Oxygen concentration distributions in biological systems can be imaged by the phosphorescence quenching method in combination with two-photon laser scanning microscopy. In this paper we identified the excitation regime in which the signal of a two-photon-enhanced phosphorescent probe1 is dependent quadratically on the excitation power (quadratic regime), and performed simulations that relate the photophysical properties of the probe to the imaging resolution. Further, we characterized polymersomes as a method of probe encapsulation and delivery. Photo-physical and oxygen sensing properties of the probe were found unchanged when the probe is encapsulated in polymersomes. Polymersomes were found capable of sustaining high probe concentrations, thereby serving to improve the signal-to-noise ratios for oxygen detection compared to the previously employed probe delivery methods. Imaging of polymersomes loaded with the probe was used as a test-bed for a new method.
phosphorescence; oxygen; two-photon microscopy; polymersome; dendrimer; porphyrin
Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis.
tissue scaffold; tissue engineering; scanning probe recognition microscopy; regenerative medicine; image processing
Atomic Force Microscopy (AFM) can be used to obtain high-resolution topographical images of bacteria revealing surface details and cell integrity. During scanning however, the interactions between the AFM probe and the membrane results in distortion of the images. Such distortions or artifacts are the result of geometrical effects related to bacterial cell height, specimen curvature and the AFM probe geometry. The most common artifact in imaging is surface broadening, what can lead to errors in bacterial sizing. Several methods of correction have been proposed to compensate for these artifacts and in this study we describe a simple geometric model for the interaction between the tip (a pyramidal shaped AFM probe) and the bacterium (Escherichia coli JM-109 strain) to minimize the enlarging effect. Approaches to bacteria immobilization and examples of AFM images analysis are also described.
Atomic force microscopy (AFM); Escherichia coli; cell dimensions; bacteria visualization
Semiconducting CrSi2 nanocrystallites (NCs) were grown by reactive deposition epitaxy of Cr onto n-type silicon and covered with a 50-nm epitaxial silicon cap. Two types of samples were investigated: in one of them, the NCs were localized near the deposition depth, and in the other they migrated near the surface. The electrical characteristics were investigated in Schottky junctions by current-voltage and capacitance-voltage measurements. Atomic force microscopy (AFM), conductive AFM and scanning probe capacitance microscopy (SCM) were applied to reveal morphology and local electrical properties. The scanning probe methods yielded specific information, and tapping-mode AFM has shown up to 13-nm-high large-area protrusions not seen in the contact-mode AFM. The electrical interaction of the vibrating scanning tip results in virtual deformation of the surface. SCM has revealed NCs deep below the surface not seen by AFM. The electrically active probe yielded significantly better spatial resolution than AFM. The conductive AFM measurements have shown that the Cr-related point defects near the surface are responsible for the leakage of the macroscopic Schottky junctions, and also that NCs near the surface are sensitive to the mechanical and electrical stress induced by the scanning probe.
Correlative microscopy is a sophisticated approach that combines the capabilities of typically separate, but powerful microscopy platforms: often including, but not limited, to conventional light, confocal and super-resolution microscopy, atomic force microscopy, transmission and scanning electron microscopy, magnetic resonance imaging and micro/nanoCT (computed tomography). When targeting rare or specific events within large populations or tissues, correlative microscopy is increasingly being recognized as the method of choice. Furthermore, this multi-modal assimilation of technologies provides complementary and often unique information, such as internal and external spatial, structural, biochemical and biophysical details from the same targeted sample. The development of a continuous stream of cutting-edge applications, probes, preparation methodologies, hardware and software developments will enable realization of the full potential of correlative microscopy.
Lateral profiles of the electron probe of scanning transmission electron microscopy (STEM) were simulated at different vertical positions in a micrometers-thick carbon sample. The simulations were carried out using the Monte Carlo method in the CASINO software. A model was developed to fit the probe profiles. The model consisted of the sum of a Gaussian function describing the central peak of the profile, and two exponential decay functions describing the tail of the profile. Calculations were performed to investigate the fraction of unscattered electrons as function of the vertical position of the probe in the sample. Line scans were also simulated over gold nanoparticles at the bottom of a carbon film to calculate the achievable resolution as function of the sample thickness and the number of electrons. The resolution was shown to be noise limited for film thicknesses less than 1 μm. Probe broadening limited the resolution for thicker films. The validity of the simulation method was verified by comparing simulated data with experimental data. The simulation method can be used as quantitative method to predict STEM performance or to interpret STEM images of thick specimens.
