The tissue of the extracellular matrix (ECM) are made up of distinct structural proteins, including different types of collagens. Intrinsic autofluorescence lifetimes of ECM proteins offer a non-contact, label-free characterization of tissues. This is especially useful in monitoring wound healing, where differentiating between different tissue matrix signatures can assist in evaluating the tissue structure underneath. Our previous work has demonstrated distinction between collagen types from bovine and human tissues. In this abstract, we are applying our frequency domain imaging technique to look at burn wounds in porcine skin.
In this abstract, we measured normal skin tissues and compared their frequency domain lifetime scans to superficial burn tissues (80°C for 20 s) that have been healing for 21 days. Hairs were plucked from all samples prior to the scans. Each sample was scanned with 5 millimeter by 5 millimeter area at 70 µm step resolution. The scans were modulated at 20 MHz and the acquired tissue intensities were then calculated using Fast Fourier Transform and fitted to a two-exponential decay model per our previous work. The autofluorescence lifetime of the normal skin sample showed a uniform lifetime of 6.9±0.5 ns (n=8). The burn wound tissue showed an increase in lifetime to 7.4±0.6 ns (n=12).
The data suggests that as wound tissue heals, in this case porcine burn model, the autofluorescence lifetime is altered, signifying potential correlation to the collagen remodeling underneath. This can be used to determine the effect of treatments on wound healing in a noninvasive nondestructive manner.
The no-slip boundary condition is a signature characteristic of laminar flow, and the Tesla turbine leverages this condition to transfer mechanical rotation into fluidic pressure. This means that the Tesla turbine is optimally driven by microfluidic flow, which maximizes the use of the boundary layer flow. To this end, we employed a lithography based high resolution 3D printing to realize an embedded micro Tesla pump smaller than the diameter of a penny, integrated to a microfluidic network. The pump was completely sealed in the PDMS (polydimethylsiloxane) device and coupled magnetically to a 3” portable stir plate for rotation. The pump was operated up to 4k rpm (verified with an optical tachometer and slow motion capturing), netting an output pressure of 125 Pa. The pressure transients over time was deconvolved with the rotor transients from the stir plate to yield a pump response function, with a decay constant around 1 second. This means that the pump was able to respond to transients as short as 1 second, and negligible shearing did not affect the rotor-stir plate coupling to ensure efficient pumping. Finally, the pump was applied to drive blue and red dyes into a microfluidic mixer network, demonstrating stable, on-chip fluidic flows without external pumps. The transient characterization of the µTesla pump can provide important insights to the scaling of Tesla pumps and the power transfer between mechanical rotation and fluidic flow, leading to better understanding of the Tesla turbine efficiencies.
Various types of collagens, e.g., type I and III, represent the main load-bearing components in biological tissues. Their composition changes during processes such as wound healing and fibrosis. When excited by ultraviolet light, collagens exhibit autofluorescence distinguishable by their unique fluorescent lifetimes across a range of emission wavelengths. Here, we designed a miniaturized spectral-lifetime detection system as a noninvasive probe for monitoring tissue collagen compositions. A sine-modulated LED illumination was applied to enable frequency domain fluorescence lifetime measurements under three wavelength bands, separated via a series of longpass dichroics at 387, 409, and 435 nm. We employed a lithography-based three-dimensional (3-D) printer with <50 μm resolution to create a custom designed optomechanics in a handheld form factor. We examined the characteristics of the optomechanics with finite element modeling to simulate the effect of thermal (from LED) and mechanical (from handling) strain on the optical system. The geometry was further optimized with ray tracing to form the final 3-D printed structure. Using this device, the phase shift and demodulation of collagen types were measured, where the separate spectral bands enhanced the differentiation of their lifetimes. This system represents a low cost, handheld probe for clinical tissue monitoring applications.
In wounds, cells secret biomolecules such as vascular endothelial growth factor (VEGF), a protein that controls many processes in healing. VEGF protein is expressed in a gradient in tissue, and its shape will be affected by the tissue injury sustained during wounding. In order to study the responses of keratinocyte cell migration to VEGF gradients and the geometric factors on wound healing, we designed a microfluidic gradient device that can generate large area gradients (1.5 cm in diameter) capable of mimicking arbitrary wound shapes. Microfluidic devices offer novel techniques to address biological and biomedical issues. Different from other gradient microfluidics, our device balances diffusion of biomolecules versus the convective clearance by a buffer flow on the opposite ends of the gradient. This allows us to create a large area gradient within shorter time scales by actively driving mass transport. In addition, the microfluidic device makes use of a porous filter membrane to create this balance as well as to deliver the resulting gradient to a culture of cells. The culture of cells are seeded above the gradient in a gasket chamber. However, Keratinocytes do not migrate effectively on filter paper. Therefore, in order to improve the motility of cells on the surface, we coated the filter paper with a 30m thick layer of gelatin type B. after observation under the microscope we found that the gelatin coated sample showed cells with more spread out morphology, with 97% viability, suggesting better adhesion than the non-coated sample.
