The present invention is directed to a method of detecting a neurodegenerative disease in a mammal by activating brain tissue of the mammal by application of radiation under conditions effective to promote a simultaneous multiphoton excitation of the brain tissue and to emit a fluorescence characteristic. The fluorescence characteristic is then compared to a standard fluorescence emitted by exciting healthy brain tissue of the mammal under the same conditions used to carry out the activating step. Brain tissue where the fluorescence characteristic differs from the standard fluorescence is identified as potentially having a neurodegenerative disease. Another aspect of the present invention is directed to a method of producing an image of brain tissue from a mammal by activating brain tissue of a mammal with radiation applied under conditions effective to promote a simultaneous multiphoton excitation of the brain tissue and to produce fluorescence. The fluorescence is then collected to produce an image of the brain tissue.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is directed to a method of detecting a neurodegenerative disease in a mammal by activating brain tissue of the mammal by application of radiation under conditions effective to promote a simultaneous multiphoton excitation of the brain tissue and to emit a fluorescence characteristic. The fluorescence characteristic is then compared to a standard fluorescence emitted by exciting healthy brain tissue of the mammal under the same conditions used to carry out the activating step. Brain tissue where the fluorescence characteristic differs from the standard fluorescence is identified as potentially having a neurodegenerative disease.

Another aspect of the present invention is directed to a method of producing an image of brain tissue from a mammal by activating brain tissue of a mammal with radiation applied under conditions effective to promote a simultaneous multiphoton excitation of the brain tissue and to produce fluorescence. The fluorescence is then collected to produce an image of the brain tissue.

The current state of the art is that diagnosis of Alzheimer's Disease occurs only after clinical manifestations of marked memory loss and other cognitive impairments occur. Neuropathological studies suggest that the disease process actually starts many years prior to these overt symptoms. An imaging technology that allows detection of the pathological process prior to clinical symptoms would allow treatments aimed at prevention of progression rather than simply treatment of symptoms. Ideally, a biomarker of this nature can be used for presymptomatic identification of patients, as a method of following the efficacy of therapy, and as a method to direct different therapies. Each of these applications would provide a substantial advance over the current state of affairs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of detecting a neurodegenerative disease in a mammal by activating brain tissue of the mammal by application of radiation under conditions effective to promote a simultaneous multiphoton excitation of the brain tissue and to emit a fluorescence characteristic. The fluorescence characteristic is then compared to a standard fluorescence emitted by exciting healthy brain tissue of the mammal under the same conditions used to carry out the activating step. Brain tissue where the fluorescence characteristic differs from the standard fluorescence is identified as potentially having a neurodegenerative disease.

Another aspect of the present invention is directed to a method of producing an image of brain tissue from a mammal by activating brain tissue of a mammal with radiation applied under conditions effective to promote a simultaneous multiphoton excitation of the brain tissue and to produce fluorescence. The fluorescence is then collected to produce an image of the brain tissue.

FIGS. 1A-C (see Original Patent) show different embodiments for imaging neurodegenerative disease in a patient in accordance with the present invention.

FIG. 1A (see Original Patent) illustrates how a patient P's skull is imaged by placing imaging device 2 against the skull.

FIG. 1B (see Original Patent) carries out imaging with a spectroscopic system. This system includes dichroic mirror 4, lens 6, spectroscopic selection device 8, and photo-detector 10. In use, multiphoton radiation L from a laser is directed against dichroic mirror 4 which redirects this radiation through lens 6. Lens 6 is placed against a thin part of the skull of patient P so that multiphoton radiation L is passed through the patient's skin, skull, duramater, arachnoid and subarachnoid, and piamater and into the cortex. Within the cortex, radiation L causes fluorescence F to occur and this fluorescence passes through lens 6, past dichroic mirror 4, and to spectroscopic selection device 8 which is provided with photodetector 10 . Photodetector 10 receives fluorescence F passed to it by spectroscopic selection device 8 and causes an image to be created. This image is then examined for the purpose of diagnosing Alzheimer's Disease or other neurodegenerative diseases.

