WO2002061405A2

WO2002061405A2 - Method and hand-held device for fluorescence detection

Info

Publication number: WO2002061405A2
Application number: PCT/CA2002/000129
Authority: WO
Inventors: Roy H. Pottier; Eva F. Dickson; Frank Fischer; James C. Kennedy
Original assignee: Pottier Roy H; Dickson Eva F; Frank Fischer; Kennedy James C
Priority date: 2001-01-31
Filing date: 2002-01-31
Publication date: 2002-08-08
Also published as: AU2002227838A1; WO2002061405A3

Abstract

The present invention provides a portable hand held device (10) for exciting and detecting fluorescence in a biological sample (16) for discriminating between normal and diseased tissue. The device includes a light source (12) to irradiate the sample with excitation light, a detector (18) to detect fluorescence emitted by the sample, the fluorescence resulting from irradiation of the sample with excitation light, and an indicator (26) coupled to the detector to output an indication of the fluorescence. This portable device can be used in a clinic or doctor's office for photodiagnosis (PD) or management of photodynamic therapy (PDT).

Description

METHOD AND HAND-HELD DEVICE FOR FLUORESCENCE DETECTION

CROSS REFERENCE TO RELATED U. S. PATENT APPLICATION

This patent application relates to United States provisional patent application Serial No. 60/265,164 filed on January 31 , 2001 , METHOD AND

DEVICE FOR FLUORESCENCE DETECTION.

FIELD OF THE INVENTION

The present invention relates to a method and hand-held device for exciting and detecting fluorescence emission from biological material, particularly for monitoring tissue properties by detecting the emission of fluorescent molecules within the tissue.

BACKGROUND OF THE INVENTION When certain substances are applied to tissue or administered to a patient, abnormal tissue or foreign organisms will fluoresce when excited by light of a specific wavelength(s). Specifically, selective fluorescent photodiagnostic compounds, including photosensitizers, are chemical compounds which, when delivered to a patient, can accumulate preferentially in abnormal tissue or foreign organisms at some time after application. In some cases, for example protoporphyrin IX, rather than administering the photodiagnostic compound itself, a precursor is administered that is subsequently converted selectively by the abnormal tissue or foreign organism to the photodiagnostic compound. Such compounds, when photosensitizers, may also be useful for selective treatment of the abnormal tissue or foreign organism. In addition, some naturally occurring substances fluoresce when excited by light of a specific wavelength. Such endogenously present compounds may also preferentially accumulate in abnormal tissue or foreign organisms. In any case, when irradiated with light of appropriate defined wavelengths, the photodiagnostic molecule can be excited, emitting characteristic fluorescence permitting enhanced or selective visualization of the abnormality. This allows one to identify abnormal tissue, such as, but not limited to cancer, skin lesions, psoriasis, actinic keratosis, and foreign organisms due to infections, amongst others. In addition, when the fluorescent molecule is a photosensitizer intended to be used for treatment using photodynamic therapy, subsequent irradiation with stronger light of an appropriate wavelength can cause local killing of abnormal tissue or foreign organisms through formation of cytotoxic oxygen species by photosensitization of molecular oxygen. Prior to the treatment, the fluorescence visualization process described above further allows the determination of whether the photosensitizer has in fact selectively accumulated in the abnormal tissue, as is required before treatment can commence.

It would be very advantageous to provide a portable and cheap device that can be used in a clinic or doctor's office for photodiagnosis (PD) or management of photodynamic therapy (PDT). It would be advantageous for such a device to enhance contrast in the measurement of abnormal human tissue compared to the normal surrounding tissue based on inducing and detecting fluorescence in abnormal tissue, either from the abnormal tissue itself or photosensitive compounds accumulated into the abnormal tissue. Present fluorescence systems presently used for this type of application are generally laser based and involve very expensive lasers and fluorescence detection and imaging systems that require extensive user training and maintenance for proper operation and data interpretation. A simple to use device for primary caregivers is current lacking.

