Session M46: Novel Instrumentation and Measurements for Biomedical Research

Investigation of cell morphology for disease diagnostics via high content screening

Ninety percent of all cancer-related deaths are caused by metastatic disease, i.e. the spreading of a subset of cells from a primary tumor in an organ to distal sites in other organs. Understanding this progression from localized to metastatic disease is essential for further developing effective therapeutic and treatment strategies. However, despite research efforts, no distinct genetic, epigenetic, or proteomic signature of cancer metastasis has been identified so far. Metastasis is a physical event: through invasion and migration through the dense, tortuous stromal matrix, intravasation, shear forces of blood flow, successful re-attachment to blood vessel walls, migration, the colonization of a distal site, and, finally, reactivation following dormancy, metastatic cells may share precise physical properties. Cell morphology is the most direct physical property that can be measured. In this work, we develop a high throughput cell phenotyping process and investigate the morphological signature of primary tumor cells and liver metastatic pancreatic cancer cells.   

Multiplexing nano-electroporation for simultaneous transfection of multiple cells

Transfection of biomolecules into cells via electrophoresis across nanochannels, or nano-electroporation, is a recently developed technique shown to deliver precisely controlled dosages with low cell mortality rates. Such advantages are due to the nanochannels used for transfection, which distinguish this technique from bulk and micro-electroporation. Recent demonstrations of nano-electroporation rely on optical tweezers for cell localization, which restrict throughput to sequential electroporation of one cell at a time. In the current work, we overcome this drawback by advancing a multiplexed approach that integrates the nano-channel device with an array of magnetic traps remotely controlled by external magnetic fields. This setup enables multiple magnetically labeled cells to be manipulated in parallel, allowing for simultaneous electroporation of many cells with precisely controlled dosages. After transfection, the cells can be moved downstream for further analysis. Such a magnetically-actuated, remotely-controlled approach for loading of cells and subsequent removal of transfected cells has the potential to transform the current device into an automated platform for simultaneous dosage-controlled biomolecule delivery to large numbers of individual cells.   

Nanopore Mass Spectrometry

We report on the design, construction, and characterization of a nanopore-based ion source for mass spectrometry. Our goal is to field-extract ions directly from solution into the high vacuum to enable unit collection efficiency and temporal resolution of sequential ion emissions for DNA sequencing. The ion source features a capillary whose tip, measuring tens to hundreds of nanometers in inner diameter, is situated in the vacuum $\sim$ 1.5 cm away from an extractor electrode. The capillary was filled with conductive solution and voltage-biased relative to the extractor. Applied voltages of hundreds of volts extracted tens to hundreds of nA of current from the tip. A mass analysis of the extracted ions showed primarily singly charged clusters comprising the cation or anion solvated by several solvent molecules. Our interpretation of these results, based on the works of Taylor and of de la Mora, is that the applied electric stresses distort the fluid meniscus into a Taylor cone, where electric fields reach $\sim$ 1V/nm and induce significant ion evaporation. Accordingly, the abundances of extracted ionic clusters resemble a Boltzmann distribution.   

Coupled External Cavity Photonic Crystal Enhanced Fluorescence

In this work we report a fundamentally new approach to enhance fluorescence in which surface adsorbed fluorophore-tagged biomolecules are excited on a photonic crystal surface that functions as a narrow bandwidth and tunable mirror of an external cavity laser. This scheme leads to $\sim$10x increase in the electromagnetic enhancement factor compared to ordinary photonic crystal enhanced fluorescence. In our experiments, the cavity automatically tunes its lasing wavelength to the resonance wavelength of the photonic crystal, ensuring optimal on-resonance coupling even in the presence of variable device parameters and variations in the density of surface-adsorbed capture molecules. We achieve $\sim$10$^5$x improvement in the limit of detection of a fluorophore-tagged protein compared to its detection on an unpatterned glass substrate. The enhanced fluorescence signal and easy optical alignment make cavity-coupled photonic crystals a viable approach for further reducing detection limits of optically-excited light emitters that are used in biological assays.   

Confocal absorption microscopy of biomolecules and single cells from the visible to the ultraviolet spectral range

We present a versatile approach for absorption spectroscopy on the micron scale that combines a broadband white light source with a confocal microscope and a multichannel detector. The attenuation of the propagating light provides a mechanism for contrast that allows spectrally resolved measurements of biomolecules in minuscule quantities and of single live cells. UV absorption spectra of aromatic amino acids, proteins, and single stranded DNA oligomers (100 bases) in solution are measured with less than 10$^7$ molecules in the probe volume. We discuss applications to spectroscopically identify heterogeneities at the single cell level and to the label-free detection of nucleic acids.   

