Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.     

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.     

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.     

Sub-Diffraction Nano Manipulation Using STED AFM

In the last two decades, nano manipulation has been recognized as a potential tool of scientific interest especially in nanotechnology and nano-robotics. Contemporary optical microscopy (super resolution) techniques have also reached the nanometer scale resolution to visualize this and hence a combination of super resolution aided nano manipulation ineluctably gives a new perspective to the scenario. Here we demonstrate how specificity and rapid determination of structures provided by stimulated emission depletion (STED) microscope can aid another microscopic tool with capability of mechanical manoeuvring, like an atomic force microscope (AFM) to get topological information or to target nano scaled materials. We also give proof of principle on how high-resolution real time visualization can improve nano manipulation capability within a dense sample, and how STED-AFM is an optimal combination for this job. With these evidences, this article points to future precise nano dissections and maybe even to a nano-snooker game with an AFM tip and fluorospheres.     

A Highlights from MBoC Selection: Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro­tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.     

Concepts in imaging and microscopy. Exploring biological structure and function with confocal microscopy

Confocal microscopy is providing new and exciting opportunities for imaging cell structure and physiology in thick biological specimens, in three dimensions, and in time. The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus providing sharp, high-contrast images of cells and subcellular structures within thick samples. Computer controlled focusing and image-capturing features allow for the collection of through-focus series of optical sections that may be used to reconstruct a volume of tissue, yielding information on the 3-D structure and relationships of cells. Tissues and cells may also be imaged in two or three spatial dimensions over time. The resultant digital data, which encode the image, are highly amenable to processing, manipulation and quantitative analyses. In conjunction with a growing variety of vital fluorescent probes, confocal microscopy is yielding new information about the spatiotemporal dynamics of cell morphology and physiology in living tissues and organisms. Here we use mammalian brain tissue to illustrate some of the ways in which multidimensional confocal fluorescence imaging can enhance studies of biological structure and function.     

Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample.     

Localization and high-resolution imaging of cortical neurotransmitter compartments using confocal laser scanning microscopy: GABA and glutamate interactions in rat cortex.

This report compares the application of confocal laser scanning fluorescence microscopy with standard epifluorescence microscopy for the simultaneous localization of the neurotransmitters gamma-aminobutyric acid and glutamate in rat cerebral cortex. With this approach, sections of fixed rat brain are treated with primary antibodies against gamma-aminobutyric acid (rabbit-derived) and glutamate (mouse-derived), followed by treatment with fluorescein isothiocyanate-tagged donkey anti-rabbit and rhodamine-tagged goat anti-mouse secondary antibodies, respectively. The results demonstrate that images from immunofluorescence localizations with a confocal laser scanning microscope have superior resolution and contrast as a result of significant reductions of background flare caused by emission from out-of-focus structures in the field of view. The confocal microscope achieves this improved image quality by optically sectioning through a specimen at narrow planes of focus and then compiling a composite image of an object of interest. The composite image can be further enhanced by using various image processing options. The combined use of double immunofluorescence and confocal laser scanning microscopy provides an important means to simultaneously study the anatomical relationships of pre- and post-synaptic elements in a complex neural system.     

Magnetic force microscopy of iron oxide nanoparticles and their cellular uptake

Magnetic force microscopy has the capability to detect magnetic domains from a close distance, which can provide the magnetic force gradient image of the scanned samples and also simultaneously obtain atomic force microscope (AFM) topography image as well as AFM phase image. In this work, we demonstrate the use of magnetic force microscopy together with AFM topography and phase imaging for the characterization of magnetic iron oxide nanoparticles and their cellular uptake behavior with the MCF7 carcinoma breast epithelial cells. This method can provide useful information such as the magnetic responses of nanoparticles, nanoparticle spatial localization, cell morphology, and cell surface domains at the same time for better understanding magnetic nanoparticle‐cell interaction. It would help to design magnetic‐related new imaging, diagnostic and therapeutic methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Conjugation of both on-axis and off-axis light in Nipkow disk confocal microscope to increase availability of incoherent light source

Laser-scanning confocal microscopy has been employed for exploring structures at subcellular, cellular and tissue level in three dimensions. To acquire the confocal image, a coherent light source, such as laser, is generally required in conventional single-point scanning microscopy. The illuminating beam must be focused onto a small spot with diffraction-limited size, and this determines the spatial resolution of the microscopy system. In contrast, multipoint scanning confocal microscopy using a Nipkow disk enables the use of an incoherent light source. We previously demonstrated successful application of a 100 W mercury arc lamp as a light source for the Yokogawa confocal scanner unit in which a microlens array was coupled with a Nipkow disk to focus the collimated incident light onto a pinhole (Saito et al., Cell Struct. Funct., 33: 133-141, 2008). However, transmission efficiency of incident light through the pinhole array was low because off-axis light, the major component of the incident light, was blocked by the non-aperture area of the disk. To improve transmission efficiency, we propose an optical system in which off-axis light is able to be transmitted through pinholes surrounding the pinhole located on the optical axis of the collimator lens. This optical system facilitates the use of not only the on-axis but also the off-axis light such that the available incident light is considerably improved. As a result, we apply the proposed system to high-speed confocal and multicolor imaging both with a satisfactory signal-to-noise ratio.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Atomic Force Microscopy of Biological Membranes

