We demonstrate the use of diffuse optical imaging via transillumination to detect cancerous tissue in a rat animal model.
In this imaging modality infrared radiation is transmitted through whole animal tissue. The radiation is nonionizing and
uses endogenous contrast: namely deoxyhemoglobin (Hb) and oxyhemoglobin (HbO). Differential image analysis is
performed to visualize the presence of cancerous tissue. Varying levels of inspired air and carbogen gases ensure a
differential response in absorption by blood due to changing levels of Hb and HbO. We believe that this response may be
sufficient to provide contrast in differential image analysis. The present method also sheds light on physiological
challenges in whole animal imaging especially with respect to significant optical signals from healthy tissue.
Specifically, we have seen strong signals from abdominal regions in normal rats indicative of diet related anomalous
transmission. We have also been able to track the changes in optical signal during animal death.
We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.
Luminescent emission from lanthanide complexes on silver island films has been studied. Silver films with and without silica spacer layers have been prepared chemically and by thermal vacuum deposition. Enhancement of emission from lanthanide complexes on silver compared to that on bare glass depends on the optical density of the silver films (silver coverage) and sample coverage. A silica spacer layer reduces the number of molecules in quenching regions of the film and leads to better enhancement. Lanthanides with lower quantum efficiencies are expected to exhibit greater enhancement factors and our preliminary results are consistent with that prediction.
We have studied the dynamic changes in tissue vasculature following the inhalation of hyperoxic gasses in rodents as a model for optical breast cancer detection. We have used a CW apparatus to measure the near infrared (NIR) optical properties of animal models immersed in a liquid tissue phantom. By looking at the transmission of different wavelengths in the NIR, we were able to qualitatively observe changes in blood oxygenation following the inhalation of different mixtures of CO2 and O2. These changes enhanced the image contrast between cancerous tissue and normal tissues of the rodents. The oxygenation dynamics of the tumors, following inhalation of the hyperoxic gases, exhibited differences from surrounding tissues in both the magnitude of the observed signal change and the dynamic response.
We demonstrate a novel method for the control of small droplets using laser-based heating. Temperature dependent interfacial surface tensions were the primary force used to move droplets. With this approach, ~1.7 μL to 14 pL droplets were moved on a bare, unmodified polystyrene surface, at speeds of up to 3 mm/s. Upon contact, droplets spontaneously fused and rapidly mixed within 33 ms. We performed an optical absorption-based protein assay using horseradish peroxidase and a chromogenic substrate (ABTS), and readily detected as little as ~125 attomoles of reacting enzyme.
We have prepared and characterized several lanthanide ion complexes of multidentate ligands or chelates in
an effort to develop new luminescent reporters that will be immune to autofluorescence and photobleaching.
Our study has involved the characterization of various chelates of Eu, Er, and Tm with respect to relative
luminescent efficiency and excited state lifetimes. Included in the list of chelates studied are TTFA, EDTA,
DPA, DOTA and DTPA as well as mixed and double chelates. In addition to determining the relative
efficiencies and luminescence lifetimes of the lanthanide chelates, we have explored various excitation
mechanisms and determined optimum excitation wavelengths. This paper will address the various hurdles
encountered in the development of this new class of reporters.
We have recently developed a new type of reporter (upconverting chelate) for biomedical diagnostics. For this reporter, the light is absorbed and emitted by a lanthanide ion, rather than an organic molecule, as is the case for a typical fluorescent dye. These materials do not photobleach and have no autofluorescent background. We focus in this paper on neodymium ions complexed with the familiar chelating agents, EDTA, DPA, DTPA and DOTA. We have performed experimental measurements with one- and two-color laser light excitation for different chelate compounds. The samples are excited using two Nd:YAG-pumped dye laser systems that provide laser light near 587 nm and 800 nm. For one-color excitation, the emitted light depends quadratically on the incident laser power, as expected. Three strongly emitting lines are observed, located near 360 nm, 387 nm, and 417 nm. We observed more efficient upconversion in EDTA although the DPA chelates show comparable ground state absorbance. We have studied the influence of temporal delay between the two laser pulses and obtained the decay lifetime of the first intermediate state in the various chelated compounds.
We are using rodent animal models to study and compare contrast mechanisms for detection of breast cancer. These measurements are performed with the animals immersed in a matching scattering medium. The matching scattering medium or liquid tissue phantom comprises a mixture of Ropaque (hollow acrylic/styrene microspheres) and ink. We have previously applied matched imaging to imaging in humans. Surrounding the imaged region with a matched tissue phantom compensates for variations in tissue thickness and geometry, provides more uniform illumination, and allows better use of the dynamic range of the imaging system. If the match is good, the boundaries of the imaged region should almost vanish, enhancing the contrast from internal structure as compared to contrast from the boundaries and surface topography. For our measurements in animals, the immersion plays two additional roles. First, we can readily study tumors through tissue thickness similar to that of a human breast. Although the heterogeneity of the breast is lost, this is a practical method to study the detection of small tumors and monitor changes as they grow. Second, the immersion enhances our ability to quantify the contrast mechanisms for peripheral tumors on the animal because the boundary effects on photon migration are eliminated. We are currently developing two systems for these measurements. One is a continuous-wave (CW) system based on near-infrared LED illumination and a CCD (charge-coupled device) camera. The second system, a frequency domain system, can help quantify the changes observed with the CW system.
