Spectrally encoded dual-mode interferometry with orthogonal scanning.

Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technique. Here, we present a method to integrate optical coherence tomography (OCT) and SECM for complementary imaging by adding orthogonal scanning to the SECM configuration. The co-registration of SECM and OCT is automatic, as all system components are shared in the same order, eliminating the need for additional optical alignment. The proposed multimode imaging system is compact and cost-effective while providing the benefits of imaging aiming and guidance. Furthermore, speckle noise can be suppressed by averaging the speckles generated by shifting the spectral-encoded field in the direction of dispersion. Using a near infrared (NIR) card and a biological sample, we demonstrated the capability of the proposed system by showing SECM imaging at depths of interest guided by the OCT in real time and speckle noise reduction. Interfaced multimodal imaging of SECM and OCT was implemented at a speed of approximately 7 frames/s using fast-switching technology and GPU processing.

Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality.

Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.

Shaping Rolling Circle Amplification Products into DNA Nanoparticles by Incorporation of Modified Nucleotides and Their Application to In Vitro and In Vivo Delivery of a Photosensitizer.

Rolling circle amplification (RCA) is a robust way to generate DNA constructs, which are promising materials for biomedical applications including drug delivery because of their high biocompatibility. To be employed as a drug delivery platform, however, the DNA materials produced by RCA need to be shaped into nanoparticles that display both high cellular uptake efficiency and nuclease resistance. Here, we showed that the DNA nanoparticles (DNPs) can be prepared with RCA and modified nucleotides that have side-chains appended on the nucleobase are capable of interacting with the DNA strands of the resulting RCA products. The incorporation of the modified nucleotides improved cellular uptake efficiency and nuclease resistance of the DNPs. We also demonstrated that these DNPs could be employed as carriers for the delivery of a photosensitizer into cancer cells to achieve photodynamic therapy upon irradiation at both the in vitro and in vivo levels.

3D Defect Localization on Exothermic Faults within Multi-Layered Structures Using Lock-In Thermography: An Experimental and Numerical Approach.

Micro-electronic devices are increasingly incorporating miniature multi-layered integrated architectures. However, the localization of faults in three-dimensional structure remains challenging. This study involved the experimental and numerical estimation of the depth of a thermally active heating source buried in multi-layered silicon wafer architecture by using both phase information from an infrared microscopy and finite element simulation. Infrared images were acquired and real-time processed by a lock-in method. It is well known that the lock-in method can increasingly improve detection performance by enhancing the spatial and thermal resolution of measurements. Operational principle of the lock-in method is discussed, and it is represented that phase shift of the thermal emission from a silicon wafer stacked heat source chip (SSHSC) specimen can provide good metrics for the depth of the heat source buried in SSHSCs. Depth was also estimated by analyzing the transient thermal responses using the coupled electro-thermal simulations. Furthermore, the effects of the volumetric heat source configuration mimicking the 3D through silicon via integration package were investigated. Both the infrared microscopic imaging with the lock-in method and FE simulation were potentially useful for 3D isolation of exothermic faults and their depth estimation for multi-layered structures, especially in packaged semiconductors.

Laser Scanning Confocal Thermoreflectance Microscope for the Backside Thermal Imaging of Microelectronic Devices.

In this paper, we report on a confocal thermoreflectance imaging system that can examine the thermal characteristics of microelectronic devices by penetrating the backside of a device through the substrate. In this system, the local reflectivity variations due to heat generation in the device are measured point by point by a laser scanning confocal microscope capable of eliminating out-of-focus reflections and the thermoreflectance is extracted via Fourier-domain signal processing. In comparison to the conventional widefield thermoreflectance microscope, the proposed laser scanning confocal thermoreflectance microscope improves the thermoreflectance sensitivity by ~23 times and the spatial resolution by ~25% in backside thermoreflectance measurements.

Automated Dielectrophoretic Tweezers-Based Force Spectroscopy System in a Microfluidic Device.