FIG. 1C (see Original Patent) shows a second embodiment for carrying out the method of the present invention. In this version of the present invention, single mode optical fiber 100, terminal lens 102, and detector 104 are used to carry out the imaging procedure of the present invention. In use, multiphoton radiation L is passed through the patient's skin, skull, duramater, arachnoid and subarachnoid, and piamater and into the cortex or some or all of these layers need to be penetrated to allow penetration of the fiber closer to the cortex surface. Within the cortex, radiation L causes fluorescence F to occur and this fluorescence passes into detector 104 where an image is created. This image is then examined for the purpose of diagnosing Alzheimer's Disease or other neurodegenerative disease.

As shown in FIGS. 1B-C (see Original Patent), the diagnostic/imaging procedure of the present invention can be carried out non-invasively by applying radiation L to the patient's head. Alternatively, the patient's skull can be thinned by drilling or abrading the skull. Alternatively, the brain can be exposed by surgically opening the skull and then subjection the brain to radiation L.

Detection of the multiphoton excited fluorescence and the second and third harmonic of the laser excitation generated in tissue can be accomplished through an optical fiber that provides the excitation and, of course, through surrounding fibers in a bundle or through thick optical tubes for efficient collection of light excited near the tip of the single mode excitation fiber or fibers. There is a significant advantage in fluorescence collection efficiency for multiphoton tissue fluorescence over single photon excitation, because the emission is localized near the fiber tip where it is most accessible to collection optics. Desirably, the present invention is carried out with a plurality of optical fibers, including a single excitation fiber surrounded by a plurality of collection fibers.

Effective multiphoton molecular excitation is made possible, in accordance with the present invention, by the combination of (a) the very high, local, instantaneous intensity and (b) the temporal concentration of a pulsed laser. A high intensity, long wavelength, monochromatic light source which is focusable to the diffraction limit such as a titanium sapphire mode locked solid state laser, with each pulse having a duration of about 100 femtoseconds (100.times.10.sup.-15 seconds) at a repetition rate of about 80 MHz. Other lasers that are also effective for multiphoton excitation and harmonic generation can also be used. Because of the high instantaneous power provided by the very short duration intense pulses focused to the diffraction limit, there is an appreciable probability that a fluorophore (a fluorescent dye), contained in the target, and normally excitable by a single high energy photon having a short wavelength, typically ultraviolet, will absorb two long wavelength photons from the laser source simultaneously. This absorption combines the energy of the two photons in the fluorophore molecule, thereby raising the fluorophore to its excited state. When the fluorophore returns to its normal state, it emits light, and this light then passes to a suitable detector.

The multiphoton excitation of fluorophores by highly intense, short pulses of light constitutes a general fluorescence microscopy technique for imaging which provides improved background discrimination and reduces photobleaching of the fluorophores. This is because the focused illumination provided in the microscope fills a converging cone as it passes into the specimen. All of the light which reaches the plane of focus at the apex of the converging cone, except the tiny fraction which is absorbed in the fluorophore, then passes out the opposite side of the specimen through a diverging cone. Only in the region of the focal point on the object plane at the waist formed by the converging and diverging cones is the intensity sufficiently high to produce multiphoton absorption in the specimen fluorophore, and this intensity dependence enables long wavelength excitation only in the small local volume of the specimen surrounding the focal point. This absorption is produced by means of a stream of fast, high intensity, femtosecond pulses of relatively long wavelength which retains a moderate average illumination intensity of long wavelength light throughout the remainder of the specimen outside the region of the focal point. As a result, photobleaching of the fluorophore outside the plane of focus is virtually eliminated. One-photon absorption of the long wavelength light is negligible, and outside the plane of focus the instantaneous intensity is too low for appreciable two-photon absorption and excitation, even though the time average illumination is in reality nearly uniform throughout the depth of the specimen. This effect also significantly reduces the damage to living cells.

