Mark E. Fauver

Three-dimensional imaging of single isolated cell nuclei using optical projection tomography

A method is presented for imaging single isolated cell nuclei in 3D, employing computed tomographic image reconstruction. The system uses a scanning objective lens to create an extended depth-of-field (DOF) image similar to a projection or shadowgram. A microfabricated inverted v-groove allows a microcapillary tube to be rotated with sub-micron precision, and refractive index matching within 0.02 both inside and outside the tube keeps optical distortion low. Cells or bare cell nuclei are injected into the tube and imaged in 250 angular increments from 0 to 180 degrees to collect 250 extended DOF images. After these images are further aligned, the filtered backprojection algorithm is applied to compute the 3D image. To estimate the cutoff spatial frequency in the projection image, a spatial frequency ratio function is calculated by comparing the extended depth-of-field image of a typical cell nucleus to the fixed focus image. To assess loss of resolution from fixed focus image to extended DOF image to 3D reconstructed image, the 10-90% rise distance is measured for a dyed microsphere. The resolution is found to be 0.9 microm for both extended DOF images and 3D reconstructed images. Surface and translucent volume renderings and cross-sectional slices of the 3D images are shown of a stained nucleus from fibroblast and cancer cell cultures with added color histogram mapping to highlight 3D chromatin structure.

Microfabricated cantilevers for measurement of subcellular and molecular forces

We present two new microfabricated cantilever-beam force transducers. The transducers were fabricated from thin silicon-nitride films, and were used respectively to measure forces generated by two small-muscle preparations: the single myofibril, and the single actin filament in contact with a myosin-coated surface. A simple resonance method was developed to characterize the transducers. Because of the high reproducibility of lever dimensions and the consistency of the modulus of elasticity, few calibration measurements sufficed to characterize the stiffness of all the levers on a single wafer.

Tissue-engineered microenvironment systems for modeling human vasculature

The high attrition rate of drug candidates late in the development process has led to an increasing demand for test assays that predict clinical outcome better than conventional 2D cell culture systems and animal models. Government agencies, the military, and the pharmaceutical industry have started initiatives for the development of novel in-vitro systems that recapitulate functional units of human tissues and organs. There is growing evidence that 3D cell arrangement, co-culture of different cell types, and physico-chemical cues lead to improved predictive power. A key element of all tissue microenvironments is the vasculature. Beyond transporting blood the microvasculature assumes important organ-specific functions. It is also involved in pathologic conditions, such as inflammation, tumor growth, metastasis, and degenerative diseases. To provide a tool for modeling this important feature of human tissue microenvironments, we developed a microfluidic chip for creating tissue-engineered microenvironment systems (TEMS) composed of tubular cell structures. Our chip design encompasses a small chamber that is filled with an extracellular matrix (ECM) surrounding one or more tubular channels. Endothelial cells (ECs) seeded into the channels adhere to the ECM walls and grow into perfusable tubular tissue structures that are fluidically connected to upstream and downstream fluid channels in the chip. Using these chips we created models of angiogenesis, the blood–brain barrier (BBB), and tumor-cell extravasation. Our angiogenesis model recapitulates true angiogenesis, in which sprouting occurs from a “parent” vessel in response to a gradient of growth factors. Our BBB model is composed of a microvessel generated from brain-specific ECs within an ECM populated with astrocytes and pericytes. Our tumor-cell extravasation model can be utilized to visualize and measure tumor-cell migration through vessel walls into the surrounding matrix. The described technology can be used to create TEMS that recapitulate structural, functional, and physico-chemical elements of vascularized human tissue microenvironments in vitro.

Automated cell analysis in 2D and 3D: A comparative study

Optical projection tomographic microscopy is a technique that allows 3D analysis of individual cells. Theoretically, 3D morphometry would more accurately capture cellular features than 2D morphometry. To evaluate this thesis, classifiers based on 3D reconstructions of cell nuclei were compared with 2D images from the same nuclei. Human adenocarcinoma and normal lung epithelium cells were used. Testing demonstrated a three-fold reduction in the false negative rate for adenocarcinoma detection in 3D versus 2D at the same high specificity. We conclude that 3D imaging will potentially expand the horizon for automated cell analysis with broad applications in the biological sciences.

Elastic properties of isolated thick filaments measured by nanofabricated cantilevers.

