Femtosecond laser-patterned nanopore arrays for surface-mediated peptide treatment.

The major goal of this study was to create easy-to-use, reusable substrates capable of storing any peptides or bioactive molecules for a desired period of time until cells uptake them without the need for bioactive molecule or peptide-specific techniques. Nanopore arrays of uniform size and distribution were machined into fused silica substrates using femtosecond laser ablation and loaded with peptides by simple adsorption. The nanopore substrates were validated by examining the effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) loaded nanopores on macrophage phagocytosis and intracellular production of reactive oxygen species (ROS) with and without the pro-inflammatory lipopolysaccharide (LPS). Our results demonstrated that nanopores were generated in a uniform array fashion. Ac-SDKP peptides were stably stored in nanopores and internalized by macrophages. Significant reductions in ROS production and phagocytosis in macrophages were observed over control substrates, even in combination with LPS stimulation, indicating that loading Ac-SDKP peptides in pores significantly improved the anti-inflammatory effects. FROM THE CLINICAL EDITOR: This team of scientists intended to create easy-to-use, reusable substrates for storing peptides or bioactive molecules for a desired period of time before cellular uptake occurs, and without the need for bioactive molecule or peptide-specific techniques. They demonstrate the successful generation of nanopores in a uniform array that stably stores Ac-SDKP peptides in the nanopores. When peptides were internalized by macrophages, significant reductions in ROS production and phagocytosis were observed, indicating improved anti-inflammatory effects.

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.

Rapid, single-molecule assays in nano/micro-fluidic chips with arrays of closely spaced parallel channels fabricated by femtosecond laser machining.

Cost-effective pharmaceutical drug discovery depends on increasing assay throughput while reducing reagent needs. To this end, we are developing an ultrasensitive, fluorescence-based platform that incorporates a nano/micro-fluidic chip with an array of closely spaced channels for parallelized optical readout of single-molecule assays. Here we describe the use of direct femtosecond laser machining to fabricate several hundred closely spaced channels on the surfaces of fused silica substrates. The channels are sealed by bonding to a microscope cover slip spin-coated with a thin film of poly(dimethylsiloxane). Single-molecule detection experiments are conducted using a custom-built, wide-field microscope. The array of channels is epi-illuminated by a line-generating red diode laser, resulting in a line focus just a few microns thick across a 500 micron field of view. A dilute aqueous solution of fluorescently labeled biomolecules is loaded into the device and fluorescence is detected with an electron-multiplying CCD camera, allowing acquisition rates up to 7 kHz for each microchannel. Matched digital filtering based on experimental parameters is used to perform an initial, rapid assessment of detected fluorescence. More detailed analysis is obtained through fluorescence correlation spectroscopy. Simulated fluorescence data is shown to agree well with experimental values.

Silica coating of polymer nanowires produced via nanoimprint lithography from femtosecond laser machined templates.

In this paper we report on the fabrication of regular arrays of silica nanoneedles by deposition of a thin layer of silica on patterned arrays of polymer nanowires (or polymer nanohair). An array of high-aspect-ratio nanoscale diameter holes of depths greater than 10 µm was produced at the surface of a fused silica wafer by an amplified femtosecond laser system operated in single-pulse mode. Cellulose acetate (CA) film was imprinted into the nanoholes and peeled off to form a patterned array of standing CA nanowires, a negative replica of the laser machined nanoholes. The cellulose acetate replica was then coated with silica in a chemical vapor deposition process using silicon tetrachloride vapor at 65 °C. Field emission scanning electron microscopy, focused ion beam sectioning, energy dispersive x-ray analysis and Fourier-transform infrared spectroscopy were used to characterize the silica nanoneedles. Precisely patterned, functionalized arrays of standing silica nanoneedles are useful for a number of applications.

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.

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.

Single-pulse ultrafast-laser machining of high aspect nano-holes at the surface of SiO2.