In order to obtain three dimensional resolution, the present invention can utilize two-photon excitation of a fluorophore which has a one-photon absorption peak at a wavelength which overlaps or exceeds one-half that of the exciting light. For three-photon excitation, the one-photon absorption overlaps one-third that of the exciting light. To accomplish this, the laser produces a very short pulsed laser beam of high instantaneous power and of a relatively long wavelength, for example in the visible red of the infrared range. This light is directed to a specimen containing a fluorophore normally excited by a single photon having a short wavelength (e.g., ultraviolet radiation) range so that two low energy (red) photons must combine their energy to provide the same excitation of the specimen that would be provided by a single high energy (ultraviolet) photon. Both the excitation and hence the fluorescence rates in the specimen are proportional to the square of the intensity of the incident light. In the focused excitation laser beam, the intensity of the long wavelength incident light becomes high enough to excite the fluorophores in the specimen only in the region of the focal point. This focal point may be adjustably positioned within the specimen so that fluorescence and/or photolysis of the specimen are produced only in a selected ellipsoidal volume around the focus. Thus, in accordance with the present invention, only long wavelength excitation light has to pass through the specimen, and this long wavelength light is focused to produce sufficient intensity to excite fluorescence only in a very small region. This fluorescence is produced even if the fluorophore normally absorbs only in the ultraviolet. Since the focal point can be selectively positioned in the specimen, three-dimensional resolution is provided in both scanning fluorescence microscopy and in photolysis, including photolysis of photon-activatable reagents which can be released by photolysis.

In accordance with the present invention, the necessary excitation intensity is provided from a radiation light source which may be, for example, a titanium sapphire mode locked laser generating pulses of light having a wavelength in the red region of the spectrum, for example about 700-1000 nm, or with the pulses having a width of 10.sup.-9 seconds to 10.sup.-15 seconds, conveniently at about 80 MHz repetition rate. Other bright pulsed lasers may also be used to produce light at different relatively long wavelengths in the infrared or visible red region of the spectrum, for example, to generate the necessary excitation photon energies which will add up to the appropriate absorption energy band required by the fluorophores in the spectrum which normally would be excited by absorption of a single photon in the spectral region having wavelengths about one-half the wavelength of the incident light. If shorter excitation wavelengths are needed, the laser wavelengths can be divided by 2, 3, or 4 by external harmonic generation. Thus, for example, two photons in the visible red region at 750 nm would combine to excite a fluorophore which normally absorbs light in the ultraviolet region at or above 375 nm, while two photons in the infrared region of, for example, 1070 nm, would excite a fluorophore which absorbs at or above 535 nm in the visible light region.

In a modified form of the invention, the single wavelength light source can be replaced by two different long wavelength laser sources so that the incident light beam consists of two superimposed pulsed light beams of high instantaneous power and of different wavelengths. The wavelengths of the incident beam are selected to excite a fluorophore which is absorbent at a short wavelength which may be described as: 1/.lamda..sub.abs=1/.lamda..sub.1+1.lamda..sub.2 where .lamda..sub.abs is the short wavelength of the absorber, and .lamda..sub.1 and .lamda..sub.2 are the laser incident beam wavelengths.

In two-photon excitation, with a typical two-photon cross section .delta. of: .delta.=10.sup.-58m.sup.4s/photon with the pulse parameters given above (100 fsec. pulses at a repetition rate of 80 MHz), and with the beam focused by a lens of numerical aperture A--1.4, the average incident laser power (P.sub.0) of approximately 50 mW saturates the fluorescence output of a fluorophore at the limit of one absorbed photon per pulse per fluorophore. The number n.sub..alpha. of photons absorbed per fluorophore per pulse depends on the following relationship -- see Original Patent.

The fluorescence emission could be increased, however, by increasing the pulse repetition frequency up to the inverse fluorescence lifetime, which typically is: .tau..sub.f.sup.-1=10.sup.9S.sup.-1 For comparison, one-photon fluorescence saturation occurs at incident powers of about 3 mW.