We reported an automated dielectrophoretic (DEP) tweezers-based force spectroscopy system to examine intermolecular weak binding interactions, which consists of three components: (1) interdigitated electrodes and micro-sized polystyrene particles used as DEP tweezers and probes inside a microfluidic device, along with an arbitrary function generator connected to a high voltage amplifier; (2) microscopy hooked up to a high-speed charge coupled device (CCD) camera with an image acquisition device; and (3) a computer aid control system based on the LabVIEW program. Using this automated system, we verified the measurement reliability by measuring intermolecular weak binding interactions, such as hydrogen bonds and Van der Waals interactions. In addition, we also observed the linearity of the force loading rates, which is applied to the probes by the DEP tweezers, by varying the number of voltage increment steps and thus affecting the linearity of the force loading rates. This system provides a simple and low-cost platform to investigate intermolecular weak binding interactions.

Real-Time Analysis of Cellular Response to Small-Molecule Drugs within a Microfluidic Dielectrophoresis Device.

Quantitative detection of the biological properties of living cells is essential for a wide range of purposes, from the understanding of cellular characteristics to the development of novel drugs in nanomedicine. Here, we demonstrate that analysis of cell biological properties within a microfluidic dielectrophoresis device enables quantitative detection of cellular biological properties and simultaneously allows large-scale measurement in a noise-robust and probeless manner. Applying this technique, the static and dynamic biological responses of live B16F10 melanoma cells to the small-molecule drugs such as N-ethylmaleimide (NEM) and [(dihydronindenyl)oxy]alkanoic acid (DIOA) were quantitatively and statistically examined by investigating changes in movement of the cells. Measurement was achieved using subtle variations in dielectrophoresis (DEP) properties of the cells, which were attributed to activation or deactivation of K(+)/Cl(-) cotransporter channels on the cell membrane by the small-molecule drugs, in a microfluidic device. On the basis of quantitative analysis data, we also provide the first report of the shift of the complex permittivity of a cell induced by the small-molecule drugs. In addition, we demonstrate interesting quantifiable parameters including the drug effectiveness coefficient, antagonistic interaction coefficient, kinetic rate, and full width at half-maximum, which corresponded to changes in biological properties of B16F10 cells over time when NEM and DIOA were introduced alone or in combination. Those demonstrated parameters represent very useful tools for evaluating the effect of small-molecule drugs on the biological properties of cells.

Colorimetric and Electrochemical Dual-Mode Detection of Thioredoxin 1 Based on the Efficient Peroxidase-Mimicking and Electrocatalytic Property of Prussian Blue Nanoparticles.

As a potent detection method for cancer biomarkers in physiological fluid, a colorimetric and electrochemical dual-mode sensing platform for breast cancer biomarker thioredoxin 1 (TRX1) was developed based on the excellent peroxidase-mimicking and electrocatalytic property of Prussian blue nanoparticles (PBNPs). PBNPs were hydrothermally synthesized using K3[Fe(CN)6] as a precursor and polyvinylpyrrolidone (PVP) as a capping agent. The synthesized spherical PBNPs showed a significant peroxidase-like activity, having approximately 20 and 60% lower Km values for 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, respectively, compared to those of horseradish peroxidase (HRP). The PBNPs also enhanced the electron transfer on the electrode surface. Based on the beneficial features, PBNPs were used to detect target TRX1 via sandwich-type immunoassay procedures. Using the strategies, TRX1 was selectively and sensitively detected, yielding limit of detection (LOD) values as low as 9.0 and 6.5 ng mL-1 via colorimetric and electrochemical approaches, respectively, with a linear range of 10-50 ng mL-1 in both strategies. The PBNP-based TRX1 immunoassays also exhibited a high degree of precision when applied to real human serum samples, demonstrating significant potentials to replace conventional HRP-based immunoassay systems into rapid, robust, reliable, and convenient dual-mode assay systems which can be widely utilized for the identification of important target molecules including cancer biomarkers.

Label-free photothermal optical coherence microscopy to locate desired regions of interest in multiphoton imaging of volumetric specimens.

