Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2002-12-18
2004-11-30
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C250S458100, C600S317000
Reexamination Certificate
active
06825928
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the invention is fluorescence spectroscopy and imaging, and in particular, the use of fluorescence to detect epithelial pre-cancers and cancers.
Fluorescence spectroscopy and imaging in the ultraviolet-visible (UV-VIS) wavelength spectrum is an exciting new modality for detecting human epithelial pre-cancer and cancer. Fluorescence spectroscopy is performed by irradiating the tissue surface with light and detecting the fluorescent light emitted by “fluorophores” in the tissue. The fluorophores may be “endogenous” molecules that absorb the impinging photons and emit photons at a different wavelength, or they may be “exogenous” fluorophores such as injected photosensitizing agents. This emerging technology has shown promising results for detecting early neoplastic growth in a variety of organ sites including the colon, bronchus, cervix, oral cavity, skin and bladder. Noninvasive and fast detection of epithelial pre-cancers and early cancers through the use of fluorescence spectroscopy can significantly improve the efficacy and reduce cost of cancer screening and diagnostic programs.
One of the most widely explored applications of fluorescence spectroscopy is the detection of endoscopically invisible, early neoplastic growth in epithelial tissue sites. Early neoplastic growth refers to pre-malignant changes such as dysplasia and carcinoma in situ (CIS), which precede malignancy, i.e., invasive carcinoma. Currently, there are no effective and commonly accepted diagnostic techniques for these early tissue transformations. Fluorescence spectroscopy is ideally suited for this application because of its ability to examine tissue surfaces, rather than tissue volumes, and the ability to deploy this technology in an endoscopic device. If fluorescence spectroscopy can be applied successfully as a diagnostic technique in this clinical context, it may increase the potential for curative treatment, and thus, reduce complications. In addition to the potential for improved patient outcome, the fast and noninvasive nature of this diagnostic technique may also reduce health care costs.
Referring to
FIG. 1
, when a biologic molecule is illuminated at an excitation wavelength, which lies within the absorption spectrum of that molecule, it will absorb photons' energy and be activated from its ground state (state of lowest energy; S
0
) to an excited state (state of higher energy; S
1
). The molecule can then relax back from the excited state to the ground state by generating energy in the form of fluorescence, at emission wavelengths, which are longer than the excitation wavelength. The phenomenon of fluorescence displays several general characteristics for a particular biologic molecule. First, due to losses in energy between absorption and emission, fluorescence occurs at emission wavelengths, which are always red-shifted, relative to the excitation wavelength. Second, the emission wavelengths of fluorescence are independent of the excitation wavelength. Third, the fluorescence spectrum of a biologic molecule is generally a mirror image of its absorption spectrum. The fluorescence of a biologic molecule is characterized by its quantum yield and its lifetime. The quantum yield is simply the ratio of the energy converted to fluorescence to the energy absorbed. The lifetime is defined as the average time the biologic molecule spends in the excited state before returning to the ground state. The fluorescence quantum yield and lifetime are modified by a number of factors that can increase or decrease the energy losses. For example, a molecule may be non-fluorescent as a result of a large rate of non-radiative decay (thermal generation).
Fluorescence spectroscopy is the measurement and analysis of various features that are related to the fluorescence quantum yield and/or lifetime of a biologic molecule (s). The fluorescence intensity of a biologic molecule is a function of its concentration, its extinction coefficient (absorbing power) at the excitation wavelength, and its quantum yield at the emission wavelength. A fluorescence emission spectrum represents the fluorescence intensity measured over a range of emission wavelengths, at a fixed excitation wavelength. Conversely, a fluorescence excitation spectrum is a plot of the fluorescence intensity at a particular emission wavelength, for a range of excitation wavelengths. A fluorescence, excitation-emission matrix (EEM) is a two dimensional contour plot, which displays the fluorescence intensities as a function of a range of excitation and emission wavelengths. Each contour represents points of equal fluorescence intensity. Finally, fluorescence lifetime measurements are represented as the fluorescence intensity distributed over a very short time scale, at a fixed excitation-emission wavelength pair.
FIGS. 2
a
-
2
d
are graphic illustrations of a fluorescence (a) emission spectrum, (b) excitation spectrum, (c) EEM and (d) decay profile.
Table 1 shows a list of biologic endogenous fluorophores and their excitation and emission maxima. These endogenous fluorophores include the amino acids, structural proteins, enzymes and coenzymes, vitamins, lipids and porphyrins. Their excitation maxima range lies between 250 and 450 nm (which spans the ultraviolet and visible spectral range), whereas their emission maxima range lies between 280 and 700 nm (which spans the ultraviolet, visible and near-infrared spectral range). Fluorophores that are believed to play a role in transformations that occur in the neoplastic process in tissue, are the amino acids, tryptophan, the structural protein, collagen, the co-enzymes, NADH and FAD and porphyrins.
TABLE 1
Endogenous
Excitation Maxima
Emission Maxima
Fluorophores
(nm)
(nm)
Amino acids
Tryptophan
280
350
Tyrosine
275
300
Phenylalanine
260
280
Structural Proteins
Collagen
325, 360
400, 405
Elastin
290, 325
340, 400
Enzymes and Coenzymes
FAD, Flavins
450
535
NADH
290, 351
440, 460
NADPH
336
464
[NADH, reduced nicotinamide dinucelotide; NAD(P)H, reduced
nicotinamide dinucleotide phosphate; FAD, flavin adenine dinucleotide.]
Vitamins
Vitamins A
327
510
Vitamins K
335
480
Vitamins D
390
480
Vitamins B
6
compounds
Pyridoxine
332, 340
400
Pyridoxamine
335
400
Pyridoxal
330
385
Pyridoxic acid
315
425
Pyridoxal
5′-330
400
phosphate
Vitamin B
12
275
305
Lipids
Phospholipids
436
540, 560
Lipofuscin
340-395
540, 430-460
Ceroid
340-395
430-460, 540
Porphyrins
400-450
630, 690
Fluorescence spectroscopy of turbid media such as tissue depends on any of a number of factors. It depends on the concentration and distribution of fluorophore(s) present in the tissue, as well as the biochemical/biophysical environment, which may alter the quantum yield and lifetime of the fluorophore(s). For example, epithelial tissues, generally have two primary sub-layers: a surface epithelium and an underlying stroma or submucosa; the specific fluorophores, as well as their concentration and distribution can vary significantly in these two tissue layers with a neoplastic change. Fluorescence spectroscopy of turbid media such as tissue also depends on the absorption and scattering that results from the concentration and distribution of nonfluorescent absorbers and scatterers, respectively, within the different sub-layers of the tissue.
The effect of the aforementioned factors on fluorescence spectroscopy of tissue is wavelength-dependent. First, the fluorophores that have absorption bands that lie in the same wavelength range as the excitation light will be excited and hence, emit fluorescence. The absorption and scattering properties of the tissue will affect light at both excitation and emission wavelengths. Therefore, only those fluorophores contained in the tissue layers to which the excitation light penetrates and from which, the emitted light can escape the tissue surface will produce measurable fluorescence. Elastic scattering events in tissue are caused by random spatial variations in the density, refractive index, and dielectric constants of extracellular, cellular and subcellular
Liu Quan
Ramanujam Nirmala
Zhu Changfang
Evans F. L.
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
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