Michael P. Houlne

DIAGNOSTIC IMAGING USING RARE-EARTH CHELATES

A new class of polyazamacrocyclic chelates of terbium is studied that have rich spectroscopic properties, tissue selectivity [1], millisecond fluorescence lifetimes, sharply spiked emission spectra (<15 nm FWHM), large Stokes shifts (>280 nm), good water solubility [1] and high fluorescence quantum yields (∼0.6 for PCTMB) [1]. We report our in vitro and ex vivo spectroscopic evaluation of these chelates. Additionally, results from cellular binding specificity studies using Sprague-Dawley rats with UMR 108 osteosarcomas are presented. Finally, endoscopic imaging of dosed tissue demonstrates the potential to use the Tb(III) chelates as contrast enhancement markers.

Endoscopic fluorescence imaging of tissue selective lanthanide chelates

Endoscopic probes have been incorporated into a fluorescent imaging system for the remote quantitation of tissue selective markers. Resolution at the level of 114 line pairs/mm allows visualization of regions that are approximately 6 mm wide. Tissue selective markers, based on polyazamacrocyclic chelates of terbium increase contrast and can be quantified in tissues at the sub-picomole level. Minimally invasive in vivo fluorescence imaging is demonstrated. The potential for application of the system to disease diagnosis and bone graft morphology quantitation is discussed.

Imaging and Quantitation of a Tissue-Selective Lanthanide Chelate Using an Endoscopic Fluorometer

Tissue spectroscopy and endoscopy are combined with a tissue site-selective fluorescent probe molecule to demonstrate in vitro, spatial, remote, quantitative imaging of the rat small intestine. The probe molecule employed, Tb-3,6,9-tris(methylene phosphonic acid n-butyl ester)-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca-1(15),11,13-triene (Tb-PCTMB), is shown to bind with the small intestine and provide improved image contrast. High sensitivity is possible due to the absorption-emission Stokess shift exhibited by the Tb-PTCMB complex. Excitation is centered near 270 nm and multifeatured emission is observed at 490, 550, 590, and 625 nm. Sprague-Dawley rats were dosed with the Tb-PTCMB complex, which shows biodistribution, leading to preferential binding to the inner surface of the small intestine. It is shown that the fluorescent image, taken at 550 nm, can be used to quantify the amount of Tb-PCTMB present in an excised tissue sample. The 3σ detection limits are found to be in the femtomole range. An optical mass balance for Tb-PCTMB-dosed small intestine is performed and along with radiotracer biodistribution, demonstrates that approximately 40% of the marker probe resides in the endothelial tissue of the small intestine inner lumen. This result is of particular interest since most adult colon cancers develop in this region. These results demonstrate the ability to perform spatial, quantitative, in vitro, endoscopic imaging of a complex biological sample using a probe marker.

Characterization of microendoscopic imaging systems for tissue spectroscopy

Micro-endoscopes based on a fiber optic conduit and thin rod lens are investigated for applications in tissue diagnostics and studies of fluid transport in tissue-like matrices. The design, evaluation and implementation of these moderate to high resolution optical instruments is presented. The optical configuration employs a micro-endoscopic probe, an objective lens and a commercial color CCD. The flexible micro-endoscope is 2.65 meters in length, with an outer diameter of 1.5 mm and is employed to quantitatively measure EBT transport through a tissue-like substrate. The determined resolution is 20 line-pairs/mm (LP/mm). Calibration is accomplished by measuring absorbance of Eriochrome Black T dye against a reflective background. Fluorescence detection of fluorescein is evaluated using a thin rod lens micro- endoscope. The rod lens is 210 mm in length, 3 mm in outside diameter and can resolve 160 LP/mm. This resolution allows imaging of cellular organelles having a diameter of 2.8 mu;m. The limit of detection of the rod lens endoscopic system is approximately 20 pmol for fluorescein.

Remote semiquantitative two-dimensional flow transport imaging using microendoscopic probes

A flexible microscope is configured, evaluated, and employed in the noninvasive investigation of fluid transport in a tissuelike model or porous, fibrous matrix. A simple optical configuration that employs microendoscopic probes, an objective lens, and a commercial CCD for detection, produces resolution performance in the range of 25 to 40 line pairs/mm (lp/mm). Remote, time-sequenced imaging is shown to be possible using this moderate resolution flexible microscope. The microendoscopes employed are 1 to 3 m in length and range in outer diameter from 1.5 to 2.5 mm; some contain a working lumen for the introduction of a tool or a fluid at the site of investigation. Target lighting is accomplished by an integral fiber bundle built into the endoscope and supplied from an external xenon discharge lamp. This instrumentation is combined with specialized image processing for quantitative, time-sequenced imaging of marker dye transport on a tissuelike model substrate. Quantitative absorbance imaging is accomplished by calibrating response for marker dye standards on the test substrate and correlating camera pixel grayscale values to solute concentration. The quantitative mapping (concentration versus time and position) of fluid transport is used to evaluate convective versus diffusive flow.