Atomic force microscopy (AFM) is an ideal method to study the surface topography of biological membranes. It allows membranes that are adsorbed to flat solid supports to be raster-scanned in physiological solutions with an atomically sharp tip. Therefore, AFM is capable of observing biological molecular machines at work. In addition, the tip can be tethered to the end of a single membrane protein, and forces acting on the tip upon its retraction indicate barriers that occur during the process of protein unfolding. Here we discuss the fundamental limitations of AFM determined by the properties of cantilevers, present aspects of sample preparation, and review results achieved on reconstituted and native biological membranes.     

Optical sectioning microscopy

Confocal scanning microscopy, a form of optical sectioning microscopy, has radically transformed optical imaging in biology. These devices provide a powerful means to eliminate from images the background caused by out-of-focus light and scatter. Confocal techniques can also improve the resolution of a light microscope image beyond what is achievable with widefield fluorescence microscopy. The quality of the images obtained, however, depends on the user's familiarity with the optical and fluorescence concepts that underlie this approach. We describe the core concepts of confocal microscopes and important variables that adversely affect confocal images. We also discuss data-processing methods for confocal microscopy and computational optical sectioning techniques that can perform optical sectioning without a confocal microscope.     

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.     

Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS

Imaging, optical mapping, and optical multisite recording of transmembrane potential (V_m) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in V_m. Accordingly, they have been used for monitoring V_m in nonratioing and both emission and excitation ratioing modes. Their changes in fluorescence are usually much less than 10% per 100 mV. Conventional ratioing increases sensitivity to between 3 and 15% per 100 mV. Low sensitivity limits the value of these dyes, especially when imaged with low light systems like confocal scanners. Here we demonstrate the improvement afforded by shifted excitation and emission ratioing (SEER) as applied to imaging membrane potential in flexor digitorum brevis muscle fibers of adult mice. SEER—the ratioing of two images of fluorescence, obtained with different excitation wavelengths in different emission bands—was implemented in two commercial confocal systems. A conventional pinhole scanner, affording optimal setting of emission bands but less than ideal excitation wavelengths, achieved a sensitivity of up to 27% per 100 mV, nearly doubling the value found by conventional ratioing of the same data. A better pair of excitation lights should increase the sensitivity further, to 35% per 100 mV. The maximum acquisition rate with this system was 1 kHz. A fast “slit scanner” increased the effective rate to 8 kHz, but sensitivity was lower. In its high-sensitivity implementation, the technique demonstrated progressive deterioration of action potentials upon fatiguing tetani induced by stimulation patterns at >40 Hz, thereby identifying action potential decay as a contributor to fatigue onset. Using the fast implementation, we could image for the first time an action potential simultaneously at multiple locations along the t-tubule system. These images resolved the radially varying lag associated with propagation at a finite velocity.     

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.     

Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.     

In situ single-molecule imaging with attoliter detection using objective total internal reflection confocal microscopy

Confocal microscopy is widely used for acquiring high spatial resolution tissue sample images of interesting fluorescent molecules inside cells. The fluorescent molecules are often tagged proteins participating in a biological function. The high spatial resolution of confocal microscopy compared to wide field imaging comes from an ability to optically isolate and image exceedingly small volume elements made up of the lateral (focal plane) and depth dimensions. Confocal microscopy at the optical diffraction limit images volumes on the order of approximately 0.5 femtoliter (10(-15) L). Further resolution enhancement can be achieved with total internal reflection microscopy (TIRM). With TIRM, an exponentially decaying electromagnetic field (near-field) established on the surface of the sample defines a subdiffraction limit dimension that, when combined with conventional confocal microscopy, permits image formation from <7 attoL (10(-18) L) volumes [Borejdo et al. (2006) Biochim. Biophys. Acta, in press]. Demonstrated here is a new variation of TIRM, focused TIRM (fTIRM) that decreases the volume element to approximately 3 attoL. These estimates were verified experimentally by measuring characteristic times for Brownian motion of fluorescent nanospheres through the volume elements. A novel application for TIRM is in situ single-molecule fluorescence spectroscopy. Single-molecule studies of protein structure and function are well-known to avoid the ambiguities introduced by ensemble averaging. In situ, proteins are subjected to the native forces of the crowded environment in the cell that are not present in vitro. The attoL fluorescence detection volume of TIRM permits isolation of single proteins in situ. Muscle tissue contains myosin at a approximately 120 microM concentration. Evidence is provided that >75% of the bleachable fluorescence detected with fTIRM is emitted by five chromophore-labeled myosins in a muscle fiber.     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)