SUMMARY OF THE INVENTION It is the general object of this invention to provide a device that can be used in a clinic or doctor's office for photodiagnosis (PD) or management of photodynamic therapy (PDT). The device enhances contrast in the measurement of abnormal human tissue compared to the normal surrounding tissue. In particular, devices based on the contrast enhancement method used are portable and quantify the total fluorescence emission signal of a defined tissue area (henceforth referred to as 'penlight device'). The portability of the apparatus, due to the incorporation of laser diodes or light emitting diodes (LEDs) or other small light sources, is an object of the invention and makes the viewing or measurement of abnormal tissue in clinical practice easier, because the apparatus can be brought to the patient, not requiring the patient to be brought to the apparatus. While a portable imaging device still has to be large enough to include a monitor, a spot device using LEDs or other small light sources in conjunction with a very small electronic light detector can be built as a light weight, battery driven, hand held device that can be carried along like a pen, which it is only used from time to time by a physician.

The invention provides a hand-held device that permits fast and inexpensive preliminary diagnosis of tissue by creating and detecting this fluoresence. The invention thus allows quantitative examination of abnormal tissue not possible with the naked eye.

In one aspect of the invention there is provided a hand-held device for detecting fluorescence in a biological sample, comprising a light source to irradiate the sample with excitation light; a detector to detect fluorescence emitted by the sample, the fluorescence resulting from irradiation of the sample with excitation light; and an indicator coupled to the detector to output an indication of the fluorescence; and wherein said device is adapted to be hand-held. In another aspect of the invention there is provided a device for detecting fluorescence in abnormal tissue, comprising: an elongated housing having a tip end; a light source within the housing to irradiate tissue with excitation light from the tip end; a detector within the housing to detect fluorescence of the tissue, resulting from the irradiation with the excitation light, at the tip end; and a circuit coupled to the detector to output an indication of the fluorescence. In another aspect of the invention there is provided a method for detecting fluorescence in a biological sample, comprising providing a hand-held device comprising a light source, a detector, and circuit coupled to the detector to output an indication of the fluorescence; irradiating a biological sample with excitation light; detecting fluorescence emitted by the sample, the fluorescence resulting from irradiation of the sample with excitation light; and obtaining an indication of the detected fluorescence.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of non-limiting examples only, reference being made to the accompanying drawings, in which:

Figure 1a shows the excitation spectrum of ALA induced protoporphyrin IX in Balb C mice; Figure 1 b shows the emission spectrum of ALA induced protoporphyrin

IX in Balb C mice;

Figure 2a shows a perspective view of a hand-held fluorescence excitation and detection device; and

Figure 2b is a view of the device of Figure 2a without showing the housing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a device that can be used in a clinic or doctor's office for photodiagnosis (PD) or management of photodynamic therapy (PDT). The device enhances contrast in the measurement of abnormal human tissue compared to the normal surrounding tissue by exciting fluorescent molecules in the tissue and then detecting fluorescence emitted from the fluorescent molecules in the tissue. In particular, devices based on the contrast enhancement method used are portable and quantify the total fluorescence emission signal of a defined tissue area (also referred to as a 'penlight device').

The portability of the apparatus, due to the incorporation of laser diodes or light emitting diodes (LEDs) or other small light sources, is an object of the invention and makes the imaging or measurement of abnormal tissue in clinical practice easier, because the apparatus can be brought to the patient, not requiring the patient to be brought to the apparatus. While a portable imaging device still has to be large enough to include a monitor, a spot device using LEDs or other small light sources in conjunction with a very small electronic light detector can be built as a light weight, battery driven, hand held device that can be carried along like a pen, which is only used from time to time by a physician. As described below, there are many variations and features of the invention. In one particular version of the invention, the invention includes a single wavelength light source, a point detector, optional filter(s), and a device for providing feedback to the user when fluorescence corresponding to the photodiagnostic molecule is detected. The feedback can include, for example, a visual (for example a liquid crystal display (LCD)) or audio indication of the amount and extent of fluorescence. The invention may be powered from an external power source, for example, via a cable connected to the pen-like housing, or from an internal power source. In one variation, the light source and/or the detector are somewhat remote from the tip and fiber optic line(s) are employed to provide excitation light to the tip and to direct fluoresce light from the tip to the detector. Suitable light sources include, for example, LEDs, laser diodes, lasers, lamps, and the like. Suitable detectors include, for example, photodiodes, photoresistors, phototransistors, optocouplers, and similar analog devices as well as fiber optical detectors and digital detectors (e.g., non-matrix).

