US 7192783 B2
The Stokes shift emission spectra were measured for various samples, including tissues containing photoactive bio-molecules such as tryptophan, elastin, collagen, NADH and flavin. This new approach allows for the extraction of new information not easily obtained from the excitation and or fluorescence spectra of the same samples. For example, Stokes shift spectroscopy of tissue samples can detect disease states in humans and animals.
1. A method of testing a sample comprising:
a. exciting the sample with excitation radiation and thereby generating emission radiation;
b. varying the excitation radiation by regulating a parameter selected from the group consisting of wavelength and frequency of the excitation radiation; and
c. synchronously scanning the excitation radiation and the emission radiation to produce a spectrum,
wherein a constant interval between the regulated parameter of the excitation radiation and that of the emission radiation is maintained during the synchronous scanning; and
wherein the constant interval is maintained to be substantially the Stokes shift of at least one component of the sample.
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29. A method of testing a sample comprising:
a. exciting the sample with excitation radiation and thereby generating emission radiation;
b. varying the excitation radiation by regulating a parameter selected from the group consisting of wavelength and frequency;
c. synchronously scanning the excitation radiation and the emission radiation to produce a first spectrum, wherein a constant interval between the regulated parameter of the excitation radiation and that of the emission radiation is maintained during the synchronous scanning;
d. adjusting the constant interval and repeating the steps of a)–c) at least one time to produce at least a second spectrum; and
wherein the constant interval is maintained or adjusted to be substantially the Stokes shift of at least one component of the sample.
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This application claims priority from U.S. Provisional Patent Application Ser. No. 60/444,869, which was filed on Feb. 5, 2003.
1. Field of the Invention
The present invention relates generally to the fields of chemical analysis and biomedical diagnostics, and more particularly, to the use of fluorescence and absorption associated with the Stokes shift to determine the molecular components.
2. Description of the Related Art
The emission and absorption of organic molecules in different hosts has been studied since Newton's work on color and dispersion. Information on the state of matter has been obtained by measuring the emission and absorption spectra. For over four decades, spectroscopy has played an important role in understanding the fundamental processes that occur in biology, chemistry, and solid-state physics. In the context of biology, the presence of intrinsic fluorophores such as tryptophan, collagen, elastin, NADH, and flavins in animal and human tissues offer potential opportunities to probe the morphological changes occurring in diseased tissues using cw and time-resolved fluorescence spectroscopy (1).
Since 1984, fluorescence spectroscopy has been used as diagnostic tool to separate cancer, benign and surrounding tissue regions (2-5), Typically, a tissue is excited at a given wavelength to emit radiation at different wavelengths, which characterize the tissue. The wavelengths from UV to blue range (280–520 nm) excite proteins in the tissue associated with changes in structure and molecular content, which gives their spectral fingerprints. The particles in the tissue include tryptophan, collagen, elastin, and NADH.
The underlying dynamics of organic biomolecules occur on the energy configuration coordinate (E-Q) diagram (6-8), where E≡energy and Q≡normalized dimensionless lattice displacement coordinate. The electronic, vibration and rotational states associated with ground and excited states of a biomolecule are denoted on the E-Q diagram. The absorption and emission transition occurs as vertical transition between the states on the E-Q diagram (see
Typically, the peaks of the absorption and the emission occur at different wavelengths. The emission band occurs at lower energy than the excitation band. The shift of the emission and absorption peaks is known as the Stokes shift given by □2Shω. The Stokes shift depends on the polarization of the host environment surrounding the emitting organic molecule. Large shifts are observed for a polar surrounding, such as water. There is dynamic change in the location of energy states associated with this interaction between dipole moment of transition and the surrounding host molecules during excitation and emission (8)
Therefore, one object of the present invention is to develop a new spectroscopic method combining fluorescence and absorption, which is associated with the Stokes shift. This method is called STOKES SHIFT SPECTROSCOPY (SSS).
Another object of the present invention is to develop a new spectroscopic method which has the potential of being much more sensitive for detecting changes associated with native biomarkers in biological tissue and cells that characterize a disease state such as a cancer, precancer, normal or atherosclerosis.
