Session V15: Focus Session: Novel Instrumentation and Measurements for Medical and Biological Systems

New impedance and electrochemical image techniques for biological applications

A method to image local surface impedance and electrochemical current optically is developed for biological applications. The principle of the impedance imaging is based on sensitive dependence of surface plasmon resonance (SPR) on local surface charge density. The technique can image local surface impedance and charge while providing simultaneously a conventional surface plasmon resonance (SPR) image. By applying a potential modulation to a sensor surface, it is possible to obtain an image of the DC component, and the amplitude and phase images of the AC component. The DC image provides local molecular binding, as found in the conventional SPR imaging technique. The AC images are directly related to the local impedance of the surface. This imaging capability may be used as a new detection platform for DNA and protein microarrays, a new method for analyzing local molecular binding and interfacial processes and a new tool for imaging cells and tissues.   

Quantification of Nanoscale Density Fluctuations in Biological Cells/Tissues: Inverse Participation Ratio (IPR) Analysis of Transmission Electron Microscopy Images and Implications for Early-Stage Cancer Detection

We report a study of the nanoscale mass density fluctuations of biological cells and tissues by quantifying their nanoscale light-localization properties. Transmission electron microscope (TEM) images of human cells and tissues are used to construct corresponding effective disordered optical lattices. Light-localization properties are studied by statistical analysis of the inverse participation ratio (IPR) of the eigenfunctions of these optical lattices at the nanoscales. Our results indicate elevation of the nanoscale disorder strength (e.g., refractive index fluctuations) in early carcinogenesis. Importantly, our results demonstrate that the increase in the nanoscale disorder represents the earliest structural alteration in cells undergoing carcinogenesis known to-date. Potential applications of the technique for early stage cancer detection will be discussed.   

MRI at 132 $\mu$T for the Detection of Tumors

We are investigating the use of magnetic resonance imaging (MRI), typically at 132 $\mu$T, with enhanced longitudinal-relaxation-time (T$_{1}$)-weighted contrast to detect tumors. We have measured T$_{1}$ of healthy and cancerous prostate tissue specimens--within a few hours of their surgical removal--from approximately 20 patients. The measurements involve a field-cycling imaging technique in which we prepolarize protons in fields up to 150 mT. After this field has been ramped down, the image of each pair of samples is encoded using magnetic field gradients, and the proton nuclear magnetic resonance signal is measured using a SQUID coupled to an untuned, second-derivative gradiometer. The observed T$_{1}$ contrast is significantly greater than that at (say) 1.5 T, suggesting that one may be able to distinguish tumors from healthy tissue without a contrast agent: average T$_{1}$ values at 132 $\mu$T for healthy and cancerous prostate tissue are 60 and 46 ms. We describe a 150-mT prepolarizing coil that will allow the system to be reconfigured in a geometry suitable for in vivo imaging of human prostates.   

Applications of atomic magnetometry in chemical and biological imaging

Atomic magnetometry has been recently developed as the most sensitive technique for detecting magnetic field, especially low-frequency magnetic signal. However, its applications in chemistry and biomedicine have not been extensively explored. In addition, the applications are often limited by the bulky size and high operating temperature of the magnetometers. We report a sensitive and compact atomic magnetometer that has an optimal operating temperature of 37 degree. The small size of the atomic sensors significantly improves the coupling between the sample and the detectors. Using this magnetometer in a scanning detection scheme, we show high-resolution, quantitative imaging of magnetically-labeled antibody binding to targeted molecules. We also show applications of this technique in nuclear magnetic resonance and magnetic resonance imaging in the Earth's magnetic field: a pH-sensitive gadolinium chelate for low magnetic field is revealed, which can be potentially used for minimum-invasive pH mapping; fluid flow in porous metallic materials is measured, which overcomes the penetration problem associated with conventional magnetic resonance imaging. Further improvement for the magnetometer and new detection schemes will be discussed.   

