Alexander Terekhov

On femtosecond micromachining of HPHT single-crystal diamond with direct laser writing using tight focusing

We investigate the formation of diversiform micro-/nano-structures in High-Pressure High-Temperature (HPHT) synthetic single-crystal diamond by tight-focusing 200 fs regeneratively amplified Ti: Sapphire laser pulses centered at lambda = 800 nm. Ablated samples of synthetic single crystal nanodiamond and their acetate replicas are analyzed using scanning electron microscopy (SEM). Using pulse energies that are significantly above the threshold for permanent change, it is shown from this work that amplified femtosecond pulses are capable of producing controlled modification of HPHT single-crystal diamond at size scales below the diffraction limit and provided negligible collateral heating and shock-wave damage. This is attributed to the low thermal losses and negligible hydrodynamic expansion of the ablated material during the femtosecond laser pulse. It is shown that low pulse energy is a key factor for the accurate and precise machining of micropattems.

Solution-cast high-aspect-ratio polymer structures from direct-write templates.

This letter presents a novel strategy for template synthesis of polymer structures with laser machined substrates. User-designed patterns of submicrometer holes with aspect ratios >10:1 and depths >10 μm were produced by focusing 160 fs, 5.2 μJ laser pulses on the surface of fused silica with a high numerical aperture microscope objective. Some holes were enlarged by chemical etching. Polymer solutions were cast into the templates to create high-aspect-ratio polymer structures using replication. Engineered polymer structures prepared by this unique method are useful for a number of applications such as high surface area electrodes and biological substrates.

On-Chip Open Microfluidic Devices for Chemotaxis Studies

Microfluidic devices can provide unique control over both the chemoattractant gradient and the migration environment of the cells. Our work incorporates laser-machined micro and nanofluidic channels into bulk fused silica and cover slip-sized silica wafers. We have designed “open” chemotaxis devices that produce passive chemoattractant gradients without an external micropipette system. Since the migration area is unobstructed, cells can be easily loaded and strategically placed into the devices with a standard micropipette. The reusable monolithic glass devices have integral ports that can generate multiple gradients in a single experiment. We also used cover slip microfluidics for chemotaxis assays. Passive gradients elicited from these cover slips could be readily adapted for high throughput chemotaxis assays.We have also demonstrated for the first time that cells can be recruited into cover slip ports eliciting passive chemoattractant gradients. This proves, in principle, that intravital cover slip configurations could deliver controlled amounts of drugs, chemicals, or pathogens as well as recruit cells for proteomic or histological analysis in living animals while under microscopic observation. Intravital cover slip fluidics will create a new paradigm for in vivo observation of biological processes.

Maximum-likelihood position sensing and actively controlled electrokinetic transport for single-molecule trapping

A freely diffusing single fluorescent molecule may be scrutinized for an extended duration within a confocal microscope by actively trapping it within the femtoliter probe region. We present results from computational models and ongoing experiments that research the use of multi-focal pulse-interleaved excitation with time-gated single photon counting and maximum-likelihood estimation of the position for active control of the electrophoretic and/or electro-osmotic motion that re-centers the molecule and compensates for diffusion. The molecule is held within a region with approximately constant irradiance until it photobleaches and/or is replaced by the next molecule. The same photons used for determining the position within the trap are also available for performing spectroscopic measurements, for applications such as the study of conformational changes of single proteins. Generalization of the trap to multi-wavelength excitation and to spectrally-resolved emission is being developed. Also, the effectiveness of the maximum-likelihood position estimates and semi-empirical algorithms for trap control is discussed.

A Microfluidic-Enabled Mechanical Microcompressor for the Immobilization of Live Single- and Multi-Cellular Specimens

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.

Patterned polymer matrix promotes stemness and cell-cell interaction of adult stem cells

BackgroundThe interaction of stem cells with their culture substrates is critical in controlling their fate and function. Declining stemness of adult-derived human mesenchymal stem cells (hMSCs) during in vitro expansion on tissue culture polystyrene (TCPS) severely limits their therapeutic efficacy prior to cell transplantation into damaged tissues. Thus, various formats of natural and synthetic materials have been manipulated in attempts to reproduce in vivo matrix environments in which hMSCs reside.ResultsWe developed a series of patterned polymer matrices for cell culture by hot-pressing poly(ε-caprolactone) (PCL) films in femtosecond laser-ablated nanopore molds, forming nanofibers on flat PCL substrates. hMSCs cultured on these PCL fiber matrices significantly increased expression of critical self-renewal factors, Nanog and OCT4A, as well as markers of cell-cell interaction PECAM and ITGA2. The results suggest the patterned polymer fiber matrix is a promising model to maintain the stemness of adult hMSCs.ConclusionThis approach meets the need for scalable, highly repeatable, and tuneable models that mimic extracellular matrix features that signal for maintenance of hMSC stemness.