Biochip-based research is currently evolving into a three-dimensional and large-scale basis similar to the in vivo microenvironment. For the long-term live and high-resolution imaging in these specimens, nonlinear microscopy capable of label-free and multiscale imaging is becoming increasingly important. Combination with non-destructive contrast imaging will be useful for effectively locating regions of interest (ROI) in large specimens and consequently minimizing photodamage. In this study, a label-free photothermal optical coherence microscopy (OCM) serves as a new approach to locate the desired ROI within biological samples which are under investigation by multiphoton microscopy (MPM). The weak photothermal perturbation in sample by the MPM laser with reduced power was detected at the endogenous photothermal particles within the ROI using the highly sensitive phase-differentiated photothermal (PD-PT) OCM. By monitoring the temporal change of the photothermal response signal of the PD-PT OCM, the hotspot generated within the sample focused by the MPM laser was located on the ROI. Combined with automated sample movement in the x-y axis, the focal plane of MPM could be effectively navigated to the desired portion of a volumetric sample for high-resolution targeted MPM imaging. We demonstrated the feasibility of the proposed method in second harmonic generation microscopy using two phantom samples and a biological sample, a fixed insect on microscope slide, with dimensions of 4 mm wide, 4 mm long, and 1 mm thick.

Single-step electropolymerization patterning of a polypyrrole nanowire by ultra-short pulses via an AFM cantilever.

Conducting polymers (CPs) have attracted a great deal of attention due to their unique properties; these properties are useful in implementing various functional devices, such as memory, and chemical and biological sensors. In particular, the nanopatterning of CPs is a key technology that will accelerate the adoption of CPs in fabricating nanoscaled multifunctional devices. This paper presents an innovative technique for forming polypyrrole nanowire (PPy-NW) patterns, without any additional pretreatment on the gold surface, using atomic force microscopy (AFM) and ultra-short pulse voltage. Applying the ultra-short pulse voltage to the AFM tip has the following advantage: since the electrochemical current is extremely localized around the tip, the successful formation of CP nanowires results. This is because the pulse width is much shorter than the resistor-capacitor (RC) time constant of the equivalent electrochemical circuit of our experimental set-up. This paper provides systematic results regarding the dimensional variation of the PPy-NW patterns produced by varying the electrical conditions of the ultra-short pulse, such as the pulse amplitude, width, and frequency. The results show that use of an ultra-short pulse is essential in fabricating PPy-NW patterns. Additionally, an ultra-short pulse offers excellent pattern controllability for both width (353 nm ∼ 3.37 µm) and height (2.0 ∼ 88.3 nm).

Quantitative Photothermal Characterization with Bioprinted 3D Complex Tissue Constructs for Early-Stage Breast Cancer Therapy Using Gold Nanorods.

Plasmonic photothermal therapy (PPTT) using gold nanoparticles (AuNPs) has shown great potential for use in selective tumor treatment, because the AuNPs can generate destructive heat preferentially upon irradiation. However, PPTT using AuNPs has not been added to practice, owing to insufficient heating methods and tissue temperature measurement techniques, leading to unreliable and inaccurate treatments. Because the photothermal properties of AuNPs vary with laser power, particle optical density, and tissue depth, the accurate prediction of heat generation is indispensable for clinical treatment. In this report, bioprinted 3D complex tissue constructs comprising processed gel obtained from porcine skin and human decellularized adipose tissue are presented for characterization of the photothermal properties of gold nanorods (AuNRs) having an aspect ratio of 3.7 irradiated by a near-infrared laser. Moreover, an analytical function is suggested for achieving PPTT that can cause thermal damage selectively on early-stage human breast cancer by regulating the heat generation of the AuNRs in the tissue.

Size-based separation and collection of mouse pancreatic islets for functional analysis.

