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Three-dimensional (3D) imaging and object tracking is critical for medical and biological research and can be achieved by multifocal imaging with diffractive optical elements (DOEs) converting depth (To precisely track microscopic fluorescent objects in biological systems in 3D with a simple low-cost DOE system.We designed a multiring spiral phase plate (SPP) generating a single-spot rotating point spread function (SS-RPSF) in a microscope. Our simple, analytically transparent design process uses Bessel beams to avoid rotational ambiguities and achieve a significant depth range. The SPP was inserted into the Nomarski prism slider of a standard microscope. Performance was evaluated using fluorescent beads and in live cells expressing a fluorescent chromatin marker.Bead localization precision wasPrecise 3D localization and tracking can be achieved with a SS-RPSF SPP in a standard microscope with minor modifications.

Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.

The study of radiation effects on biological tissues is a diverse field of research with direct applications to improve human health, in particular in the contexts of radiation therapy and space exploration. Understanding the DNA damage response following radiation exposure, which is a key determinant for mutagenesis, requires reproducible methods for delivering known doses of ionizing radiation (IR) in a controlled environment. Multiple IR sources, including research X-ray and gamma-ray irradiators are routinely used in basic and translational research with cell and animal models. These systems are however not ideal when a high temporal resolution is needed, for example to study early DNA damage responses with live cell microscopy. Here, we characterize the dose rate and beam properties of a commercial, miniature, affordable, and versatile X-ray source (Mini-X). We describe how to use Mini-X on the stage of a fluorescence microscope to deliver high IR dose rates (up to 29 Gy/min) or lower dose rates (≤ 0.1 Gy/min) in live cell imaging experiments. This article provides a blueprint for radiation biology applications with high temporal resolution, with a step-by-step guide to implement a miniature X-ray system on an imaging platform, and the information needed to characterize the system.

Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation. ispartof: ONCOGENE vol:38 issue:20 pages:3855-3870 ispartof: location:England status: published

Image-based particle tracking is an essential tool to answer research questions in cell biology and beyond. A major challenge of particle tracking in living systems is that low light exposure is required to avoid phototoxicity and photobleaching. In addition, high-speed imaging used to fully capture particle motion dictates fast image acquisition rates. Short exposure times come at the expense of tracking accuracy. This is generally true for quantitative microscopy approaches and particularly relevant to single molecule tracking where the number of photons emitted from a single chromophore is limited. Image restoration methods based on deep learning dramatically improve the signal-to-noise ratio in low-exposure datasets. However, it is not clear whether images generated by these methods yield accurate quantitative measurements such as diffusion parameters in (single) particle tracking experiments. Here, we evaluate the performance of two popular deep learning denoising software packages for particle tracking, using synthetic datasets and movies of diffusing chromatin as biological examples. With synthetic data, both supervised and unsupervised deep learning restored particle motions with high accuracy in two-dimensional datasets, whereas artifacts were introduced by the denoisers in 3D datasets. Experimentally, we found that, while both supervised and unsupervised approaches improved the number of trackable particles and tracking accuracy, supervised learning generally outperformed the unsupervised approach, as expected. We also highlight that with extremely noisy image sequences, deep learning algorithms produce deceiving artifacts, which underscores the need to carefully evaluate the results. Finally, we address the challenge of selecting hyper-parameters to train convolutional neural networks by implementing a frugal Bayesian optimizer that rapidly explores multidimensional parameter spaces, identifying networks yielding optional particle tracking accuracy. Our study provides quantitative outcome measures of image restoration using deep learning. We anticipate broad application of the approaches presented here to critically evaluate artificial intelligence solutions for quantitative microscopy.

Electrospun nanofibers manufactured from biocompatible materials are used in numerous bioengineering applications, such as tissue engineering, creating organoids or dressings, and drug delivery. In many of these applications, the morphological and mechanical properties of the single fiber affect their function. We used a combined atomic force microscope (AFM)/optical microscope technique to determine the mechanical properties of nanofibers that were electrospun from a 50:50 fibrinogen:PCL (poly-&epsilon, caprolactone) blend. Both of these materials are widely available and biocompatible. Fibers were spun onto a striated substrate with 6 &mu, m wide grooves, anchored with epoxy on the ridges and pulled with the AFM probe. The fibers showed significant strain softening, as the modulus decreased from an initial value of 1700 MPa (5&ndash, 10% strain) to 110 MPa (&gt, 40% strain). Despite this extreme strain softening, these fibers were very extensible, with a breaking strain of 100%. The fibers exhibited high energy loss (up to 70%) and strains larger than 5% permanently deformed the fibers. These fibers displayed the stress&ndash, strain curves of a ductile material. We provide a comparison of the mechanical properties of these blended fibers with other electrospun and natural nanofibers. This work expands a growing library of mechanically characterized, electrospun materials for biomedical applications.