Various types of collagens, e.g. type I and III, represent the main load-bearing components in biological tissues. Their composition changes during processes like wound healing and fibrosis. Collagens exhibit autofluorescence when excited by ultra-violet light, distinguishable by their unique fluorescent lifetimes across a range of emission wavelengths. Therefore, we designed a miniaturized spectral-lifetime detection system for collagens as a non-invasive probe for monitoring tissue in wound healing and scarring applications. A sine modulated LED illumination was applied to enable frequency domain (FD) fluorescence lifetime measurements under different wavelengths bands, separated via a series of longpass dichroics at 387nm, 409nm and 435nm. To achieve the minute scale of optomechanics, we employed a stereolithography based 3D printer with <50 μm resolution to create a custom designed optical mount in a hand-held form factor. We examined the characteristics of the 3D printed optical system with finite element modeling to simulate the effect of thermal (LED) and mechanical (handling) strain on the optical system. Using this device, the phase shift and demodulation of collagen types were measured, where the separate spectral bands enhanced the differentiation of their lifetimes.
Microfluidic devices offer novel techniques to address biological and biomedical issues. Standard microfluidic fabrication uses photolithography to pattern channels on silicon wafers with high resolution. Even the relatively straightforward SU8 and soft lithography in microfluidics require investing and training in photolithography, which is also time consuming due to complicated thick resist procedures, including sensitive substrate pretreatment, coating, soft bake, expose, post-exposure bake, and developing steps. However, for applications where low resolution (>200 μm) and high turn-around (> 4 designs/day) prototyping are met with little or no lithography infrastructure, robotic cutters [1] offer flexible options for making glass and PDMS microfluidics. We describe the use of robotics cutters for designing microfluidic geometries, and compliment it with safe glass etching, with depths down to 60 μm. Soft lithography patterning of 200 μm thick PDMS membrane was also explored. Without high equipment investment and lengthy student training, both glass and PDMS microfluidics can be achieved in small facilities using this technique.
The overall objective this work is the development of a miniaturized fluorescence spectroscopy analyzer realized via microfabrication technology. Previously, we reported a MEMS micro grating actuated by a piezoelectric cantilever. For such device to be used in a spectroscopic system, optical characterization of the grating's efficiency and the system's stray light are required. We report here the characterization of the grating cantilever with a MEMS micro lens with the intention of fitting into a packaged micro spectroscopic system. This packaging is accomplished by multi-wafer (silicon) bonding of strategically aligned crystalline planes in order to form the basic geometry of a miniaturized spectroscopy setup. One of these crystalline planes, <111> of silicon, is used as a mirror for folding and compacting the optics at the specific angle of 54.74° (with wafer plane normal). The packaging, microlens, and grating cantilever are position in the designed geometry to accept a self-aligned fiber input from a flash lamp source. The microlens component is presented with beam profilometry of its focusing at a focal length of 7.7 mm. The diffraction is interrogated by a monochromator for quantifying the above said characteristics. The relative efficiency of the grating was 40-70% in the 400-600 nm range. Together these characterized components define the geometry and performance of our micro fluorescence spectroscopy system.
Fluorescence spectroscopy plays a key role in a broad area of biological and medical applications. Development of fluorescence spectroscopy micro-devices will enable construction of fully integrated platforms for clinical diagnostics. We report the design, microfabrication and testing of a piezoelectric MEMS micro-grating as a part of the development of a combined spectral/time-resolved fluorescence biosensor for tissue characterization. For the design of the device, we simulated its theoretical performance using a piezoelectric multi-morph model with appropriate diffraction geometry. The microfabrication process was based on a SiN diaphragm (formed via KOH bulk-micromachining) on which the supporting layer of the micro-cantilevers was patterned. Piezoelectric ZnO was then magnetron sputtered and patterned on the cantilever as the physical source for linear actuation with low voltage (>32V). E-beam evaporation of aluminum formed the final reflective diffraction pattern as well as the electrode connections to the device units. The device actuation and displacement were characterized using LDDM (Laser Doppler Displacement Meter). Current cantilevers designed with 500 μm wide gratings (20 μm spacing) produced a maximum 38 μm bi-polar deflection at 3.5 kHz, with scanning from 350-650 nm at 26 nm resolution (10 nm with new 10 μm period prototype). The MEMS device was designed to be integrated with a fast response photomultiplier, and thus can be used with time-resolved fluorescence detection. Because in the case of time-resolved measurements, spectral resolution is not a crucial element, this configuration allows for the compensation of the geometric limitations (linear dispersion) of a micro-scale device that require wavelength differentiation and selection.
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