In accordance with one embodiment of the present invention, a method of testing a sample comprises:
In accordance with another embodiment of the present invention, a method of testing a sample comprises:
The Stokes shift emission is measured at a fixed wavelength shift between the absorption and emission wavelengths, Δλ=λE−λA giving the convolution of absorption and emission spectral profiles. The intensity emission is measured and scanned at emission wavelength fixed Δλ=λE−λA. The Stokes shift Δλ=λE−λA can be written in frequency domain Δν=νA−νE, and expressed in units of Hz, THz or cm−1. In this manner, the key molecules involved in the material such as tissue will be highlighted.
In Stokes shift emission spectrum, the excitation and emission wavelength is scanned synchronously with a fixed value of Δλ between excitation and emission wavelength. The intensity of emission is measured at λE. The different molecules in the sample can be excited and revealed, which is much different than fluorescence spectrum excited at a fixed wavelength. The intensity of Stokes shift emission depends on the value of Δλ for each molecular component present in the sample. The maximum intensity occurs when Δλ is near equal to Stokes Shift of particular molecule type excited. The fluorescence peaks of key molecules in tissue can be obtained in one scan by using Stokes shift emission spectrum. The position of peak can be determined by using multiple peaks fit to the SS spectrum. SS spectrum will play an important role in the disease detection, such as cancer and atherosclerosis detection.
In one preferred embodiment, at least one optical fiber is used to propagate the excitation radiation to the sample, or to propagate the emission radiation from the sample to a video system for scanning, or both. The optical fiber may be used as one component of an endoscopic system.
The various features of novelty, which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawing and descriptive matter in which there are illustrated and described preferred embodiments of the invention.
In the drawings:
In accordance with the present invention, the tissue to be tested may be breast, urinary, colon, stomach, brain, prostate, or GYN tissue. The tissue or cell may be in a state selected from the group consisting of normal, cancer, precancer, and atherosclerosis. The wavelength of the excitation may be varied in the range of 200 to 800 nm.
For testing biological molecules of tryptophan, NADH, and flavin, samples of these molecules were obtained from Sigma Company. The tissues were supplied from National Disease Research Interchange (NDRI) under IRB. It was fresh and neither chemically treated nor frozen prior to spectroscopic
A mixture solution containing tryptophan, NADH and flavin was made for the test measurements. The concentration of each component in this composite solution was adjusted to make the fluorescence intensity of each component similar. The Stokes Shift emission spectrum was performed using an automated dual lamp-based spectrophotometer (Mediciscience Technology Corp. CD scanner) by selecting synchronizes scan mode. The wavelength interval between excited and emission monochromators were adjusted to Δλ=λhd E−λA=10, 20, 30, . . . 200 nm or in frequency Δν=νA−νE. The value should be calculated according to corresponding Δλ value or Δν value.
The relationship between wavelength shift Δλ(nm), frequency shift Δν(THz) and frequency shift Δν(cm−1) are shown in the Table 1: (1THz)=33.3564 cm−1
The combined emission and absorption spectrum of biomolecules solution of tryptophan, NADH and flavin was measured and displayed in
The range of Δλ gives a measure of the overlap resolution and correlation of two spectra. The measured Stokes Shift values Δλ=λE−λA for different molecules are listed in Table 2 as follows.
If the regulated parameter in accordance with the present invention is wavelength, the constant interval maintained or adjusted may be within the range of about 40 to 145 nm, preferably about 40 to 120 nm. When the regulated parameter is frequency, the constant interval may be within the range of about 5 to 500 THz or 150 to 15,000 cm−1, preferably 10 to 500 THz or 300 cm−1 to 15,000 cm−1.
The Stokes shift emission spectrum of a tryptophan solution with different Δλ value from 10 nm to 100 nm is shown in
The Stokes shift emission spectrum of normal breast tissue with different Δλ value is displayed in
For spectrum with a different Δλ value, the fit to the curve peaks is nearly the same since the Stokes shift is dependent on the molecular components present (tryptophan, NADH, collagen, flavin and porphyrin).
In conventional fluorescence spectrum, the excited wavelength is fixed in a scan. If the sample includes more than one molecular component, the fluorescence spectrum cannot locate all the peaks in one scan as illustrated in
The invention is not limited by the embodiments described above which are presented as examples only but can be modified in various ways within the scope of protection defined by the appended patent claims.