The relationship between stress and alcohol-drinking behaviors has been intensively explored; however, neuronal substrates and neurotransmitter dynamics responsible for a causal link between these conditions are still unclear. Here, we optogenetically manipulated locus coeruleus (LC) norepinephrine (NE) activity by applying distinct stimulation protocols in order to explore how phasic and tonic NE release dynamics control alcohol-drinking behaviors. Our results clearly demonstrate contrasting behavioral consequences of LC-NE circuitry activation during low and high frequency stimulation. Specifically, applying tonic stimulation during a standard operant drinking session resulted in increased intake, while phasic stimulation decreased this measure. Furthermore, stimulation during extinction probe trials, when the lever press response was not reinforced, did not significantly alter alcohol-seeking behavior if a tonic pattern was applied. However, phasic stimulation substantially suppressed the number of lever presses, indicating decreased alcohol seeking under the same experimental condition. Given the well-established correlative link between stress and increased alcohol consumption, here we provide the first evidence that tonic LC-NE activity plays a causal role in stress-associated increases in drinking.

The epithelium forms a protective barrier against external biological, chemical and physical insults. So far, AFM-based, micro-mechanical measurements have only been performed on single cells and confluent cells, but not yet on cells in the physiologically relevant, mature epithelial layer.Using a combination of atomic force, fluorescence and confocal microscopy, we determined the changes in stiffness, morphology and actin distribution of human mammary epithelial cells (HMECs) as they transition from single cells to confluency to a mature epithelial layer.Single cells have a tall, round (planoconvex) morphology, have actin stress fibers at the base, have diffuse cortical actin, and have a stiffness of 1 kPa. Confluent cells become flatter, basal actin stress fibers start to disappear, and actin accumulates laterally where cells abut. Overall stiffness is still 1 kPa with two-fold higher stiffness in the abutting regions. Cells in an epithelial layer are flat on top and seven times stiffer (average, 7 kPa) than single and confluent cells. Epithelial layer cells show strong actin accumulation in the regions where cells adjoin and in the apical regions. Stiffness is significantly enhanced in the regions of adjoining cells, compared to the central regions of cells.Physiologically, this previously unrecognized, drastic stiffness increase may be important to the protective function of the epithelium.

Summary Despite many years of work on dopaminergic mechanisms of alcohol addiction, much of the evidence remains mostly correlative in nature. Fortunately, recent technological advances have provided the opportunity to explore the causal role of alterations in neurotransmission within circuits involved in addictive behaviors. Here, we address this critical gap in our knowledge by integrating an optogenetic approach and an operant alcohol self-administration paradigm to assess directly how accumbal dopamine (DA) release dynamics influences the appetitive (seeking) component of alcohol-drinking behavior. We show that appetitive reward-seeking behavior in rats trained to self-administer alcohol can be shaped causally by ventral tegmental area-nucleus accumbens (VTA-NAc) DA neurotransmission. Our findings reveal that phasic patterns of DA release within this circuit enhance a discrete measure of alcohol seeking, whereas tonic patterns of stimulation inhibit this behavior. Moreover, we provide mechanistic evidence that tonic-phasic interplay within the VTA-NAc DA circuit underlies these seemingly paradoxical effects., Graphical Abstract, Highlights • VTA-NAc DA transmission can bidirectionally modulate motivated behavior • Optogenetic increases in phasic DA release in the NAc enhance alcohol seeking • Optogenetic increases in tonic DA release in the NAc inhibit alcohol seeking • Phasic DA release can be decreased by the concurrent tonic activation, Animal Physiology; Neuroscience; Behavioral Neuroscience

Activity in the mesolimbic dopamine (DA) pathway is known to have a role in reward processing and related behaviors. The mesolimbic DA response to reward has been well-examined, while the response to aversive or negative stimuli has been studied to a lesser extent and produced inconclusive results. However, a brief increase in the DA concentration in terminals during nociceptive activation has become an established but not well-characterized phenomenon. Consequently, the interpretation of the significance of this neurochemical response is still elusive. The present study was designed to further explore these increases in subsecond DA dynamics triggered by negative stimuli using voltammetry in anesthetized rats. Our experiments revealed that repeated exposure to a tail pinch resulted in more efficacious DA release in rat nucleus accumbens. This fact may suggest a protective nature of immediate DA efflux. Furthermore, a sensitized DA response to a neutral stimulus, such as a touch, was discovered following several noxious pinches, while a touch applied before these pinches did not trigger DA release. Finally, it was found that the pinch-evoked DA efflux was significantly decreased by ethanol acutely administrated at an analgesic dose. Taken together, these results support the hypothesis that subsecond DA release in the nucleus accumbens may serve as an endogenous antinociceptive signal.