Electron probe broadening; Monte Carlo simulations; nanoparticles; noise-limited resolution; STEM; spatial resolution; thick specimen
We report a simple technique for mapping Electrostatic Force Microscopy (EFM) bias sweep data into 2D images. The method allows simultaneous probing, in the same scanning area, of the contact potential difference and the second derivative of the capacitance between tip and sample, along with the height information. The only required equipment consists of a microscope with lift-mode EFM capable of phase shift detection. We designate this approach as Scanning Probe Potential Electrostatic Force Microscopy (SPP-EFM). An open-source MATLAB Graphical User Interface (GUI) for images acquisition, processing and analysis has been developed. The technique is tested with Indium Tin Oxide (ITO) and with poly(3-hexylthiophene) (P3HT) nanowires for organic transistor applications.
The Chemistry Department at the University of Nebraska – Lincoln (UNL) is located in Hamilton Hall on the main campus of UNL in Lincoln, NE, USA. This department houses the primary graduate and research program in chemistry in the state of Nebraska. This program includes the traditional fields of analytical chemistry, biochemistry, inorganic chemistry, organic chemistry and physical chemistry. However, this program also contains a great deal of multidisciplinary research in fields that range from bioanalytical and biophysical chemistry to nanomaterials, energy research, catalysis and computational chemistry. Current research in bioanalytical and biophysical chemistry at UNL includes work with separation methods such as HPLC and CE, as well as with techniques such as MS and LC–MS, NMR spectroscopy, electrochemical biosensors, scanning probe microscopy and laser spectroscopy. This article will discuss several of these areas, with an emphasis being placed on research in bioanalytical separations, binding assays and related fields.
The important role that surface chemical analysis methods can and should play in the characterization of nanoparticles is described. The types of information that can be obtained from analysis of nanoparticles using Auger electron spectroscopy (AES); X-ray photoelectron spectroscopy (XPS); time of flight secondary ion mass spectrometry (TOF-SIMS); low energy ion scattering (LEIS); and scanning probe microscopy (SPM), including scanning tunneling microscopy (STM) and atomic force microscopy (AFM), are briefly summarized. Examples describing the characterization of engineered nanoparticles are provided. Specific analysis considerations and issues associated with using surface analysis methods for the characterization of nanoparticles are discussed and summarized, along with the impact that shape instability, environmentally induced changes, deliberate and accidental coating, etc., have on nanoparticle properties.
The study discussed the synthesis of silica sol using the sol-gel method, doped with two different amounts of Cu nanoparticles. Cotton fabric samples were impregnated by the prepared sols and then dried and cured. To block hydroxyl groups, some samples were also treated with hexadecyltrimethoxysilane. The average particle size of colloidal silica nanoparticles were measured by the particle size analyzer. The morphology, roughness, and hydrophobic properties of the surface fabricated on cotton samples were analyzed and compared via the scanning electron microscopy, the transmission electron microscopy, the scanning probe microscopy, with static water contact angle (SWC), and water shedding angle measurements. Furthermore, the antibacterial efficiency of samples was quantitatively evaluated using AATCC 100 method. The addition of 0.5% (wt/wt) Cu into silica sol caused the silica nanoparticles to agglomerate in more grape-like clusters on cotton fabrics. Such fabricated surface revealed the highest value of SWC (155° for a 10-μl droplet) due to air trapping capability of its inclined structure. However, the presence of higher amounts of Cu nanoparticles (2% wt/wt) in silica sol resulted in the most slippery smooth surface on cotton fabrics. All fabricated surfaces containing Cu nanoparticles showed the perfect antibacterial activity against both of gram-negative and gram-positive bacteria.
cotton; superhydrophobicity; antibacterial; sol-gel method; contact angle
Cell volume determination plays a pivotal role in the investigation of the biophysical mechanisms underlying various cellular processes. Whereas light microscopy in principle enables one to obtain three dimensional data, the reconstruction of cell volume from z-stacks is a time consuming procedure. Thus, three dimensional topographic representations of cells are easier to obtain by scanning probe microscopical measurements.