Using newly developed nanofabricated cantilever force transducers, we have measured the mechanical properties of isolated thick filaments from the anterior byssus retractor muscle of the blue mussel Mytilus edulis and the telson levator muscle of the horseshoe crab Limulus polyphemus. The single thick filament specimen was suspended between the tip of a flexible cantilever and the tip of a stiff reference beam. Axial stress was placed on the filament, which bent the flexible cantilever. Cantilever tips were microscopically imaged onto a photodiode array to extract tip positions, which could be converted into force by using the cantilever stiffness value. Length changes up to 23% initial length (Mytilus) and 66% initial length (Limulus) were fully reversible and took place within the physiological force range. When stretch exceeded two to three times initial length (Mytilus) or five to six times initial length (Limulus), at forces approximately 18 nN and approximately 7 nN, respectively, the filaments broke. Appreciable and reversible strain within the physiological force range implies that thick-filament length changes could play a significant physiological role, at least in invertebrate muscles.

Microfabrication of fiber optic scanners

A cantilevered optical fiber is micromachined to function as a miniature resonant opto-mechanical scanner. By driving the base of the cantilevered fiber at a resonance frequency using a piezoelectric actuator, the free end of the cantilever beam becomes a scanned light source. The fiber scanners are designed to achieve wide field-of-view (FOV) and high scan frequency. We employ a non-linearly tapered profile fiber to achieve scan amplitudes of 1 mm at scan frequencies above 20 KHz. Scan angles of over 120 degree(s) (full angle) have been achieved. Higher order modes are also employed for scanning applications that require compactness while maintaining large angular FOV. Etching techniques are used to create the non-linearly tapered sections in single mode optical fiber. Additionally, micro-lenses are fabricated on the tips of the etched fibers, with lens diameters as small as 15 microns. Such lenses are capable of reducing the divergence angle of the emitted light to 5 degree(s) (full angle), with greater reduction expected by employing novel lens shaping techniques. Microfabricated optical fiber scanners have display applications ranging from micro-optical displays to larger panoramic displays. Applications for micro-image acquisition include small barcode readers to medical endoscopes.

Direct Measurement of Single Synthetic Vertebrate Thick Filament Elasticity Using Nanofabricated Cantilevers

Thick filaments are generally thought to be effectively inextensible. Here we use novel nanofabricated cantilevers to carry out the first direct force-elongation measurements on single vertebrate thick filaments. Cantilevers are ideal for these experiments: force ranges are from pico- to micronewtons, specimens can be visualized during the experiment, and attachment surfaces are in the same plane as the filament. Synthetic thick filaments from rabbit myosin were suspended between two cantilevers and stretched. With stretch, stiffness increased gradually and then became nearly constant after approximately 100 pN. Stretch rate had little or no effect on force-elongation behavior. Under physiological loads (approximately 240 pN axially averaged with full activation) filaments elongated by 1.1 +/- 0.3%. Previous x-ray diffraction results showed a 1.0 to 1.5% increase in myosin head spacing with activation; however, this increase in spacing has been interpreted as change in the state of the cross-bridges, not as elasticity in the thick filament backbone. Comparison with our data suggests that changes in the myosin x-ray reflections seen during activation may be due to elongation of the thick filament backbone. Recognition of thick filament elasticity is important because it affects the interpretation of mechanical experiments and inferences drawn on the molecular mechanism of contraction.

Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system

Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.

Microfabricated optical fiber with microlens that produces large field-of-view video-rate optical beam scanning for microendoscopy applications

Our goal is to produce a micro-optical scanner at the tip of an ultrathin flexible endoscope with an overall diameter of 1 mm. Using a small diameter piezoelectric tube actuator, a cantilevered optical fiber can be driven in mechanical resonance to scan a beam of light in a space-filling, spiral scan pattern. By knowing and/or controlling the fiber position and acquiring backscattered intensity with a photodetector, an image is acquired. A microfabrication process of computer-controlled acid etching is used to reduce the mass along the fiber scanner shaft to allow for high scan amplitude and frequency. A microlens (<1 mm diameter) is fabricated on the end of the optical fiber to reduce divergence of the scanned optical beam. This added mass of the microlens at the free end of the fiber causes the location of the second vibratory node to shift to near the focal length of the microlens. The result is a microlens undergoing angular rotation along two axes with minimal lateral microlens displacement. Preliminary experimental results indicate that this method of optical beam scanning can deliver laser energy over wide fields of view (>50 degrees full angle), up to video scan rates (>10 KHz), while maintaining a scanner diameter of 1 mm. A comparison can be made to bi-axial mirror scanners being fabricated as a MEMS device (micro-electro-mechanical system). Based on the opto-mechanical performance of these microlensed fiber scanners, flexible catheter scopes are possible for new microendoscopies that combine imaging with laser diagnoses.

P-37: Optical Fiber scanning as a Microdisplay source for a Wearable Low Vision Aid

Optical fibers are microfabricated and piezoelectrically vibrated to produce rectilinear scan patterns. By coupling a laser to the optical fiber and projecting the collimated light directly to the viewers eye, the result is a proposed low-cost, spectacle-mounted, wearable low vision aid.