Recent optogenetic studies demonstrated that phasic dopamine release in the nucleus accumbens may play a causal role in multiple aspects of natural and drug reward-related behaviors. The role of tonic dopamine release in reward consummatory behavior remains unclear. The current study used a combinatorial viral-mediated gene delivery approach to express ChR2 on mesolimbic dopamine neurons in rats. We used optical activation of this dopamine circuit to mimic tonic dopamine release in the nucleus accumbens and to explore the causal relationship between this form of dopamine signaling within the ventral tegmental area (VTA)-nucleus accumbens projection and consumption of a natural reward. Using a two bottle choice paradigm (sucrose vs. water), the experiments revealed that tonic optogenetic stimulation of mesolimbic dopamine transmission significantly decreased reward consummatory behaviors. Specifically, there was a significant decrease in the number of bouts, licks and amount of sucrose obtained during the drinking session. Notably, activation of VTA dopamine cell bodies or dopamine terminals in the nucleus accumbens resulted in identical behavioral consequences. No changes in the water intake were evident under the same experimental conditions. Collectively, these data demonstrate that tonic optogenetic stimulation of VTA-nucleus accumbens dopamine release is sufficient to inhibit reward consummatory behavior, possibly by preventing this circuit from engaging in phasic activity that is thought to be essential for reward-based behaviors.

Due to its low cost, biocompatibility and slow bioresorption, poly-ε-caprolactone (PCL) continues to be a suitable material for select biomedical engineering applications. We used a combined atomic force microscopy (AFM)/optical microscopy technique to determine key mechanical properties of individual electrospun PCL nanofibers with diameters between 440–1040 nm. Compared to protein nanofibers, PCL nanofibers showed much lower adhesion, as they slipped on the substrate when mechanically manipulated. We, therefore, first developed a novel technique to anchor individual PCL nanofibers to micrometer-sized ridges on a substrate, and then mechanically tested anchored nanofibers. When held at constant strain, tensile stress relaxed with fast and slow relaxation times of 1.0 ± 0.3 s and 8.8 ± 3.1 s, respectively. The total tensile modulus was 62 ± 26 MPa, the elastic (non-relaxing) component of the tensile modulus was 53 ± 36 MPa. Individual PCL fibers could be stretched elastically (without permanent deformation) to strains of 19–23%. PCL nanofibers are rather extensible; they could be stretched to a strain of at least 98%, and a tensile strength of at least 12 MPa, before they slipped off the AFM tip. PCL nanofibers that had aged for over a month at ambient conditions became stiffer and less elastic. Our technique provides accurate nanofiber mechanical data, which are needed to guide construction of scaffolds for cells and other biomedical devices.

Background Adenosine serves many functions within the CNS, including inhibitory and excitatory control of neurotransmission. The understanding of adenosine dynamics in the brain is of fundamental importance. The goal of the present study was to explore subsecond adenosine fluctuations in the rat brain in vivo . Method Long Evans rats were anesthetized and a carbon fiber electrode was positioned in the motor cortex or dorsal striatum. Real time electrochemical recordings were made at the carbon fiber electrodes every 100 ms by applying a triangular waveform (−0.4 to +1.5 V, 400 V/s). Adenosine spikes were identified by the background-subtracted cyclic voltammogram. Results The frequency of detected adenosine spikes was relatively stable in both tested regions, and the time intervals between spikes were regular and lasted from 1 to 5 s within an animal. Spike frequency ranged from 0.5 to 1.5 Hz in both the motor cortex and the dorsal striatum. Average spike amplitudes were 85 ± 11 and 66 ± 7 nM for the motor cortex and the dorsal striatum, respectively. Comparison with existing methods The current study established that adenosine signaling can operate on a fast time scale (within seconds) to modulate brain functions. Conclusions This finding suggests that spontaneous adenosine release may play a fast, dynamic role in regulating an organism's response to external events. Therefore, adenosine transmission in the brain may have characteristics similar to those of classical neurotransmitters, such as dopamine and norepinephrine.

We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of [Formula: see text] , which simultaneously photoactivate a [Formula: see text] matrix pattern of GFP-labeled histones, with spots [Formula: see text] apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.