We present a method of separating the cell soma volume of bipolar cells in adherent cell cultures from the contributions of the cell processes from data obtained by scanning ion conductance microscopy. Soma volume changes between successive scans obtained from the same cell can then be computed even if the cell is changing its position within the observed area. We demonstrate that the estimation of the cell volume on the basis of the width and the length of a cell may lead to erroneous determination of cell volume changes.
We provide a new algorithm to repeatedly determine single cell soma volume and thus to quantify cell volume changes during cell movements occuring over a time range of hours.
Scanning X-ray microscopy focuses radiation to a small spot and probes the sample by raster scanning. It allows information to be obtained from secondary signals such as X-ray fluorescence, which yields an elemental mapping of the sample not available in full-field imaging. The analysis and interpretation from these secondary signals can be considerably enhanced if these data are coupled with structural information from transmission imaging. However, absorption often is negligible and phase contrast has not been easily available. Originally introduced with visible light, Zernike phase contrast1 is a well-established technique in full-field X-ray microscopes for visualization of weakly absorbing samples2–7. On the basis of reciprocity, we demonstrate the implementation of Zernike phase contrast in scanning X-ray microscopy, revealing structural detail simultaneously with hard-X-ray trace-element measurements. The method is straightforward to implement without significant influence on the resolution of the fluorescence images and delivers complementary information. We show images of biological specimens that clearly demonstrate the advantage of correlating morphology with elemental information.
The present study introduces a method for determining the labile iron pool (LIP) in human lymphocytes. It is measured using the probe CP655, the fluorescence of which is stoichiometrically quenched by the addition of iron. The intracellular CP655 fluorescence in lymphocytes was quenched by increasing intracellular iron concentrations using the highly lipophilic 8-hydroxyquinoline iron complex. Intracellular fluorescence quenching, mediated by the physiological intracellular labile iron, can be recovered on the addition of excess membrane-permeable iron chelator, CP94. The intracellular probe concentration was measured using laser scanning microscopy. An ex situ calibration was performed in a “cytosolic” medium based on the determined intracellular CP655 concentration and probe fluorescence quenching in the presence of iron. The concentration of the LIP of healthy human lymphocytes was determined to be 0.57 ± 0.27 μM. The use of the fluorescent probe CP655 renders it possible to record the time course of iron uptake and iron chelation by CP94 in single intact lymphocytes.
lymphocytes; intracellular chelatable iron; fluorescence microscopy; fluorescent probe
Self-assembled iron-silicide nanostructures were prepared by reactive deposition epitaxy of Fe onto silicon. Capacitance-voltage, current-voltage, and deep level transient spectroscopy (DLTS) were used to measure the electrical properties of Au/silicon Schottky junctions. Spreading resistance and scanning probe capacitance microscopy (SCM) were applied to measure local electrical properties. Using a preamplifier the sensitivity of DLTS was increased satisfactorily to measure transients of the scanning tip semiconductor junction. In the Fe-deposited area, Fe-related defects dominate the surface layer in about 0.5 μm depth. These defects deteriorated the Schottky junction characteristic. Outside the Fe-deposited area, Fe-related defect concentration was identified in a thin layer near the surface. The defect transients in this area were measured both in macroscopic Schottky junctions and by scanning tip DLTS and were detected by bias modulation frequency dependence in SCM.