In general, a device according to the present invention comprises a light source to irradiate a biological sample with excitation light; a detector to detect fluorescence emitted by the sample, the fluorescence resulting from irradiation of the sample with excitation light; and a circuit coupled to the detector to output an indication of the fluorescence. In accordance with the invention, the biological sample can be tissue such as skin, in which case the device can be used with an in vivo or in vitro sample, or the sample can be a solution such as a cell suspension or a solution comprising one or more compounds of interest. Preferred compounds are protoporphyrin IX or a precursors) thereof. In particular, the invention encompasses, for example, illumination of a biological sample in admixture with a fluor such as, for example, protoporphyrin IX, and detection of resulting fluorescence of such fluor, in this case protoporphyrin IX.

The excitation and emission wavelength spectra of protoporphyrin IX are shown in Figures 1a and 1 b. From Figure 1a it can be seen that protoporphyrin IX has multiple excitation peaks. A device according to the invention will work with any of those excitation peaks, provided that the excitation wavelength used is in the range of the excitation peak of interest. The invention preferably uses the peaks at about 408 nm or about 634 nm. Excitation at about the 408 nm peak is desirable for detection of fluorescence near the surface of the biological sample (e.g., tissue), as this wavelength does not penetrate deeply into tissue (penetration of about 1 to 2 mm). (This is due to pigments of the biological sample absorbing much of the light in this energy range.) Thus, the excitation wavelength should be at a wavelength of about 408 nm or near 408 nm, for example, in the range of about 380 nm to about 440 nm. Excitation at about the 634 nm peak is desirable for detection of fluorescence below the surface of the biological sample (e.g., tissue), as this wavelength penetrates deeper into tissue (penetration of about 10 mm). In this case, the excitation wavelength should be at a wavelength of about 634 nm or near 634 nm, for example, in the range of about 600 nm to about 660 nm. Detection of the emitted fluorescence of protoporphyrin IX is preferentially carried out at the lower emission wavelength, about 636 nm (see Figure 1b), because this peak is the highest. However, when using an excitation wavelength of about 634 nm, it will be appreciated that the longer emission wavelength, about 708 nm should be detected. It should be noted that the wavelengths of these emission peaks vary somewhat depending on the type of biological sample under investigation. For example, the emission wavelengths mentioned pertain to human skin, but will vary according to species, sample type, etc. Accordingly, the detector used to detect the fluorescence should be chosen with sensitivity at the appropriate wavelength or range of wavelengths. This is important where one or more optical filters, for example, band pass filters, are used in conjunction with the detector, as such filters are available in very narrow bandwidths.

Referring to Figures 2a and 2b, a portable hand held fluorescence excitation and detection device is shown generally at 10. Portable fluorescence excitation/detection device 10 includes light sources 12 mounted on a housing 14 so that the light emitted from the source 12 is projected onto a tissue target 16 located in front of the device. A photodetector 18 is mounted in the housing 14 to detect fluorescence emitted from either the tissue itself or from photosensitizer molecules present in the tissue sample 16. An indicator circuit 26 is coupled to the detector 18 to output an indication of the fluorescence and may be any one or combination of visual or audio indicators. For example, a green/orange/red color bar graph indicator such as Model #MV5A1564, Newark Electronics, Mississauga ON, Canada, may be used. For low excitation light levels, more than one photodetector 18 may be used in parallel to give higher signals at low fluorescence light intensity levels. In accordance with the invention, the light source 12 can be, for example, a white light source, a light-emitting diode (LED), a laser diode, or the like. The light source 12 can comprise one or more such sources. In cases where the light source produces a broad spectrum of light, an optical filter 22, such as a band pass filter, can be used to restrict the light to a range encompassing the desired excitation wavelength. A suitable LED light source is produced by SUN, model LUB 53W, which produces light at 435 nm + 35 nm (in the blue range). Although more expensive, laser diodes are desirable . because they produce light within a very narrow bandwidth (e.g., 635 nm ± 2 nm). Clearly, any light source can be used, provided that such light source produces light at any of the desired excitation peaks of protoporphyrin IX, or that an optical filter can be used to restrict the light produced to the desired peak and bandwidth. In accordance with the invention, the detector 18 can be any suitable photodetector, such as a photodiode, photoresistor, phototransistor, and the like. A photodarlington is advantageously used because of the high gain of such device. As such detector devices are often broad spectrum, it is desirable to use an optical filter, such as a band pass filter 23, to restrict the light detected to a range encompassing the desired emission wavelength. For example, to detect the emission peak of 636 nm, a bandwidth of about 610 nm to about 660 nm is suitable, preferably about 620 nm to about 650 nm, whereas to detect the emission peak of 708 nm, a bandwidth of about 680 nm to about 740 nm is suitable, preferably about 690 nm to about 730 nm.