Reflectance spectrometry of placental vessels in cases of twin-twin transfusion syndrome: experiments and modeling

A stochastic photon transport model in multilayer skin tissue combined with reflectance spectroscopy measurements is used to study placental vessels in cases of twin-twin transfusion syndrome (TTTS). TTTS occurs in about 12{\%} of monozygotic (identical) twin pregnancies wherein flow within placental vessels linking the twins together becomes unbalanced, leading to dual mortality. Endoscopic laser ablation can halt the syndrome by occluding the anastomoses connecting the two fetuses. The objective of this study is to develop a technique to determine hemoglobin (Hb) content through spectral analysis of diffuse reflectance spectra of placental vessels to aid in identification of the anastomoses. Previous work by researchers at Brown University has shown that the reflectance spectra of the donor twin and recipient twin are considerably different in the wavelengths for Hb absorbance. This presentation will give preliminary results for a Monte Carlo model adapted to fit the physiology of the placenta that can be used to quantitative determine the Hb levels. The reflectance spectra of the vessels are simulated for different values of Hb as well oxygenation and water concentration with the vessel and placental mass. The preliminary results will be shown to be in good approximation with the prior experimental data. The combination of modeling with spectroscopic measurement will provide a new tool for detailed prenatal study.   

Microwave Spectrometry for the Assessment of the Structural Integrity and Restenosis Degree of Coronary Stents

Cardiovascular disease is the main cause of death worldwide. Coronary stents are one of the most important improvements to reduce deaths from cardiovascular disorders. Stents are prosthetic tube-shaped devices which are used to rehabilitate obstructed arteries. Despite their obvious advantages, reocclusion occurs in some cases arising from restenosis or structural distortions, so stented patients require chronic monitoring (involving invasive or ionizing procedures). We study microwave scattering spectra (between 2.0 - 18.0 GHz) of metallic stents in open air, showing that they behave like dipole antennas in terms of microwave scattering. They exhibit characteristic resonant frequencies in their microwave absorbance spectra that are univocally related to their length and diameter. This fact allows one to detect stent fractures or collapses. We also investigate the ``dielectric shift'' in the frequency of the resonances mentioned above due to the presence of different fluids along the stent lumen. This shift could give us information about the restenosis degree of implanted stents.   

Higher Resolution and Faster MRI of $^{31}$Phosphorus in Bone

Probing the internal composition of bone on the sub-100 $\mu$m length scale is important to study normal features and to look for signs of disease. However, few useful non-destructive techniques are available to evaluate changes in the bone mineral chemical structure and functional micro-architecture on the interior of bones. MRI would be an excellent candidate, but bone is a particularly challenging tissue to study given the relatively low water density, wider linewidths of its solid components leading to low spatial resolution, and the long imaging time compared to conventional $^1$H MRI. Our lab has recently made advances in obtaining high spatial resolution (sub-400 $\mu$m)$^3$ three-dimensional $^{31}$Phosphorus MRI of bone through use of the quadratic echo line-narrowing sequence (1). In this talk, we describe our current results using proton decoupling to push this technique even further towards the factor of 1000 increase in spatial resolution imposed by fundamental limits. We also discuss our work to speed up imaging through novel, faster reconstruction algorithms that can reconstruct the desired image from very sparse data sets. (1) M. Frey, et al. \textit{PNAS} \textbf{109}: 5190 (2012).   

Accelerated Acquisition of 2D NMR Spectra using Iterative Projections

Typically, in 2D NMR (or 2D MRI), only one ``row'' of the time-dependent (or k-dependent) signal is sampled $N$ times per $\sim T_1$ (spin-lattice relaxation time). Thus, filling a 2D Cartesian grid of $M \times N$ data points requires $M$ additional experiments, for a total spectral acquisition time $T_\textrm{acq} \approx M \times T_1$. Measuring fewer ``rows'' than required for Fourier reconstruction decreases $T_\textrm{acq}$, but this results in a low-quality spectrum (unless more complicated, computationally slower reconstruction techniques are used). Here, we show that a new approach to this problem, using iterative projections, can work on actual 2D NMR data. This approach is built upon the Fast Fourier Transform, so it can handle large data sets (2D, 3D, 4D). Moreover, this approach is expected to work even better in higher dimensions, yielding greater speed ups. Finally, we will discuss how the accelerated acquisition may also improve signal-to-noise and frequency resolution.   