There is a clear need for new approaches in the field of microbial community analyses, since the methods used can be severely biased. We have developed a DNA array-based method that targets16S ribosomal DNA (rDNA), enabling the direct detection and quantification of microorganisms from complex communities without cultivation. The approach is based on the construction of specific probes from the 16S rDNA sequence data retrieved directly from the communities. The specificity of the assay is obtained through a combination of DNA array hybridization and enzymatic labeling of the constructed probes. Cultivation-dependent assays (enrichment and plating) and cultivation-independent assays (direct fluorescence microscopy and scanning electron microscopy) were used as reference methods in the development and evaluation of the method. The description of microbial communities in ready-to-eat vegetable salads in a modified atmosphere was used as the experimental model. Comparisons were made with respect to the effect of storage at different temperatures for up to 12 days and with respect to the geographic origin of the crisphead lettuce (Spanish or Norwegian), the main salad component. The conclusion drawn from the method comparison was that the DNA array-based method gave an accurate description of the microbial communities. Pseudomonas spp. dominated both of the salad batches, containing either Norwegian or Spanish lettuce, before storage and after storage at 4°C. The Pseudomonas population also dominated the batch containing Norwegian lettuce after storage at 10°C. On the contrary, Enterobacteriaceae and lactic acid bacteria dominated the microbial community of the batch containing Spanish lettuce after storage at 10°C. In that batch, the Enterobacteriaceae also were abundant after storage at 4°C as well as before storage. The practical implications of these results are that microbial communities in ready-to-eat vegetable salads can be diverse and that microbial composition is dependent both on the origin of the raw material and on the storage conditions.
The trajectories of differently shaped nanoparticles manipulated by atomic force microscopy are related to the scan path of the probing tip. The direction of motion of the nanoparticles is essentially fixed by the distance b between consecutive scan lines. Well-defined formulas are obtained in the case of rigid nanospheres and nanowires. Numeric results are provided for symmetric nanostars. As a result, orienting the fast scan direction perpendicular to the desired direction of motion and reducing b well below the linear size of the particles turns out to be an efficient way to control the nanomanipulation process.
atomic force microscopy; nanomanipulation; nanoparticles
Micro-cantilever arrays with different dimensions are fabricated by micromachining technique onto silicon <1 0 0> substrate. These sputtered Gold-Coated micro-cantilevers were later surface functionalized. Scanning Electron Microscopy, Atomic Force Microscopy and Optical SWLI using LASER probe are employed to characterize the morphology and image measurement of the micro-cantilever arrays, respectively. Compared with conventional AFM and SPM measurement technique, the proposed method has demonstrated sufficient flexibility and reliability. The experimental results have been analyzed and presented in this paper for MEMS Micro-cantilevers. The scanning White Light Interferometry based two point high resolution optical method is presented for characterizing Micro-cantilevers and other MEMS micro-structures. The repeatable error and the repeatable precision produced in the proposed image measurement method is nanometre confirmable. In this piece of work, we investigate the micro-structure fabrication and image measurement of Length, Width and Step-Height of micro-cantilever arrays fabricated using bulk micromachining technique onto Silicon <100> substrate.
Scanning Electron Microscopy; Atomic Force Microscopy; Micro-cantilever; Optics; Image Measurement; Silicon (100), Scanning White Light Interferometry
The human large intestine is covered with a protective mucus coating, which is heavily colonized by complex bacterial populations that are distinct from those in the gut lumen. Little is known of the composition and metabolic activities of these biofilms, although they are likely to play an important role in mucus breakdown. The aims of this study were to determine how intestinal bacteria colonize mucus and to study physiologic and enzymatic factors involved in the destruction of this glycoprotein. Colonization of mucin gels by fecal bacteria was studied in vitro, using a two-stage continuous culture system, simulating conditions of nutrient availability and limitation characteristic of the proximal (vessel 1) and distal (vessel 2) colon. The establishment of bacterial communities in mucin gels was investigated by selective culture methods, scanning electron microscopy, and confocal laser scanning microscopy, in association with fluorescently labeled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin-degrading enzymes and measurements of residual mucin sugars. Mucin gels were rapidly colonized by heterogeneous bacterial populations, especially members of the Bacteroides fragilis group, enterobacteria, and clostridia. Intestinal bacterial populations growing on mucin surfaces were shown to be phylogenetically and metabolically distinct from their planktonic counterparts.
