The invention relates to a method for noninvasive imaging of skin and other tissues using spectroscopy. The invention relates to administering a deuterated imaging agent to a tissue of interest prior to spectroscopic probing. Chemically specific imaging can be achieved by use of specific imaging agents.
There is a need for reliable and precise methods for diagnosing medical abnormalities and for assessing the general condition of body tissues. While any approach that offers early and reliable warning of medical problems has some utility, noninvasive methods offer many advantages. Anticipation by a patient of pain and scarring associated with invasive procedures can cause delays in seeking medical attention. There is also a myriad of inconveniences, risks and difficulties associated with direct collection and contact with patient body fluids. For these reasons, there has been intense scientific and engineering research into devising noninvasive approaches to assessment and diagnosis of medical conditions.
Use of spectroscopic methods, while of considerable use in direct in vitro application to fluids, has not found equal in vivo application. In vivo sampling is substantially more complicated for a variety of reasons, although some of the challenges can be handled by reference to in vitro procedures. Even in vitro procedures require at least some sample preparation before spectroscopic interrogation. And in vivo samples cannot be handled with nearly the ease of in vitro samples.
All chemometric analyses benefit from the availability of samples having known composition of various analytes and having favorable light propagation properties, allowing a straightforward application of Beer""s law. Selectively modulated in vitro samples, or xe2x80x9cexemplarsxe2x80x9d, are much easier to synthesize or otherwise obtain than in vivo exemplars. Thus, samples for chemometric interpretation of in vivo samples can be expected to require specialized approaches to sample preparation and specifically designed methods for obtaining modulated samples of known composition.
Perhaps the most important approach to detecting tissue abnormalities is by direct observation. This requires specialized stains to produce images and in this regard, there is considerable ongoing research. Neither magnetic resonance, radioactive tracers nor fluorescent imaging agents are ideal for in vivo applications because the former lacks sufficient spatial resolution and the latter two have potential toxicity problems.
Long data collection times are needed to extract small signals from some samples, but in vivo sampling requires the patient to endure the waiting. Prolonged data collection is not always practical. Moreover, applying too much excitation light to in vivo samples can lead to catastrophic results. Thus, there remains a need for non-invasive methods for producing spatially resolved images of living tissues.
To address the above-described needs, the invention provides methods and materials for obtaining spatially resolved images of specific types of tissues. In a preferred embodiment, the method for imaging tissue comprises administering to the tissue a deuterated imaging agent and performing spectroscopy, preferably Raman spectroscopy. Electromagnetic radiation, such as a near infrared laser beam, is directed to a tissue of interest. The radiation can be scanned across and within the tissue of interest. When used in combination with a light collection system, it is possible to map out a specific volume of tissue, obtaining information regarding the distribution of specific endogenous chemical species through the imaging of exogenously applied deuterated agents. In some embodiments, specific imaging agents are employed to impart contrast between chemically and physically different types of tissues.
In one embodiment, the analyzing comprises determining a surface fractal dimension of a portion of the tissue having an area and a perimeter. Given suitable images, determining surface fractal dimension involves determining the scaling of the area with the perimeter. This scaling can be measured using a xe2x80x9cbox countingxe2x80x9d procedure from a single image or directly measured using several images obtained on different spatial scales. The method of determining a surface fractal dimension can comprise dividing the area of the portion of tissue by the perimeter of the portion of tissue. It is also possible to obtain a fractal dimension for non-closed loops (e.g., cracks, masses having indistinct borders), also using box counting. The portion of tissue can be a cell, a mass of cells or a tumor.
In another embodiment, a surface fractal dimension is derived from first and second iterations of irradiating the tissue and collecting and analyzing spectra emitted by the tissue. These iterations are performed with first and second regions of the tissue, respectively, wherein the first region of the tissue comprises a portion of the second region of the tissue. The analyzing can then further comprise comparing a quantity of Raman spectra emitted by the first and second regions of the tissue. In one embodiment, the comparing comprises determining a slope of a line connecting first and second points, wherein the first point is a logarithm of total Raman spectra emitted by the first region of the tissue plotted as a function of a logarithm of area of the first region of the tissue, and the second point is a logarithm of total Raman spectra emitted by the second region of the tissue plotted as a function of a logarithm of area of the second region of the tissue. In this manner, multiple regions (e.g., first, second, third, fourth regions, etc.) of overlapping portions of tissue can be probed and analyzed to obtain a surface fractal dimension.
In preferred embodiments, a class of Raman specific agents is employed. The invention provides deuterated imaging agents that are suitable for use with Raman spectroscopy. The general class includes, but is not limited to, water, organic solvents, phospholipids, simple alkyl esters, long chain alkyl esters, long chain alkyl alcohols, fatty acids, urea and its derivatives, and pyrrolidones. Within this general class are preferred compounds such as partially deuterated and perdeutero- {stearic acid, palmitic acid, linoleic acid, oleic acid, mono-, di- and tri-glycerides and glycerol, cholesterol, propylene glycol, 1-8 cineol, 2-n-nonyl-1,3-dioxolane (U.S. Pat. No. 4,861,764), 1-dodecylazacycloheptan-2-one (AZONE), and 4-decycloxazolidin-2-one PERMAC SR-39), and ceramides 1-6: sphinganine, 4-hydroxysphinganine, N-acetylated sphinganine and N-acetylated 4-hydroxysphinganine (N-acetylated by different fatty acids)}. The degree of deuteration of the imaging molecule need not be exhaustive, such that one can balance the ease of obtaining a particular isotopic substitution against the sensitivity and selectivity afforded by that particular molecule. The method can be executed with spatial resolution well below 100 microns (e.g., 5-10 microns), and can provide meaningful information on a one-second time scale. Expect for the use of a benign imaging agent, the method can be performed noninvasively.
In preferred embodiments, the tissue of interest is skin. The tissue can be finger tip, ear lobe, neck, back, leg, arm, shoulder, or other skin regions of interest. Other tissues can be used, including, but not limited to, biopsied tissue and deep tissues accessed surgically. The tissue can be living or dead. Preferably, the tissue is human, or of other mammalian origin.
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