Islet size has recently been demonstrated to be an important factor in determining human islet transplantation outcomes. In this study, a multi-layered microfluidic device was developed and quantified for size-based separation of a heterogeneous population of mouse islets. The device was fabricated using standard soft lithography and polydimethylsiloxane (PDMS). Size-based separation was first demonstrated via injection of a heterogeneous population of glass beads between 50-300 microm in diameter which were separated into five sub-populations based on their diameter. Next, a heterogeneous population of mouse pancreatic islets, between 50-250 microm in diameter was separated into four sub-populations. Throughout this process the islets remained intact without any signs of damage, as indicated by cell viability staining. Islet glucose-stimulated insulin secretion of each sub-population of islets was also evaluated demonstrating that islets smaller than 150 microm have superior stimulation indexes (SI) compared to islets larger than 150 microm. In this study, we found that islets between 100 microm and 150 microm in diameter had the greatest SI value in a heterogeneous population of islets.

Experimental and numerical study of electrochemical nanomachining using an AFM cantilever tip.

We fabricated nanopatterns on Cu thin films via an electrochemical route using an atomic force microscope (AFM). Experimental results were compared with an equivalent electrochemical circuit model representing an electrochemical nanomachining (ECN) technique. In order to precisely construct the nanopatterns, an ultra-short pulse was applied onto the Cu film through the AFM cantilever tip. The line width of the nanopatterns (the lateral dimension) increased with increased pulse amplitude, on-time, and frequency. The tip velocity effect on the nanopattern line width was also investigated. The study described here provides important insight for fabricating nanopatterns precisely using electrochemical methods with an AFM cantilever tip.

Probing Collective Mechanoadaptation in Cardiomyocyte Development by Plasma Lithography Patterned Elastomeric Substrates.

Understanding how the mechanical microenvironment affects cardiomyocyte development is crucial to the creation of in vitro models for studying heart physiology and pathophysiology. This knowledge will also facilitate the design of biomaterials and tissue scaffolds utilized in the generation of functional tissue constructs for regenerative medicine and drug screening applications. Here, plasma lithography patterning of elastomeric substrates is exploited for creating microtissues composed of neonatal cardiomyocytes and investigating their attributes in different mechanical microenvironments. Restriction of the cellular outgrowth in line patterns results in cardiomyocytes developing into multicellular clusters and collectively adapting to geometric confinement and substrate stiffness. Immunofluorescence microscopy, video microscopy, and force spectroscopy show that the size and shape of the cardiomyocyte clusters, as well as sarcomere length, fiber alignment, beating amplitude, and beating frequency of the cardiomyocytes, are regulated by the microenvironmental cues. Computational analysis reveals that the mechanical stress at the cluster-substrate interface strongly correlates with the characteristics of the cardiomyocytes. Taken together, our results underscore a collective mechanoadaptation scheme in cardiac development.

Identifying DNA mismatches at single-nucleotide resolution by probing individual surface potentials of DNA-capped nanoparticles.

Here, we demonstrate a powerful method to discriminate DNA mismatches at single-nucleotide resolution from 0 to 5 mismatches (χ0 to χ5) using Kelvin probe force microscopy (KPFM). Using our previously developed method, we quantified the surface potentials (SPs) of individual DNA-capped nanoparticles (DCNPs, ∼100 nm). On each DCNP, DNA hybridization occurs between ∼2200 immobilized probe DNA (pDNA) and target DNA with mismatches (tDNA, ∼80 nM). Thus, each DCNP used in the bioassay (each pDNA-tDNA interaction) corresponds to a single ensemble in which a large number of pDNA-tDNA interactions take place. Moreover, one KPFM image can scan at least dozens of ensembles, which allows statistical analysis (i.e., an ensemble average) of many bioassay cases (ensembles) under the same conditions. We found that as the χn increased from χ0 to χ5 in the tDNA, the average SP of dozens of ensembles (DCNPs) was attenuated owing to fewer hybridization events between the pDNA and the tDNA. Remarkably, the SP attenuation vs. the χn showed an inverse-linear correlation, albeit the equilibrium constant for DNA hybridization exponentially decreased asymptotically as the χn increased. In addition, we observed a cascade reaction at a 100-fold lower concentration of tDNA (∼0.8 nM); the average SP of DCNPs exhibited no significant decrease but rather split into two separate states (no-hybridization vs. full-hybridization). Compared to complementary tDNA (i.e., χ0), the ratio of no-hybridization/full-hybridization within a given set of DCNPs became ∼1.6 times higher in the presence of tDNA with single mismatches (i.e., χ1). The results imply that our method opens new avenues not only in the research on the DNA hybridization mechanism in the presence of DNA mismatches but also in the development of a robust technology for DNA mismatch detection.