The most valuable property of stem cells (SCs) is their potential to differentiate into many or all cell types of the body. So far, monitoring SC differentiation has only been possible after cells were fixed or destroyed during sample preparation. It is, however, important to develop nondestructive methods of monitoring SCs. Scanning ion conductance microscopy (SICM) is a unique imaging technique that uses similar principles to the atomic force microscope, but with a pipette for the probe. This allows scanning of the surface of living cells noninvasively and enables measurement of cellular activities under more physiological conditions than is possible with other high-resolution microscopy techniques. We report here the novel use of the SICM for studying SCs to assess and monitor the status of SCs and various cell types differentiated from SCs.
Exposure of cryptic, functional sites on fibrinogen upon its adsorption to hydrophobic surfaces of biomaterials have been linked to inflammatory response and fibrosis. Such adsorption also induces ordered fibrinogen aggregation which is poorly understood.
To investigate hydrophobic surface-induced fibrinogen aggregation. Methods: Contact and lateral force scanning probe microscopy, yielding topography, image dimensions, and fiber elastic modulus measurements were used along with transmission and scanning electron microscopy. Fibrinogen aggregation was induced under non-enzymatic conditions by adsorption on a trioctyl-surface monolayer (trioctylmethylamine) grafted onto silica clay plates.
A more than one molecule thick coating was generated by adsorption on the plate from 100–200 μg/ml fibrinogen solutions, and three-dimensional networks formed from 4 mg/ml fibrinogen incubated with uncoated or fibrinogen-coated plates. Fibrils appeared laterally assembled into branching and overlapping fibers whose heights from surface ranged from ~3 to 740 nm. The elastic modulus of fibrinogen fibers was 1.55 MPa. No fibrils formed when fibrinogen lacking αC-domains was used as coating or was incubated with intact fibrinogen-coated plates, or when the latter plates were sequentially incubated with anti-Aα529–539 mAb and intact fibrinogen. When an anti-Aα241–476 mAb was used instead, fine, long fibers formed. Similarly, sequential incubations of fibrinogen-coated plates with recombinant αC-domain (Aα392–610 fragment) or αC-connector (Aα221–372 fragment) and fibrinogen resulted in distinctly fine fiber networks.
Adsorption-induced fibrinogen self-assembly is initiated by a more than one molecule-thick surface layer and eventuates in three-dimensional networks whose formation requires fibrinogen with intact αC-domains.
Fibrinogen; Fibrinogen self-assembly; Fibrinogen adsorption; Fibrinogen fibers; Fibrinogen αC; Fibrinogen fiber stiffness
Super-resolution optical microscopy is opening a new window to unveil the unseen details on the nanoscopic scale. Current far-field super-resolution techniques rely on fluorescence as the read-out1–5. Here, we demonstrate a scheme for breaking the diffraction limit in far-field imaging of non-fluorescent species by using spatially controlled saturation of electronic absorption. Our method is based on a pump-probe process where a modulated pump field perturbs the charge-carrier density in a sample, thus modulating the transmission of a probe field. A doughnut shape laser beam is then added to transiently saturate the electronic transition in the periphery of the focal volume, thus the induced modulation in the sequential probe pulse only occurs at the focal center. By raster scanning the three collinearly aligned beams, high-speed sub-diffraction-limited imaging of graphite nano-platelets was performed. This technique potentially enables super-resolution imaging of nano-materials and non-fluorescent chromophores, which may remain out of reach for fluorescence-based methods.
Thrombus and secondary thrombosis plays a key role in stroke. Recent molecular imaging provides in vivo imaging of activated factor XIII (FXIIIa), an important mediator of thrombosis or fibrinolytic resistance. The present study was to investigate the fibrin deposition in a thromboembolic stroke mice model by FXIIIa–targeted near-infrared fluorescence (NIRF) imaging.
Materials and Methods
The experimental protocol was approved by our institutional animal use committee. Seventy-six C57B/6J mice were subjected to thromboembolic middle cerebral artery occlusion or sham operation. Mice were either intravenously injected with the FXIIIa-targeted probe or control probe. In vivo and ex vivo NIRF imaging were performed thereafter. Probe distribution was assessed with fluorescence microscopy by spectral imaging and quantification system. MR scans were performed to measure lesion volumes in vivo, which were correlated with histology after animal euthanasia.