Experimental \textit{In Vitro} Test of Differential `Femton' Oximetry (`DFO') Principle for Noninvasive Quantitative Diagnosis of Hypoxia

We report a high-resolution measurement of feasibility of noninvasive diagnosis of hypoxia \textit{via} difference in $\gamma $ rates from n + O --$>$ O + $\gamma $ + n between tumor, O$_{1}$, and normal tissue, O$_{2, }$using $\lambda _{DB }\approx $ 1 fm neutrons (`femtons'), which `count' atoms unaffected by molecular bonds. Q = (O$_{1 }$-O$_{2})$/O$_{1}$ quantifies hypoxia ($<$0), oxia ($>$0), and healthy tissue (=0). Hypoxic breast/prostate tumors have -0.80$>$ Q $<$-0.96. DNA nucleotides dAdenosine, dCytosine and Thymidine, differing by 1 O atom, were irradiated with 14 MeV n's. We observed 1 atom as 0.012 $\pm $ .004 (3$\sigma ) \quad \gamma $ rate difference. This implies that DFO would diagnose \textit{stand alone} hypoxia Q = -0.10 and -0.25 with specificity 95{\%} and 99{\%}, respectively. As benchmark, we measured relative genome lengths of 2 mammal tissues to be Q = -0.12 $\pm $ .02, vs. 0.1 expected. These data suggest Femton Onco Physics as path to needle-less biopsy.   

Computer Simulation of Specificity for \textit{In Vivo} Breast Cancer Diagnosis by Carbogen-Enhanced Differential `Femton' Oximetry (`DFO')

We performed computer simulation study of noninvasive diagnosis by DFO of hypoxia in a 1 cm tumor in 10 cm breast. Unlike \textit{in vitro} cases (previous Abstract), background $\gamma $'s from non hypoxic tissue will mask tumor $\gamma $ signal, thus true hypoxia Q = -0.9 will be observed as apparent Q' = - 0.1. We propose to amplify Q' by replacing air breathing with carbogen (O$_{2}$ 95{\%}, CO$_{2}$ 5{\%}) thus making use of vasco-constrictive property that carbogen breathing increases O in normal tissue 3-fold, while O in subcutaneous hypoxic tumors remains const. This enhances apparent Q' to -0.25, shown to be detectable by DFO with specificity 99{\%}. Principle of Femto Onco Diagnostics with and w/o carbogen, will be tested on R3230 tumors in Fischer rats at UCI Center for Functional Onco Imaging. Our probe (`OncoSensor') requires imager guidance, except for palpable tumors.   

Detection of benign epithelia, prostatic intraepithelial neoplasia, and cancer regions in radical prostatectomy tissues using Raman spectroscopy

We have studied benign epithelia (BE), prostatic intraepithelial neoplasia (PIN), adenocarcinoma, and cancerous tissues of different Gleason scores in human prostrate bulk tissues using Raman spectroscopy. The data shows two main differences in the Raman spectral features of BE, PIN and cancerous tissues: ($i)$ A strong variations in the peak intensities, (\textit{ii}) shift in certain peak positions. In order to quantify these variations, Raman data were analyzed using chemometric methods of principal component analysis (PCA) and discriminant function analysis (DFA). The PCA and DFA clearly separated the data into three main distinct pathological groups representing BE, PIN and cancer. Similarly the analysis of different Gleason scores shows that the data can be categorized into three distinct groups representing Gleason score 6, 7, and 8. The results demonstrate that Raman spectroscopy can be used to distinguish different stages of the prostrate cancer.   

\emph{In vitro} microfluidic model of sickle cell disease

The pathophysiology of sickle cell disease is complicated by the multiscale processes that link the molecular genotype to the organismal phenotype: hemoglobin polymerization occurring in milliseconds, microscopic cellular sickling in a few seconds or less, and macroscopic vessel occlusion over a time scale of minutes. The rheology of sickle blood, which captures many of these processes, can be studied \emph{in vitro} using physical tools and insights. We present a minimal microfluidic device in which blood flow dynamics can be directly manipulated by modulating physical factors such as oxygen concentration, capillary size, and fluid shear. We have used this system to map out the phase space of blood flow with respect to a combination of geometric, physical, chemical, and biological parameters. We show that morphological changes in erythrocytes due to sickle hemoglobin polymerization and melting are alone sufficient to change blood rheology. We characterize whole blood from many patients in this device and correlate \emph{in vitro} performance to clinical outcomes, suggesting the potential utility of such a device for patient monitoring. Our experimental study integrates the dynamics of many of the processes associated with vasoocclusion and provides a potential tool for optimizing and individualizing treatment, and identifying new therapies.   