Spectroscopic imaging of tissues using micro-endoscopy

Recent advances in optics and CCD imaging technology have made possible the advent of small, position sensitive, articulating endoscopes allowing visual access to small cavities within the human body. We present our results for the design, construction and evaluation of a spectrometer capable of measuring molecular fluorescence phenomena as a function of wavelength, position and intensity in small cavities. State-of-the-art micro-endoscope technology is combined with wavelength sorting and CCD imaging to accomplish remote imaging. Presently, images can be magnified by 50x with visual resolution of about 50 LP/mm by direct observation of a USAF resolution target. We detail our biological cell imaging results where a special class of fluorescent molecules (chelate complexes of lathinide (III) polyazamacrocyclic acetates containing pyridine) are introduced into osteosarcoma tissue (a bone cancer tissue). It is shown that the unique molecular site selectivity of the Tb-+3 compound combined with the microendoscopic spectrometer is a useful tool for osteosarcoma rat host investigations in-vivo. We demonstrate that direct observation of the probe molecules in the tissue can yield physiological information regarding interstitial fluid flow in the osteosarcoma tumors.<<ETX>>

Transport investigations of terbium chelate complexes in a type 1 collagen tissue model using quantitative micro-endoscopic imaging

The diffusive transport characteristics of a unique class of small fluorescent molecular probes in an interstitial tissue model are investigated using micro-endoscopy. The probes employed in the present work are organo-metallic complexes of polyazamacrocycles chelated to Terbium. These particular molecules have large Stokes shifts, making them amendable to tissue analysis. The delocalized electronic structure of the organic chelate absorbs ultra-violate light (approximately 270 nm) and, after inter-molecular transfer, the lanthanide cation fluoresces in the visible region (550 nm). The diffusive transport properties of the probe molecules are related to their chemical structure, which governs their affinity toward the components of the interstitial model. The basic polyazamacrocycle is functionalized with three phosphate groups. Presently, methyl, ethyl, propyl and butyl alkyl chains are added to the phosphate groups on the polyazamacrocycle to modify the affinity of the probes toward the components of the interstitial model. Micro-endoscopy coupled with digital imaging allows remote, quantitative analysis of the transport process in near real time. Cross sectional analysis of the images yields the concentration profile of the probe as it diffuses through the gel. The concentration profile is fit to Ficks second law of diffusion to determine the diffusion coefficient, D, for each of the problem molecules. Presently the measured D values for each of the compounds are typical for small molecules in water (approximately 10-6 cm2/sec), however, D is observed to increase with decreasing hydrocarbon chain length which demonstrates interstitial transport is structurally dependent.

Structure-dependent spectroscopic properties of europium and terbium chelate complexes used in biomedical imaging analysis

Quantum efficiency, (Phi) , and molar absorption coefficients, (epsilon) , are determined for Terbium and Europium polyazamacrocylic, phosophonic acid chelating agents that have been shown to be used in biomedical imaging applications.

Refractive-index-based calorimetric studies of RNAse T1 unfolding in small volumes using microinterferometric backscatter

Micro-interferometry, a novel technique developed by the authors, employs a linearly polarized laser, a fused silica capillary tube housing for the sample and a charged coupled device as a detector. A back scattered interference pattern, observed as a high contrast fringes, is produced when the laser is directed onto the capillary containing the sample. The positional change of the fringe pattern is a function of the refractive index of the media in the capillary. In the present work, the RNA enzyme RNase T1 is heated in the sample cell over a temperature range of 30 degrees C to 60 degrees C. Over this temperature range the molecule unfolds form the quaternary to the tertiary structure. This structure change is manifested as a refractive index change and is observed by monitoring the fringe position while ramping the cell temperature in a controlled fashion. From the refractive index response over the temperature range, the Gibbs free energy associated with unfolding is calculated. The authors show milli-degree temperature stability with a 0.1 micro-liter probe volume, thus demonstrating the application of this device in micro- calorimetric investigations.

Spectroscopic evaluation of polyazamacrocyclic Tb(III) chelates for application in diagnostic imaging

The use of fluorescence probes for in-vivo diagnostics is at the forefront of medical science. To transition this technique into the clinical environment, quantitative spectral analysis and knowledge of the cellular interactions of the marker probes is vital. Furthermore, fluorescence intensity and lifetime changes, as a function of physiological environment, represents a diagnostic opportunity. A new class of polyazamacrocyclic chelates of Terbium have been identified with rich spectroscopic properties. These chelates are tissue selective, have fluorescence lifetimes on the order of milliseconds, sharply spiked emission spectra (< 15 nm FWHM), large Stokes shifts (> 280 nm), good water solubility and high quantum yields (approximately 0.6 for PCTMB). We will present our in-vitro and in-vivo spectroscopic evaluation of the chelates. In addition to the spectral investigations, results from cellular binding specificity studies using Sprague-Dawley rats with UMR 108 osteosarcomas will be presented. The potential to use the Tb(III) chelates as neoplastic tissue markers will be discussed.