Our objective was to characterize the elasticity of hydrogel formulations intended to mimic physical properties that cells and tissues experience in vivo. Using atomic force microscopy (AFM), we tested a variety of concentrations in a variety of biomaterials, including agarose, alginate, the collagens, fibrin, hyaluronic acid, kerateine, laminin, Matrigel, polyacrylamide, polyethylene glycol diacrylate (PEGDA) and silicone elastomer (polydimethylsiloxane). Manipulations of the concentration of biomaterials were detectable in AFM measurements of elasticity (Young's modulus, E), and E tended to increase with increased concentration. Depending on the biomaterials chosen, and their concentrations, generation of tunable biocompatible hydrogels in the physiologic range is possible.

Due to their low immunogenicity, biodegradability and native cell-binding domains, fibrinogen fibers may be good candidates for tissue engineering scaffolds, drug delivery vehicles and other medical devices. We used a combined atomic force microscope (AFM)/optical microscope technique to study the mechanical properties of individual, electrospun fibrinogen fibers in dry, ambient conditions. The AFM was used to stretch individual fibers suspended over 13.5 μm wide grooves in a transparent substrate. The optical microscope, located below the sample, was used to monitor the stretching process. Electrospun fibrinogen fibers (diameter, 30–200 nm) can stretch to 74% beyond their original length before rupturing at a stress of 2.1 GPa. They can stretch elastically up to 15% beyond their original length. Using incremental stress–strain curves the viscoelastic behavior of these fibers was determined. The total stretch modulus was 4.2 GPa while the relaxed elastic modulus was 3.7 GPa. When held at constant strain, fibrinogen fibers display stress relaxation with a fast and slow relaxation time of 1.2 s and 11 s. In comparison to native and electrospun collagen fibers, dry electrospun fibrinogen fibers are significantly more extensible and elastic. In comparison to wet electrospun fibrinogen fibers, dry fibers are about 1000 times stiffer.

Cellular interiors present media that are very complex as they encompass a mixture of proteins, organelles, viscoelastic filaments, and chromatin structures in the nuclear interior. The behavior of proteins in such environments reflects the cellular complexity. Here we present the results of studies on the mobility, or diffusion, of free eGFP proteins, and of the important nuclear mismatch repair protein, MSH2. MSH2 was labeled with eGFP and the diffusion of both the free eGFP and MSH2-eGFP was measured using Fluorescence Recovery After Photobleaching (FRAP) on four distinct cell types that act as a model for the neoplastic cancer transformation of human mammary epithelial cells from non-cancerous to metastatic. We obtained several interesting results, such as a significant change in behavior for the first stage of cancer (immortal) compared to other stages. We also observed a significantly lower value for MSH2 diffusion (in both cytoplasm and nucleus) than what would be expected by scaling from the eGFP values (using appropriate size ratios of the two proteins). Diffusion coefficients ranged from 14 to 24 μm2/s for EGFP and from 3 to 7 μm2/s for EGFP-MSH2. This material is based upon work supported by the National Science Foundation under Grant Number 1106105 (KB, KS, and MG). We also thank Glen Marrs, director of the WFU Microscopic Imaging Core Facility, and Anita McCauley, for their help with the confocal microscopy setup.

Cocaine/heroin combinations (speedball) induce a synergistic elevation in extracellular dopamine concentrations ([DA](e)) in the nucleus accumbens (NAc) that can explain the increased abuse liability of speedball. To further delineate the mechanism of this neurochemical synergism, in vivo fast-scan cyclic voltammetry (FSCV) was used to compare NAc DA release and reuptake kinetic parameters following acute administration of cocaine, heroin and speedball in drug-naïve rats. These parameters were extracted from accumbal DA overflow induced by electrical stimulation of the ventral tegmental area. Evoked DA efflux was increased following both cocaine and speedball delivery, whereas heroin did not significantly change evoked DA release from baseline. DA efflux was significantly greater following cocaine compared to speedball. However, DA transporter (DAT) apparent affinity (K(m)) values were similarly elevated following cocaine and speedball administration, but unaffected by heroin. Neither drug induced substantial changes in the maximal reuptake rate (V(max)). These data, combined with published microdialysis and electrophysiological results, indicate that the combination of cocaine-induced competitive inhibition of DAT and the increase in the DA release elicited by heroin is responsible for the synergistic increase in ([DA](e)) induced by speedball.