Fast Spectral Reconstruction of Noisy, Sparse Time Domain Data through Iterative Projections

We discuss here an approach for reconstructing spectra from sparse time domain data, by way of iterated projections, and more specifically by alternating projections or by use of the difference map algorithm developed by Veit Elser. This is done in a purely deterministic way, by reformulating any a priori knowledge or constraints into projections, and then iterating. This method is extremely flexible, can be applied to a variety of different signals, and is robust enough to handle real data (with noise and artifacts). In this talk we explain the motivation behind this approach, the formulation of the specific projections, and various methods for handling noise. We will demonstrate the approach using 2D NMR spectra and will compare and contrast this approach with existing methods, such as Maximum Entropy reconstruction.   

Tether-free endoscopic biopsy with self-assembled micro-surgical tools

Feynman's futuristic vision of ``swallowing the surgeon'' or a truly non-invasive surgery relies on the invention and utilization of tetherless, stimuli-responsive and miniaturized surgical tools. We propose a step in this direction by the use of sub-millimeter scale, untethered, self-assembled endoscopic tools by designing and deploying microgrippers ($\mu $-grippers) for effective mucosal sampling from large surface-area organs and for tissue retrieval from hard to reach places in the body. Due to their small size, tether-free actuation, parallel fabrication and deployment, $\mu $-grippers can be dispersed in large numbers (hundreds or thousands) to collect tissue samples and allow statistical sampling of large mucosal areas. Monte Carlo simulations showed that using large number of biopsy tools increases the sampling coverage for screening procedures and hence the chance of detecting the malignant lesions. To establish the feasibility of using $\mu $-grippers for sampling large organs we used with ex-vivo colon and in-vivo esophagus models. Our results showed that it is possible to retrieve high quality tissue samples which are suitable for either conventional cytologic or genetic analyses by using $\mu $-grippers.   

Towards Truly Quiet MRI: animal MRI magnetic field gradients as a test platform for acoustic noise reduction

Clinical MRI acoustic noise, often substantially exceeding 100 dB, causes patient anxiety and discomfort and interferes with functional MRI (fMRI) and interventional MRI. MRI acoustic noise reduction is a long-standing and difficult technical challenge. The noise is basically caused by large Lorentz forces on gradient windings---surrounding the patient bore---situated in strong magnetic fields (1.5 T, 3 T or higher). Pulsed currents of 300 A or more are switched through the gradient windings in sub-milliseconds. Experimenting with hardware noise reduction on clinical scanners is difficult and expensive because of the large scale and weight of clinical scanner components (gradient windings $\sim$ 1000 kg) that require special handling equipment in large engineering test facilities. Our approach is to produce a Truly Quiet (\textless 70 dB) small-scale animal imager. Results serve as a test platform for acoustic noise reduction measures that can be implemented in clinical scanners. We have so far decreased noise in an animal scale imager from 108 dB to 71 dB, a 37 dB reduction. Our noise reduction measures include: a gradient container that can be evacuated; inflatable antivibration mounts to prevent transmission of vibrations from gradient winding to gradient container; vibration damping of wires going from gradient to the outside world via the gradient container; and a copper passive shield to prevent the generation of eddy currents in the metal cryostat inner bore, which in turn can vibrate and produce noise.   

Extracellular recording of \textit{Hirudo medicinalis} neurons using high density, nanocoax neurointerface array

We describe the development of a nanocoax-based neuroelectronic array with submicron pixelation with potential for recording and stimulation with high spatial and temporal resolution. Our device is composed of an array of nanoscale open-ended coaxial electrodes addressed in either a group or individual configuration. As a neuroelectronic interface, our device is characterized by noninvasive real-time coupling to the ganglion sac located along the main nerve cord of the \textit{Hirudo medicinalis}. This allows for extracellular recording of interneural synaptic activity, while also showing the capability of actuating precisely-localized stimulation (faradaic regime). We report on initial results from measurements of electrical signals associated with induced and spontaneous synapse firing in pre- and post-synaptic somata.