In vivo significant higher fluorescence intensity over the ischemia-affected hemisphere, compared to the contralateral side, was detected in mice that received FXIIIa-targeted probe, but not in the controlled mice. Significantly NIRF signals showed time-dependent processes from 8 to 96 hours after injection of FXIIIa-targeted probes. Ex vivo NIRF image showed an intense fluorescence within the ischemic territory only in mice injected with FXIIIa-targeted probe. The fluorescence microscopy demonstrated distribution of FXIIIa-targeted probe in the ischemic region and nearby micro-vessels, and FXIIIa-targeted probe signals showed good overlap with immune-fluorescent fibrin staining images. There was a significant correlation between total targeted signal from in vivo or ex vivo NIRF images and lesion volume.
Non-invasive detection of fibrin deposition in ischemic mouse brain using NIRF imaging is feasible and this technique may provide an in vivo experimental tool in studying the role of fibrin in stroke.
The concentration of salt in the thin layer of fluid at the surface of large airways, the airway-surface liquid (ASL), is believed to be of central importance in airway physiology and in the pathophysiology of cystic fibrosis. Invasive sampling methods have yielded a wide range of ASL [NaCl] from 40 to 180 mM. We have developed novel fluorescent probes and microscopy methods to measure ASL thickness, salt concentration, and pH quantitatively in cell-culture models and in the trachea in vivo. By rapid z-scanning confocal microscopy, ASL thickness was 21 ± 4 μm in well-differentiated cultures of bovine tracheal epithelial cells grown on porous supports at an air-liquid interface. By ratio imaging fluorescence microscopy using sodium, chloride, and pH-sensitive fluorescent indicators, ASL [Na+] was 97 ± 5 mM, [Cl–] was 118 ± 3 mM, and pH was 6.94 ± 0.03. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 45 ± 5 μm, [Na+] was 115 ± 4 mM, [Cl–] was 140 ± 5 mM, and pH was 6.95 ± 0.05. Similar ASL tonicity and pH were found in cystic fibrosis (CFTR-null) mice. In freshly harvested human bronchi, ASL thickness was 55 ± 5 μm, [Na+] was 103 ± 3 mM, [Cl–] was 92 ± 4 mM, and pH was 6.78 ± 0.2. These results establish by a noninvasive approach the key properties of the ASL and provide direct evidence that the ASL is approximately isotonic and not saltier in cystic fibrosis.
Full-field optical coherence microscopy (FFOCM) utilizes coherence gating to obtain high-resolution optical sections in thick tissues. FFOCM is an attractive technology for endoscopic microscopy at the cellular level since it does not require a high NA objective lens or beam scanning and is therefore particularly amenable to miniaturization. In this manuscript, we present a novel scheme for conducting FFOCM that utilizes spectrally modulated, spatially incoherent illumination and a static Linnik interferometer. This approach is advantageous for endoscopic microscopy since it allows FFOCM to be conducted through a single multimode fiber optic imaging bundle and does not require moving parts in the endoscope probe. Images acquired from biological samples in free space demonstrate that this new method provides the same detailed microscopic structure as that of conventional FFOCM. High-resolution images were also obtained through a multimode fiber bundle, further supporting the potential of this method for endoscopic microscopy.
Nanoparticles are often measured using atomic force microscopy or other scanning probe microscopy methods. For isolated nanoparticles on flat substrates, this is a relatively easy task. However, in real situations, we often need to analyze nanoparticles on rough substrates or nanoparticles that are not isolated. In this article, we present a simple model for realistic simulations of nanoparticle deposition and we employ this model for modeling nanoparticles on rough substrates. Different modeling conditions (coverage, relaxation after deposition) and convolution with different tip shapes are used to obtain a wide spectrum of virtual AFM nanoparticle images similar to those known from practice. Statistical parameters of nanoparticles are then analyzed using different data processing algorithms in order to show their systematic errors and to estimate uncertainties for atomic force microscopy analysis of nanoparticles under non-ideal conditions. It is shown that the elimination of user influence on the data processing algorithm is a key step for obtaining accurate results while analyzing nanoparticles measured in non-ideal conditions.