Here we create a series of optical corrals and calculate their potential energy profile. A standing-wave Bessel beam is used to form traps in 1D (along the optical axis) and corrals in 2D, in planes perpendicular to the optical axis at the antinodal regions of the standing waves. These optical corrals are formed by an axicon-generated Bessel beam that is retro-reflected back onto itself. We report on Mie calculations of the 2D optical corrals and then compare the resulting probability distributions to those observed for latex particles of diameters 100, 200, and 300 nm. The experimental radial probability density function of tracked particles closely mimics the theoretical optical structure of a Bessel standing-wave corral. The Bessel standing-wave corrals we have characterized are being developed to measure rotational diffusion and torques on micro- and nanorods to help understand microfluidic behavior. The maximum forces on our small beads in the diffraction-free central zone of the Bessel beam standing wave are F||=0.5 pN and F⊥=0.1 pN.

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab-on-Bead™ library, a one-bead, one-sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy-aided identification of single target-bound beads and the extraction – wet or dry – of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target-binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target-binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead-based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers.

In this paper we present a theory of optical forces on particles of arbitrary shape and size and with fields of arbitrary spatial profiles. This means the theory applies to particle–light interactions in every optical regime: Rayleigh, Mie and geometrical. The theory has the advantage of conceptual simplicity and can handle the real, focused Gaussian beams typically found in optical tweezer experiments. We present optical force calculations on spheres and cubes, and the results obtained with our theory agree well with benchmark calculations obtained using other techniques.

We develop a method, which utilizes a combined atomic force microscope (AFM)/fluorescence microscope and small copy number polymerase chain reaction (PCR), to affinity-select individual aptamer species in a single cycle from a small pool of random-sequence oligonucleotides (oligos). In this method, a library of small beads, each of which is functionalized with fluorescent oligos of different sequences, is created. This library of oligo-functionalized beads is flowed over immobilized target molecules on a glass cover slip. High-affinity target-specific aptamers bind tightly to the target for prolonged periods and resist subsequent washes, resulting in a strong fluorescence signal on the substrate surface. This signal is observed from underneath the sample via fluorescence microscopy. The AFM tip, situated above the sample, is then directed to the coordinates of the fluorescence signal and is used to capture a three-dimensional high-resolution image of the surface-bound bead and to extract the bead (plus attached oligo). The extracted oligo is PCR-amplified, sequenced, and may then be subjected to further biochemical analysis. Here, we describe the underlying principles of this method, the required microscopy instrumentation, and the results of proof-of-principle experiments. In these experiments, we selected aptamers in eight trials from a binary pool containing a 1:1 mixture of thrombin aptamer oligo and a nonsense oligo. In each of the eight trials, the positive control aptamer was successfully detected, imaged, extracted, and characterized by PCR amplification and sequencing. In no case was the nonsense oligo selected, indicating good selectivity at this early stage of technology development.

Blood clots, whose main structural component is a mesh of microscopic fibrin fibers, experience mechanical strain from blood flow, clot retraction and interactions with platelets and other cells. We developed a transparent, striated and highly stretchable substrate made from fugitive glue (a styrenic block copolymer) to investigate how mechanical strain affects lysis of single, suspended fibrin fibers. In this suspended fiber assay, lysis manifested itself by fiber elongation, thickening (disassembly), fraying and collapse. Stretching single fibrin fibers significantly hampered their lysis. This effect was seen in uncrosslinked and crosslinked fibers. Crosslinking (without stretching) also hampered single fiber lysis. Our data suggest that strain is a novel mechanosensitive factor that regulates blood clot dissolution (fibrinolysis) at the single fiber level. At the molecular level of single fibrin molecules, strain may distort, or hinder access to, plasmin cleavage sites and thereby hamper lysis. Statement of significance Fibrin fibers are the major structural component of a blood clot. We developed a highly stretchable substrate made from fugitive glue and a suspended fibrin fiber lysis assay to investigate the effect of stretching on single fibrin fibers lysis. The key findings from our experiments are: 1) Fibers thicken and elongate upon lysis; 2) stretching strongly reduces lysis; 3) this effect is more pronounced for uncrosslinked fibers; and 4) stretching fibers has a similar effect on reducing lysis as crosslinking fibers. At the molecular level, strain may distort plasmin cleavage sites, or restrict access to those sites. Our results suggest that strain may be a novel mechanobiological factor that regulates fibrinolysis.

The present study was designed to characterize adenosine effluxes in the brain using fast scan cyclic voltammetry and explore how spontaneous adenosine release may be affected by a tail pinch. Long...