In addition to measurement of intrinsic tissue fluorescence (also known as autofluorescence) with multiphoton excitation, it is possible to utilize photoactive agents, including fluorescent dyes, in conjunction with multiphoton microscopy to image properties of cells and tissues. Suitable photoactive agents include dyes which are excited by multiphoton excitation such as, organic molecules whose fluorescence changes when they bind metal ions such as Ca.sup.2+, Mg.sup.2+, Na.sup.+ or K.sup.+ or H.sup.+, including dyes like DAPI (4',6-diamidino-2-phenylindole, dihydrochloride). Many such dyes are suitable for application in vivo. Such photoactive agents fluoresce upon binding to lesions of a neurodegenerative disease or other neuroanomalies. In carrying out the first embodiment of the present invention, the standard fluorescence is determined prior to treating the brain tissue with at least one photo-active agent.

The multiphoton imaging technique of the present invention can be used to observe plaques and neurons in the brains of living mammals. In addition, tangles can also be observed.

There are several possible approaches to observing the tangles and plaques: tangles are made up highly structured aggregation of tau protein, which under an electron microscope adopt a confirmation called perihelical filaments. It has been discovered that this protein confirmation is autofluorescent using wavelengths utilized for multiphoton microscopy (approximately 700 nanometer excitation, emission approximately 450 nanometer). This observation would allow detection of neurofibrillary tangles in the brain, either through imaging using devices designed to provide scanning capabilities or through spectroscopy, in which an atomic resolution is lost but the unique biochemical signature of protein structure can still be detected. The autofluorescence due to tangles can be discriminated from other brain structures due to the unique wavelength properties that have been discovered for the tangle protein, as well as ultimately using other techniques such as fluorescence lifetime imaging to discriminate from the low-level autofluorescence in the background. Thus, one could envision applying the long wavelength light through the skull to the brain substance and measuring the emitted autofluorescence. The long wavelength light passes readily through the soft tissue and overlying bone in systems currently in use to measure hemoglobin saturation in the brain, suggesting the feasibility of this approach.

A separate approach will be taken for amyloid deposition. To date, amyloid does not appear to be autofluorescent so that to visualize the amyloid, a contrast agent needs to be applied. It has been found that a commonly used reagent, thioflavine S, can be applied directly to the cortical surface or into the spinal fluid. This dye is intensely fluorescent only when bound to amyloid plaques. Thus, in another embodiment of the invention, thioflavine S or a dye with similar amyloid-binding properties could be given to patients either introduced into the spinal fluid or, using compounds that cross the blood brain barrier, systemically injected, and the brain subsequently imaged using long wavelength light. Again, either spectroscopy or direct imaging would allow for the detection and quantitation of the amount of amyloid present. Such a technique, in which long wavelength near infrared light is used to generate fluorescent markers of neurofibrillary change or amyloid deposition could be utilized for diagnosis as well as to determine the amount of these changes present in the brain, providing a quanitative readout for therapeutic interventions.

In addition to diagnosing/imaging Alzheimer's Disease, the present can also be used diagnose/image other neurodegenerative diseases. Examples of such diseases include Parkinson's Disease, Huntington's Disease, and Lou Gehrig's Disease.

Claim 1 of 28 Claims

1. A method of detecting a neurodegenerative disease in a mammal comprising: activating brain tissue of the mammal by application of radiation from a laser through an opening or a thinned portion of the mammal's skull under conditions effective to promote a simultaneous multiphoton excitation of the brain tissue and to emit a fluorescence characteristic, wherein the radiation is at an intensity level capable of being achieved by a titanium sapphire mode locked solid state laser and has a wavelength in the visible red to the infrared region of the light spectrum and is pulsed at a pulse width between about 10.sup.-9 to 10.sup.-15 second, said fluorescence characteristic being achieved by combining photons; comparing the fluorescence characteristic to a standard fluorescence emitted by exciting healthy brain tissue of the mammal under the same conditions used to carryout said activating; and identifying the brain tissue where the fluorescence characteristic differs from the standard fluorescence as potentially having a neurodegenerative disease.

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