Ultrathin Polymer Membranes with Patterned, Micrometric Pores for Organs-on-Chips

The basal lamina or basement membrane (BM) is a key physiological system that participates in physicochemical signaling between tissue types. Its formation and function are essential in tissue maintenance, growth, angiogenesis, disease progression, and immunology. In vitro models of the BM (e.g., Boyden and transwell chambers) are common in cell biology and lab-on-a-chip devices where cells require apical and basolateral polarization. Extravasation, intravasation, membrane transport of chemokines, cytokines, chemotaxis of cells, and other key functions are routinely studied in these models. The goal of the present study was to integrate a semipermeable ultrathin polymer membrane with precisely positioned pores of 2 μm diameter in a microfluidic device with apical and basolateral chambers. We selected poly(l-lactic acid) (PLLA), a transparent biocompatible polymer, to prepare the semipermeable ultrathin membranes. The pores were generated by pattern transfer using a three-step method coupling femtosecond laser machining, polymer replication, and spin coating. Each step of the fabrication process was characterized by scanning electron microscopy to investigate reliability of the process and fidelity of pattern transfer. In order to evaluate the compatibility of the fabrication method with organs-on-a-chip technology, porous PLLA membranes were embedded in polydimethylsiloxane (PDMS) microfluidic devices and used to grow human umbilical vein endothelial cells (HUVECS) on top of the membrane with perfusion through the basolateral chamber. Viability of cells, optical transparency of membranes and strong adhesion of PLLA to PDMS were observed, thus confirming the suitability of the prepared membranes for use in organs-on-a-chip devices.

The tower nozzle solid freeform fabrication technique

Purpose – The purpose of this paper is to develop a simple, easy to implement powder delivery strategy for solid freeform fabrication (SFF) processing.Design/methodology/approach – A specially designed “tower nozzle” located at the center of the processing area dispenses the feedstock powders continuously and uniformly onto the processing area, where powders accumulate progressively as a flat powder bed. During the dispensing, powders are selectively consolidated by melting and solidification using a laser beam which was scanned in a predefined pattern using a galvo‐mirror scan head.Findings – Experiments performed with AISI H13 steel show that the tower nozzle powder delivery strategy is suitable for SFF processing.Practical implications – Both powder delivery and laser consolidation are performed simultaneously and without interruption with simple apparatus. No powder delivery scrapers or rollers are used.Originality/value – The main characteristics of a prototype tower nozzle and the typical processing...

Abstract B45: Mitochondrial Ca2+ transport fine-tunes functional cross-talk between antitumor CD8+T lymphocytes and natural killer cells

Tumor antigen-specific T cell and natural killer (NK) cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of T lymphocyte responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/- and RAG-/-gamma chain-/- mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. The goal of present study was to dissect the molecular mechanisms underlying functional cross-talk between activated CD8+T lymphocytes and naive NK cells. In 2D and 3D co-culture systems, we observed that activated CD8+T cells form multiple intercellular contacts with naive NK lymphocytes. Moreover, co-culture of activated T cells with naive NK cells results in the selective down-regulation of CD25 molecule in T cells while elevating CD25 and CD69 expression on naive NK cells. Further, CD8+T and NK cells cross-regulate their mitochondrial homeostasis including calcium transport. This effect is dependent on both cytokines and intercellular contacts, and partially involves natural killer group 2 member D (NKG2D) receptor activation. Data also indicate that activated CD8+T cells may exchange mitochondria and activation molecules such as CD25 and CD69 with naive NK cells. Alterations in phosporylation status of multiple signaling proteins during CD8+T-NK interaction suggest a cellular remodeling whereby NK cells shift activated CD8+T cells towards T central-memory (TCM) phenotype and activated CD8+T lymphocytes alter the naive state of NK cells towards effector/regulatory phenotype. Inhibition of mitochondrial Ca2+ uptake (mCU) or Na+/Ca2+ exchanger (mNCE) with Ru360 and CGP37157, respectively, mimicked observed alterations in CD8+ T and NK cell phenotypes upon their interaction. These results highlight a novel role of mitochondrial Ca2+ homeostasis in the re-modeling of memory differentiation and activation signaling of interacting CD8+T cells and NK cells, respectively. Improved mechanistic understanding of CD8+T-NK interplay will help refine cellular immunotherapy approach against cancer. Citation Format: Roman V. Uzhachenko, J Shawn Goodwin, Lino Costa, Alexander Terekhov, Menaka C. Thounaojam, William H. Hofmeister, Anil Shanker. Mitochondrial Ca2+ transport fine-tunes functional cross-talk between antitumor CD8+T lymphocytes and natural killer cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B45.

Intravital Microfluidic Windows for Delivery of Chemicals, Drugs and Probes

Intravital imaging, the microscopic visualization of cellular events in living animals, is becoming a powerful tool for the study of many disease processes. The development of the mammary imaging window has been shown to be advantageous for high resolution imaging for the study of cell movements within an organism and can be used to allow the precise observation of cells and tumor angiogenesis for many days [1,2] [3]. A major disadvantage of intravital imaging, however, is that the imaging windows are typically sewn into the skin of the animal, and therefore, the imaging area is not amendable to chemical or drug treatments. The addition of local agents by syringe needle is problematic as injection can elicit unwanted wound responses that complicate elucidation of cellular-response pathways to both secreted factors and pharmacological agents. Systemic treatments can be harmful to the animal and reagents may not make their way to the area under observation. We have designed microfluidic glass windows that will alleviate these complications and provide real time spatial and temporal addition of chemical treatments to cells and tissues.