Up converting chelates are a new type of optical reporter with multiple advantages of biomedical diagnostics. We describe these materials and report spectroscopic properties relevant to their use.
We examine the ability to recover the optical properties of a two-layer turbid medium using multi-distance frequency domain reflectance measurements and a hybrid Monte Carlo-- diffusion model. Frequency domain measurements are performed on two-layer liquid tissue phantoms simulating skin on muscle and skin on fat. Particular care to systematic effects in the photomultiplier is required for the measurements at short source-detectors distances. The model converges when fitting five free parameters (the optical properties of the upper and lower layers and the upper layer thickness). However, discrepancies between experimental and model yield insufficient accuracy for the absorption coefficient of the upper layer.
We measure the optical properties of turbid media by performing a spatial scan for homogeneous liquid samples and by immersion into a known reference medium for small secondary samples. We demonstrate that both types of measurements have a precision of better than 1% (standard deviation) and an absolute accuracy of better than 3% (mean error). Furthermore we conduct an experiment measuring the absorption and scattering coefficients of a human hand immersed into a reference medium for a changing level of blood oxygenation.
We have measured the spectroscopic properties of upconverting phosphors relevant to their use of in-vitro diagnostics and other application. These novel reporters have a number of useful properties for biological and medical applications, including no autofluorescent background, no photobleaching, excitation with inexpensive and compact cw diode laser sources, and narrow emission bands. Properties we have measured include the intensity dependence of the absolute phosphorescence power per phosphor particle, the intensity-dependent temporal response, and histograms of phosphorescence intensities on a single particle basis.
We show that the optical properties of a sample with moderate scatter can be obtained by performing frequency domain measurements in a reflection geometry. In experiments and Monte Carlo simulations we show that absorption and scatter produce opposing trends in the amplitude signal and common trends in the phase signal. Therefore a measured amplitude and phase signal correspond to a unique combination of optical properties for a given phase function.
We used frequency-domain measurements with immersion in a matched scattering medium to measure the optical properties of tissue. This approach reduces the effects of boundaries and geometry on the measurements. The diffusion equation, typically used as the basis for determining tissue optical properties, is not valid near a boundary. By immersing the tissue in a scattering medium with nearly the same scattering and absorption coefficients, the boundary effects are greatly reduced and the measurement conditions nearly approximate that in an infinite medium. Also, the measurement is made as a differential against a true homogeneous medium for which the scattering and absorption coefficients can be obtained quite accurately. We demonstrated this approach, using frequency- domain measurements to determine absorption and scattering coefficients of living tissue immersed in diluted intralipid, Ropaque, or milk. Previous approaches to determine optical properties in the frequency-domain used multiple path lengths or multiple frequencies. Immersion is a third frequency-domain approach.
We examined the spectroscopic properties of a novel class of reporters for biomedical diagnostics called upconverting phosphors. These materials exhibit high emission efficiencies, extremely low background levels, no photobleaching, minimal phototoxicity and a variety of emission bands suitable for multi-analyte assays. In addition, they are buffer insensitive and can be excited with inexpensive and compact diode lasers. These properties are very useful for immunoassays, nucleic acid assays, in vivo diagnostics, and other applications. We synthesized submicron monodisperse phosphors and measured the emission spectra, excitation spectra, and power dependencies for a variety of phosphor materials containing different active rare earth ions and host materials. In this paper, we discuss the relation between spectral properties and diagnostic applications.
Performing imaging in the wavelength region of 1 to 1.3 micrometers is attractive because the scattering coefficient is up to a factor of 2 less in this wavelength region than at wavelengths commonly used near 800 nm. While hemoglobin absorption cannot be used for contrast at the longer wavelengths, the requirements for contrast are reduced, and other contrast sources are available. Higher numbers of photons may be used for imaging in this longer wavelength region. Longer wavelength imaging is challenging because the detectors operating beyond 1 micrometers , we have developed upconverting time gates that upconvert images from longer wavelengths (1-1.3 micrometers ) to wavelengths below 1 micrometers where good detectors exist.
Mercury is the only atmospheric pollutant that is present in the atmosphere
in atomic form. Range-resolved Hg mapping can be performed using the
differential absorption lidar (dial) technique employing the 254 nm Hg resonance
line. We have used the lidar technique both for mapping of industrial
plumes and for background concentration measurements. Our studies also include
mercury of geophysical origin. A field test has been performed in Icelandic
geothermal fields. We have also attempted studies of Hg emission from lake
surfaces.
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