Use of high numerical aperture focusing with negative longitudinal spherical aberration is shown to enable deep (> microm), high aspect ratio, nano-scale-width holes to be machined into the surface of a fused-silica (SiO(2)) substrate with single pulses from a 200 fs, 4 microJ Ti-Sapphire laser source. The depths of the nano-holes are characterized by use of a non-destructive acetate replication technique and are confirmed by imaging of sectioned samples with a dual focused ion beam/scanning electron microscope.

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.

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

BACKGROUND: The 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. RESULTS: We 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. CONCLUSION: This approach meets the need for scalable, highly repeatable, and tuneable models that mimic extracellular matrix features that signal for maintenance of hMSC stemness.

Cell interaction study method using novel 3D silica nanoneedle gradient arrays.

Understanding cellular interactions with culture substrate features is important to advance cell biology and regenerative medicine. When surface topographical features are considerably larger in vertical dimension and are spaced at least one cell dimension apart, the features act as 3D physical barriers that can guide cell adhesion, thereby altering cell behavior. In the present study, we investigated competitive interactions of cells with neighboring cells and matrix using a novel nanoneedle gradient array. A gradient array of nanoholes was patterned at the surface of fused silica by single-pulse femtosecond laser machining. A negative replica of the pattern was extracted by nanoimprinting with a thin film of polymer. Silica was deposited on top of the polymer replica to form silica nanoneedles. NIH 3T3 fibroblasts were cultured on silica nanoneedles and their behavior was studied and compared with those cultured on a flat silica surface. The presence of silica nanoneedles was found to enhance the adhesion of fibroblasts while maintaining cell viability. The anisotropy in the arrangement of silica nanoneedles was found to affect the morphology and spreading of fibroblasts. Additionally, variations in nanoneedle spacing regulated cell-matrix and cell-cell interactions, effectively preventing cell aggregation in areas of tightly-packed nanoneedles. This proof-of-concept study provides a reproducible means for controlling competitive cell adhesion events and offers a novel system whose properties can be manipulated to intimately control cell behavior.

Poly(ε-caprolactone)-carbon nanotube composite scaffolds for enhanced cardiac differentiation of human mesenchymal stem cells.

AIM: To evaluate the efficacy of electrically conductive, biocompatible composite scaffolds in modulating the cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs). MATERIALS & METHODS: Electrospun scaffolds of poly(ε-caprolactone) with or without carbon nanotubes were developed to promote the in vitro cardiac differentiation of hMSCs. RESULTS: Results indicate that hMSC differentiation can be enhanced by either culturing in electrically conductive, carbon nanotube-containing composite scaffolds without electrical stimulation in the presence of 5-azacytidine, or extrinsic electrical stimulation in nonconductive poly(ε-caprolactone) scaffolds without carbon nanotube and azacytidine. CONCLUSION: This study suggests a first step towards improving hMSC cardiomyogenic differentiation for local delivery into the infarcted myocardium.

An amplified femtosecond laser system for material micro-/nanostructuring with an integrated Raman microscope.

In order to obtain new insights into laser-induced chemical material modifications, we introduce a novel combined approach of femtosecond pulsed laser-direct writing and in situ Raman microscopy within a single experimental apparatus. A newly developed scanning microscope, the first of its kind, provides a powerful tool for micro-/nanomachining and characterization of material properties and allows us to relate materials' functionality with composition. We address the issues of light delivery to the photomodification site and show the versatility of the system using tight focusing. Amplified femtosecond pulses are generated by a Ti:sapphire laser oscillator and a chirped-pulse regenerative amplifier, both pumped by a diode-pumped frequency doubled neodymium-doped yttrium orthovanadate (Nd:YVO(4)) laser operating at 532 nm. Results of Raman spectroscopy and scanning electron microscopy images of femtosecond laser micro-/nanomachining on the surface and in the bulk of single-crystal diamond obtained from first trials of this instrument are also presented. This effective combination could help to shed light on the influence of the local structure fluctuations on controllability of the laser processing and the role of the irradiation in the ablation processes ruling out possible imprecisions coming from the use of the two independent techniques.