Metastasis is the process by which cancer spreads from a primary tumor site to other sites in the body. Once a cancer has metastasized it is much more difficult to treat, and a better understanding of metastasis is, thus, a critical goal in cancer research. Metastasis involves cell movement through the body, and the aim of our study was to explore the differences in motility between metastatic cells and noncancerous cells. Toward this end, we investigated cell motility using migration assays, in which cells are grown in two adjacent, but separate zones to confluency. After confluency is reached, the barrier separating the two cell zones on a glass bottom dish is removed, and the motion of the two edges of the cells separated across the septum gap is recorded. We hypothesized that cancerous human breast cells (MDA-MB-231 cells) would exhibit a higher rate of migration and a lower degree of coordinated movement than normal breast cells (HMEC cells). Migration assays were performed on both types of cells, and the migration into the gap between cell populations was recorded using time-lapse DIC microscopy. The resulting videos were processed using areal analysis and particle image velocimetry (PIV), which allowed extraction of areal migration rates and velocity vector fields. An average migration rate of 242μm2/min was measured for HMEC cells, and a lower degree of coordination was observed in MDA cells compared to HMEC cells.

The mechanical properties of cells play an important role in cellular processes such as cell migration, division, and mechanotransduction. Stiffness is an important mechanical property and can be measured using an atomic force microscope (AFM) (to determine the Young's modulus). Our previous studies showed that normal breast cells (human mammary epithelial cells, HMEC) are stiffer than highly metastatic breast cancer cells (MDA-MB-231). Also, normal breast cells in the center of a monolayer micro-colony are stiffer than cells on the periphery and isolated cells which means that the microenvironment significantly affects the mechanical properties of cells. However, the effect on stiffness when normal cells and cancer cells grow together has not been well established. In this study, we investigate how the mechanical properties of HMEC and MDA-MB-231 cells change in co-culture. Our preliminary results show that both HMEC and MDA-MB-231 cells get significantly stiffer in co-culture (HMEC 1.3 kPa -> 15 kPa; MDA-MB-231 0.59 kPa -> 1.48 kPa). This study will offer an understanding of normal-to-cancer cell interactions affect stiffness, and consequently how it might be correlated to metastasis.

The interior of cells is a highly complex medium, containing numerous organelles, a matrix of different fibers and a viscous, aqueous fluid of proteins and small molecules. The interior of cells is also a highly dynamic medium, in which many components move, either by active transport or passive diffusion. The mobility and localization of proteins inside cells can provide important insights into protein function and also general cellular properties, such as viscosity. Neoplastic transformation affects numerous cellular properties, and our goal was to investigate the diffusional and binding behavior of the important mismatch repair (MMR) protein MSH2 in live human cells at various stages of neoplastic transformation. Toward this end, noncancerous, immortal, tumorigenic, and metastatic mammary epithelial cells were transfected with EGFP and EGFP-tagged MSH2. MSH2 forms two MMR proteins (MutSα and MutSβ) and we assume MSH2 is in the complex MutSα, though our results are similar in either case. Unlike the MutS complexes that bind to nuclear DNA, EGFP diffuses freely. EGFP and MutSα-EGFP diffusion coefficients were determined in the cytoplasm and nucleus of each cell type using fluorescence recovery after photobleaching. Diffusion coefficients were 14–24 μm2/s for EGFP and 3–7 μm2/s for MutSα-EGFP. EGFP diffusion increased in going from noncancerous to immortal cells, indicating a decrease in viscosity, with smaller changes in subsequent stages. MutSα produces an effective diffusion coefficient that, coupled with the free EGFP diffusion measurements, can be used to extract a pure diffusion coefficient and a pseudo-equilibrium constant K*. The MutSα nuclear K* increased sixfold in the first stage of cancer and then decreased in the more advanced stages. The ratio of nuclear to cytoplasmic K*for MutSα increased almost two orders of magnitude in going from noncancerous to immortal cells, suggesting that this quantity may be a sensitive metric for recognizing the onset of cancer.

