorescence microscopy will be given.In structured illumination the sample is illuminated with a number of different patterns of light. In our case this is a series of sinusoidal grids at different grid positions and orientations generated by a programmable spatial light modulator or a physical phase grating. Experimental datasets acquired under these conditions and reconstructed results from these data, demonstrating a resolution improvement of up to a factor of two over standard widefield microscopy are presented.The non-linear approach of saturating optical transitions (for structured illumination as well as beam-scanning approaches) has a great potential especially in combination with photo-switchable dyes such as the recently described IrisFP protein from Ulrich Nienhaus’group or the Cy3-Alexa647 system used in Xiaowei Zhuang's group. An interesting approach is to push molecules into dark states in a patterned way shortly before imaging and exploiting the saturation of this transition.Finally a method will be presented in which the emitted fluorescence of a confocal microscope passes through two separate paths. These paths are interferometrically recombined in such a way that the images undergo a mutual rotation of 180 degrees. The self-interference of the fluorescent light is only constructive, if it originated from the optical axis of the scanning laser beam, thus leading to an efficient detection of a high resolution fluorescence images.K. Wicker, S. Sindbert, R. Heintzmann, Characterisation of resolution enhancing image inversion interferometers, Optics Express 17, 15491-15501, 2009L. Hirvonen, K. Wicker., O. Mandula and R. Heintzmann, Structured illumination microscopy of a living cell, Europ. Biophys. J. 38, 807-812, 2009

Quantitative molecular imaging of atherosclerotic endothelial dysfunction with perfluorocarbon (19F) nanoparticle magnetic resonance imaging and spectroscopy

Methods Five NZW rabbits were fed a high fat diet for 9-12 months (cholesterol: 1200-1700 mg/dL). Fluorescently-labeled, nontargeted NP were injected (2 ml/kg) intravenously into rabbit ear vein. After circulation in vivo for 1, 6 or 24 hours, aortas were excised for 19F MRI and spectroscopy (Varian 11.7 T scanner); and whole mount fluorescence imaging (Xenogen IVIS system). Two human carotid endarterectomy tissues were collected from operation room. After pretreatment with plasmin to digest fibrin on the endothelial surface and incubation with nontargeted NP for 6 hours, tissues were rinsed and formalin fixed for 19F MRI and spectroscopy. A perfluorooctyl bromide standard enabled MRS-based quantification of NP concentration in each imaged voxel. Results In rabbit aortas, MRI (19F/1H overlay) revealed abundant 19F signal from intact NP that were localized heterogeneously in the plaque interstitium (Fig. 1A) but not in unaffected areas. The average tissue concentration of NP calculated from MR spectroscopy (Fig. 1B) was 2.36 ± 0.42 × 109 /g aorta. The accumulation of NP is distinct from macrophage uptake, as demonstrated by high resolution fluorescence microscopy (Fig. 1C). Fluorescence imaging (Fig. 1D) also confirmed the presence of NP. In human carotid arterectomy tissues, the detected 19F signals were primary located on the endothelial/luminal side (Fig. 2). Quantitative analysis showed that average NP concentration in carotid arterectomy tissue was 47.1 ± 18.3 × 109 /g tissue.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Recent Developments in Super Resolution Fluorescence Microscopy

Recent Developments in Super Resolution Fluorescence Microscopy

Quantum Dot Triexciton Imaging with Three-Dimensional Subdiffraction Resolution

We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.

Correlation of 4Pi and Electron Microscopy to Study Transport Through Single Golgi Stacks in Living Cells with Super Resolution

Two problems have hampered the use of light microscopy for structural studies of cellular organelles for a long time: the limited resolution and the difficulty of obtaining true structural boundaries from complex intensity curves. The advent of modern high-resolution light microscopy techniques and their combination with objective image segmentation now provide us with the means to bridge the gap between light and electronmicroscopy in cell biology applications. In this study, we provide the first comparative correlative analysis of three-dimensional structures obtained by 4Pi microscopy and segmented by a zero-crossing procedure with those of transmission electron microscopy (TEM). The distribution within the cisternae of isolated Golgi stacks of the cargo protein procollagen 3 was mapped by both 4Pi microscopy and TEM for a detailed comparative analysis of their imaging capabilities. A high correlation was seen for the structures, indicating the particular accuracy of the 4Pi microscopy. Furthermore, for the first time, transport of a cargo molecule (vesicular stomatitis virus G protein-pEGFP) through individual Golgi stacks (labeled by galactosyl transferase-venus-YFP) was visualized by 4Pi microscopy. Following the procedures validated by the correlative analysis, our transport experiments show that (i) VSVG-pEGFP rapidly enter/exit individual Golgi stacks, (ii) VSVG-pEGFP never fills the GalT-venusYFP compartments completely and (iii) the GalT-venusYFP compartment volume increases upon VSVG-pEGFP arrival. This morphological evidence supports some previous TEM-based observations of intra-Golgi transport of VSVG-pEGFP and provides new insights toward a better understanding of protein progression across Golgi stacks. Our study thus demonstrates the general applicability of super resolution fluorescence microscopy, coupled with the zero-crossing segmentation procedure, for structural studies of suborganelle protein distributions under living cell conditions.

Actin Cytoskeleton Controls Movement Of Intracellular Organelles In Epithelia

Background: Intracellular organelles move along microtubules and filamentous actin (F-actin) using molecular motors. Previously we reported that movement of secretory granules was hindered by Ca2+-dependent formation of a dense cytoplasmic F-actin meshwork in pancreatic duct epithelial cells (PDEC). Aim: Here we addressed whether other organelles such as mitochondria and lysosomes also are controlled by the same mechanism. Methods: Mitochondria and lysosomes were labeled with fluorescent MitoTracker Red and LysoTracker Red, respectively. Their movements were monitored every 1.2 s with high resolution fluorescence microscopy and measured by comparing subsequent images. Cells were stimulated with UTP to activate an endogenous P2Y2 G-protein coupled receptor to increase intracellular Ca2+. Results: Mitochondria and lysosomes moving actively at rest stopped rapidly (τ ∼ 2.6 s) after Ca2+ rise. This organelle ‘freezing’ was accompanied by the formation of F-actin in the whole cytoplasm as stained with phalloidin-Alexa 488. EGFP-actin binding domain 2 (ABD2) expressed in PDEC, indicated a rapid formation of cytoplasmic F-actin (τ ∼ 7.8 s). In addition, the freezing of the organelles was blocked by latrunculin B, an inhibitor of F-actin formation. Conclusion: Ca2+-dependent formation of a fine F-actin meshwork reduces the movement of intracellular organelles physically in PDEC. The freezing mechanism controls exocytosis of secretory granules and may affect local ATP supply by controlling mitochondrial distribution in PDEC.Supported by UW Royalty Research Fund and an NIH grant (GM083913).