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.
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 Stoke's 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 Fick's 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.
Flexible micro-endoscopes with dimensions of as small as 1.0 mm in outer diameter and as long as 3.0 m in length produce images that have a 'honeycomb' pattern due to the spaces between the individual collection optical fibers contained in the imaging conduit. This pattern is found to exhibit a definable spatial frequency that is discrete from that of the desired information or the actual images of interest. By applying a filter and by sharpening the contrast between adjacent pixels, it was possible to remove the honeycomb pattern without significant degradation to the visual quality of the image. The technique described employs Fourier analysis to analyze the image so as to define the 'noise' component. Then a discrete band-reject frequency filter was applied to both the original and sharpened images, resulting in the effective removal of the honeycomb pattern. The advantages and limitations of the image processing technique are discussed.
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.
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.
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.
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