A cell’s mechanical properties are important in determining its adhesion, migration, and response to the mechanical properties of its microenvironment and may help explain behavioral differences between normal and cancerous cells. Using fluorescently labeled peroxisomes as microrheological probes, the interior mechanical properties of normal breast cells were compared to a metastatic breast cell line, MDA-MB-231. To estimate the mechanical properties of cell cytoplasms from the motions of their peroxisomes, it was necessary to reduce the contribution of active cytoskeletal motions to peroxisome motion. This was done by treating the cells with blebbistatin, to inhibit myosin II, or with sodium azide and 2-deoxy- $$\textsc {d}$$ -glucose, to reduce intracellular ATP. Using either treatment, the peroxisomes exhibited normal diffusion or subdiffusion, and their mean squared displacements (MSDs) showed that the MDA-MB-231 cells were significantly softer than normal cells. For these two cell types, peroxisome MSDs in treated and untreated cells converged at high frequencies, indicating that cytoskeletal structure was not altered by the drug treatment. The MSDs from ATP-depleted cells were analyzed by the generalized Stokes–Einstein relation to estimate the interior viscoelastic modulus $$G^{*}$$ and its components, the elastic shear modulus $$G^{\prime }$$ and viscous shear modulus $$G^{\prime \prime }$$ , at angular frequencies between 0.126 and 628 rad/s. These moduli are the material coefficients that enter into stress–strain relations and relaxation times in quantitative mechanical models such as the poroelastic model of the interior regions of cancerous and non-cancerous cells.

We report on a light-force technique to make absolute measurements of cluster polarizabilities in the gas phase. The technique relies on several important experimental components and methods, including a front-ablation cluster source to produce cold clusters, a position-sensitive time-of-flight spectrometer, glass collimating slits, and a reliable technique for timing a particular slice of the cluster beam. The cluster beam is collimated with a pair of slits and the ideal locations of the slits to maximize the observable light force is discussed. To date, the polarizability of C60 at the fundamental wavelength of a Nd:YAG laser (1.064µμm) has been measured.

We report on a new optical technique that uses light forces and a time-of-flight spectrometer to make absolute measurements of cluster polarizabilities. This is also the first accurate report of an ac polarizability measurement of a condensable cluster in the gas phase. We have determined the optical polarizability of C60 at the fundamental wavelength of a Nd:YAG laser (1.064 μm) to be α=79±4 A3.

In the present voltammetric study, we have characterized cocaine-induced changes in evoked dopamine release and uptake in the striatum of freely moving mice in real time. Cocaine induced marked dopamine uptake inhibition measured as apparent Km changes, producing a maximal effect 20 min following a single injection (15 mg/kg, i.p.). Changes in uptake were paralleled by increases in evoked dopamine release per stimulus pulse, revealing a high correlation between these two parameters following cocaine administration. This initial characterization of cocaine effects on striatal dopamine transmission in the commonly used C57BL/6 mouse strain provides a basis for future voltammetric studies using genetic mouse models.

There is compelling evidence that acute ethanol exposure stimulates ventral tegmental area (VTA) dopamine cell activity and that VTA-dependent dopamine release in terminal fields within the nucleus accumbens plays an integral role in the regulation of ethanol drinking behaviors. Unfortunately, due to technical limitations, the specific temporal dynamics linking VTA dopamine cell activation and ethanol self-administration are not known. In fact, establishing a causal link between specific patterns of dopamine transmission and ethanol drinking behaviors has proven elusive. Here, we sought to address these gaps in our knowledge using a newly developed viral-mediated gene delivery strategy to selectively express Channelrhodopsin-2 (ChR2) on dopamine cells in the VTA of wild-type rats. We then used this approach to precisely control VTA dopamine transmission during voluntary ethanol drinking sessions. The results confirmed that ChR2 was selectively expressed on VTA dopamine cells and delivery of blue light pulses to the VTA induced dopamine release in accumbal terminal fields with very high temporal and spatial precision. Brief high frequency VTA stimulation induced phasic patterns of dopamine release in the nucleus accumbens. Lower frequency stimulation, applied for longer periods mimicked tonic increases in accumbal dopamine. Notably, using this optogenetic approach in rats engaged in an intermittent ethanol drinking procedure, we found that tonic, but not phasic, stimulation of VTA dopamine cells selectively attenuated ethanol drinking behaviors. Collectively, these data demonstrate the effectiveness of a novel viral targeting strategy that can be used to restrict opsin expression to dopamine cells in standard outbred animals and provide the first causal evidence demonstrating that tonic activation of VTA dopamine neurons selectively decreases ethanol self-administration behaviors.

In this study, the first in-depth analysis of optically induced dopamine release using fast-scan cyclic voltammetry on striatal slices from rat brain was performed. An adeno-associated virus that expresses Channelrhodopsin-2 was injected in the substantia nigra. Tissue was collected and sectioned into 400 μm-thick coronal slices four weeks later. Blue laser light (473 nm) was delivered through a fiber optic inserted into slice tissue. Experiments revealed some difference between maximal amplitudes measured from optically and electrically evoked dopamine effluxes. Specifically, there was an increase in the amplitude of dopamine release induced by electrical stimulation in comparison with light stimulations. However, we found that dopamine release is more sensitive to changes in the pulse width in the case of optical stimulation. Light-stimulated dopamine was increased as the stimulation pulse widened. There was no difference with repeated stimulations at five minute intervals between stimulation sources and dopamine signal was stable during recording sessions, while one minute intervals resulted in a decline in the amplitude from both sources. Optical stimulation can also produce an artifact that is distinguishable from dopamine by the cyclic voltammogram. These results confirm that optical stimulation of dopamine is a sound approach for future pharmacological studies in slices.