An arrayed nanocavity structure for novel spectroscopic chem/bio-sensing

Chemical sensors based on coaxial nanocavity arrays are demonstrated. The high throughput array is developed by overlaying porous Al2O3 and Al layers on vertically aligned carbon nanotube arrays. Adsorption of molecules, such as vapors of water, methanol and ethanol, into the porous Al2O3 layer (the cavity) changes the dielectric properties of the device and leads to detectable changes in the capacitance/impedance of the nanocoax sensor. The detections were conducted in both time and frequency domains with impedance spectroscopy (1Hz-1MHz). Different chemicals exhibited different dose responses and response dynamics. A potentially characteristic 14 kHz shift of peak frequencies was observed between the spectra of acetone and ethanol. This demonstrates the concept of spectroscopic chemical sensing using high density arrays of vertical nanocavity structures. Such techniques will also be applied to biological sensing.   

Characterization of a nanospring-based biosensor by ac impedance spectroscopy

In this study, a process for developing nanospring-based electrical biosensors is presented. Impedance spectroscopy is used to characterize silica (SiO$_{2})$ nanospring-based biosensors. The sensor is a capacitor consisting of two conducting surfaces with silica nanosprings as the dielectric spacer layer. The nanosprings are grown on glass substrates coated with indium tin oxide (ITO) to form one of the electrodes of the capacitor. The top electrode is an ITO coated glass substrate. Placing one slide on top of the other slide produces a capacitor with nanosprings as the dielectric spacer layer. The initial phase of biosensor development is to characterize the response of the device with an aqueous solution consisting of sodium chloride in a phosphate buffer. The experimental impedance data is analyzed using a model equivalent resistor-inductor-capacitor (RLC) circuit. Analysis of the impedance spectra of the nanospring-based biosensor requires a much more complex equivalent circuit relative to the blank biosensor where nanosprings are not present.   

Field-Friendly Tuberculosis Biosensor

Tuberculosis is a fading threat in the United States, but in the developing world it is still a major health-care concern. With the rising number of cases and lack of resources, there is a desperate need for an affordable, portable detection system. Here, we demonstrate the feasibility of a field-friendly immunological biosensor that utilizes florescence and specialized surface chemistries. We observe fluorescently labeled antibodies as they bind to a glass slide. Slides are treated with biotinylated polyethylene glycol to inhibit non-specific interactions and facilitate the binding of primary antibodies allowing for a high degree of specificity. Solutions of tuberculosis-specific antigens where mixed with fluorescently labeled secondary antibodies and incubated on the treated surfaces. An array of different concentrations of antigens bound to fluorescent tags is then read in an epifluorescnece microscope. This assay was used in the portable detector to show that higher concentrations of bound labeled antigens produce a greater emission when excited by a HeNe laser. Home-built electronics, off-the-shelf optics, and a Si photodiode (PD) were used. The data collected from multiple concentrations show a measurable photocurrent. Work is now underway to incorporate a avalanche (PD), flow-cell technology, in a portable box.   

Nanowires as AFM cantilevers: A detection scheme to gently image and interact with soft materials in fluids

Performing AFM on soft materials in fluids (e.g., living cells) is challenging due to their easy deformation by the tip. The thermal force-noise of the cantilever is the principal limitation to reducing sample deformation and minimizing a cantilever's cross- section reduces its noise significantly. However, the minimum size of the cantilever is currently limited by a conventional deflection detection scheme, which requires a large surface area for laser specular reflection. Here we develop an optical technique to use nanowires as cantilevers, and show that we achieve a force noise in water that is orders of magnitude gentler than conventional AFM. This is a significant milestone towards non-invasive scanning probe imaging of biological processes on the surfaces of vesicles and cell membranes.   

A precision force microscope for biophysics

In a typical force spectroscopy experiment, an atomic force microscope (AFM) tip is coupled to a surface-adsorbed protein and force-extension curves are generated by retracting the tip using a piezoelectric (PZT) stage. Force is measured by cantilever deflection; extension is deduced from the PZT stage position used to control the vertical tip position. This deduced extension is sensitive to vertical mechanical drift of the AFM assembly. We have previously developed an ultrastable AFM in which the tip and the sample positions are independently measured by, and stabilized with respect to, a pair of laser foci in three dimensions. These lasers establish a local reference frame that is insensitive to long-term mechanical drift of the AFM assembly. We have now extended the ultrastable AFM capabilities into liquid and can mechanically unfold proteins very slowly, which allows averaging to increase precision. We can also reduce the pulling velocity to zero and stabilize the tip-sample separation while measuring force. Using these techniques, we are studying the unfolding and re-folding of bacteriorhodopsin, a model transmembrane protein.