Multiscale Cues Drive Collective Cell Migration.

To investigate complex biophysical relationships driving directed cell migration, we developed a biomimetic platform that allows perturbation of microscale geometric constraints with concomitant nanoscale contact guidance architectures. This permits us to elucidate the influence, and parse out the relative contribution, of multiscale features, and define how these physical inputs are jointly processed with oncogenic signaling. We demonstrate that collective cell migration is profoundly enhanced by the addition of contract guidance cues when not otherwise constrained. However, while nanoscale cues promoted migration in all cases, microscale directed migration cues are dominant as the geometric constraint narrows, a behavior that is well explained by stochastic diffusion anisotropy modeling. Further, oncogene activation (i.e. mutant PIK3CA) resulted in profoundly increased migration where extracellular multiscale directed migration cues and intrinsic signaling synergistically conspire to greatly outperform normal cells or any extracellular guidance cues in isolation.

Biaxial Dielectrophoresis Force Spectroscopy: A Stoichiometric Approach for Examining Intermolecular Weak Binding Interactions.

The direct quantification of weak intermolecular binding interactions is very important for many applications in biology and medicine. Techniques that can be used to investigate such interactions under a controlled environment, while varying different parameters such as loading rate, pulling direction, rupture event measurements, and the use of different functionalized probes, are still lacking. Herein, we demonstrate a biaxial dielectrophoresis force spectroscopy (BDFS) method that can be used to investigate weak unbinding events in a high-throughput manner under controlled environments and by varying the pulling direction (i.e., transverse and/or vertical axes) as well as the loading rate. With the BDFS system, we can quantitatively analyze binding interactions related to hydrogen bonding or ionic attractions between functionalized microbeads and a surface within a microfluidic device. Our BDFS system allowed for the characterization of the number of bonds involved in an interaction, bond affinity, kinetic rates, and energy barrier heights and widths from different regimes of the energy landscape.

Biomimetic 3D Tissue Models for Advanced High-Throughput Drug Screening.

Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately re-create the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when using such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models that accurately mimic the physiological properties of native tissue samples and highlight the advantages of using such 3D microtissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models.

Probing mechanoregulation of neuronal differentiation by plasma lithography patterned elastomeric substrates.

Cells sense and interpret mechanical cues, including cell-cell and cell-substrate interactions, in the microenvironment to collectively regulate various physiological functions. Understanding the influences of these mechanical factors on cell behavior is critical for fundamental cell biology and for the development of novel strategies in regenerative medicine. Here, we demonstrate plasma lithography patterning on elastomeric substrates for elucidating the influences of mechanical cues on neuronal differentiation and neuritogenesis. The neuroblastoma cells form neuronal spheres on plasma-treated regions, which geometrically confine the cells over two weeks. The elastic modulus of the elastomer is controlled simultaneously by the crosslinker concentration. The cell-substrate mechanical interactions are also investigated by controlling the size of neuronal spheres with different cell seeding densities. These physical cues are shown to modulate with the formation of focal adhesions, neurite outgrowth, and the morphology of neuroblastoma. By systematic adjustment of these cues, along with computational biomechanical analysis, we demonstrate the interrelated mechanoregulatory effects of substrate elasticity and cell size. Taken together, our results reveal that the neuronal differentiation and neuritogenesis of neuroblastoma cells are collectively regulated via the cell-substrate mechanical interactions.