Genome instability is an underlying factor of all cancers. Defects in DNA repair mechanisms contribute significantly to this factor. Recent reports indicate the mechanical properties of cells change as they advance through different stages of neoplastic transformation. The main goal of this study is to investigate how changes in the intracellular environment and changes in DNA repair protein distribution are altered in cells undergoing neoplastic transformation. For this study we have chosen to use human mammary epithelial (HME) cells from the Weinberg line. Lipofection was used to transiently transfect enhanced green fluorescent protein (egfp) fusions to MSH2 mismatch repair proteins. We have measured the localization and diffusion of these fusions using fluorescence recovery after photobleaching (FRAP) and raster image correlation spectroscopy (RICS) on live cells maintained under appropriate environmental conditions. We have tested normal, immortal, and tumorigenic cells in this study. The acquired data was analyzed using custom written Matlab programs as well as commercially available software. Preliminary results show localization of the fusions to the nucleolus in all cell types tested. The surrounding nucleus generally has the weakest localization signal. We have also observed smaller diffusion coefficients in the nucleus of tumorigenic cells compared to normal cells. This work is supported by NSF Materials and Surface Engineering grant CMMI-1152781.

We report on our effort to characterize the mechanical changes in cells that undergo neoplastic transformation. The cells we are studying include normal human mammary epithelial cells, as well as immortalized and tumorigenic versions of the same cells. We are studying three different aspects of cell behavior using different tools. The measurements include determining the elastic moduli of the different cells (in both the nuclear and cytoplasmic regions) using an Atomic Force Microscope (AFM) with a 5.3 um diameter spherical probe; measuring the diffusion and binding of mismatch repair proteins in the different cells using a confocal microscope to perform Fluorescence Recovery After Photobleaching (FRAP) measurements as well as Raster Imaging Correlation Spectroscopy (RICS); and finally measuring the diffusion and transport of different natural organelles such as lysosomes and peroxisomes using particle tracking microscopy. In this presentation, we plan to provide a summary of results in all three areas to date, and to put our measurements in the context of related work published by others.This work is supported by NSF Materials and Surface Engineering grant CMMI-1152781.

This article reviews the main theoretical methods and experimental techniques for determining electric-dipole polarizabilities. This review only considers the lowest-order polarizability which describes the linear response of a particle to an electric field, i.e. the induced electric-dipole moment depends linearly on the applied electric field. The scope of the review is broad: it encompasses atomic, molecular and cluster polarizabilities. Both static and dynamic polarizabilities are covered. Calculational techniques discussed in detail include variational perturbational theory, the local density approximation, and semi-empirical additivity methods. Special attention is paid to basis sets used in polarizability calculations—especially the increasingly popular Gaussian basis sets. Extensive tabulations of polarizability calculations are provided for helium and sodium atoms, the diatomic nitrogen molecule, the fullerene C60, and sodium and potassium clusters. For each of these cases, the most reliable experimental results are also provided for comparison to theory. Many traditional experimental techniques are reviewed including dielectric constant, refractive index, Rayleigh scattering, electro-optic Kerr effect, beam deflection, E—H gradient balance, and the beam resonance technique. New experimental methods for determining polarizabilities such as the light-force method and atom interferometry are also extensively discussed. In addition, a novel detection scheme that is elegant and universal—the position-sensitive time-of-flight spectrometer—is thoroughly described.

We report the measurement of the scalar [alpha][sub 0] and tensor [alpha][sub 2] polarizabilities of uranium using a light-force technique, the first accurate measurement of the polarizabilities of a refractory element. At the fundamental wavelength of a Nd:YAG laser (1.064 [mu]m) we measure [alpha][sub 0]=24.9[plus minus]1.5 A[sup 3] and [alpha][sub 2]=0.7[plus minus]1.7 A[sup 3]. From these values we determine the dc polarizabilities to be [alpha][sub 0]=20.3[plus minus]1.4 A[sup 3] and [alpha][sub 2]=0.7[plus minus]1.8 A[sup 3]. Calculations of [alpha][sub 0](dc) are within 30% of this measurement.