In accordance with the invention, the detector 18 provides to the user an indication of the detected fluorescence. Visual indicators, for example, a display comprising one or more LEDs, liquid crystal displays (LCDs), or the like, are suitable, as are audible indicators such as buzzers, chimes, and the like, and tactile indicators, such as vibrators. Further, a combination of visual and/or audible and/or tactile indicators can also be used. A device according to the invention is hand-held and can be provided in a housing of various forms such as, for example, "pen light" or pistol-grip, or any other form that affords portability and ease of hand use. The invention also contemplates an embodiment in which the light source and/or the detector are disposed in a separate housing and connected to the rest of the device via optical cables (e.g., fiber optics) and/or electrical cables. The device can be powered from an external power source, via suitable wires and connectors, or via internal batteries, such as rechargeable batteries. The invention also provides a method for detecting fluorescence in a biological sample, comprising: providing a hand-held device comprising a light source, a detector, and circuit coupled to the detector to output an indication of the fluorescence; irradiating a biological sample with excitation light; detecting fluorescence emitted by the sample, the fluorescence resulting from irradiation of the sample with excitation light; and obtaining an indication of the detected fluorescence.

Different photodiagnostic compounds can be used. The best choice for the diagnostic imaging of a special kind of abnormal tissue depends on the kind of abnormal tissue, since different compounds accumulate in different amounts in different types of abnormal tissue. Furthermore, individual patients will respond differently, and in addition, the amount of compound present in the abnormal tissue depends on photosensitizer application times, and its fluorescence yield as well as on the optical properties of the compound and tissue.

The hand-held device disclosed herein for excitation and detection of fluorescence in tissue such as human skin is very advantageous for several reasons. First, there is no imaging required which reduces the need for expensive image processors and software analysis. The device can be adapted for any wavelength of interest in the ultraviolet-visible-near infrared wavelength region so that different fluorescent drugs may be used. The device can be used for rapidly and economically monitoring fluorescence during treatment of different tissue related conditions. Little or no specialized training is required to operate the device. Additionally, the portability and low power consumption make it very useful in environments outside the hospital environment.

The method and device disclosed herein is very advantageous for allowing one to identify abnormal tissue, such as, but not limited to cancer, skin lesions, psoriasis, actinic keratosis, and foreign pathogenic organisms due to infections, viral or bacterial, amongst others.

The foregoing description of the preferred embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated. It is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims and their equivalents.

Claims

WE CLAIM

1. A hand-held device for detecting fluorescence in a biological sample, comprising a light source to irradiate the sample with excitation light; a detector to detect fluorescence emitted by the sample, the fluorescence resulting from irradiation of the sample with excitation light; and an indicator coupled to the detector to output an indication of the fluorescence; and wherein said device is adapted to be hand-held.

2. The device of claim 1 , wherein the light source produces excitation light at a wavelength the same as or near an excitation peak wavelength of protoporphyrin IX.

3. The device of claim 1 , wherein the light source produces excitation light at a wavelength of 408 nm or near 408 nm.

4. The device of claims 1 , 2 or 3 wherein the light source is a white light source in combination with a filter.

5. The device of claims 1 , 2, 3 or 4 wherein the light source is an LED.

6. The device of claims 1 , 2, 3 or 4 wherein the light source is a laser diode.

7. The device of claim 1 , wherein the light source produces excitation light at a wavelength of 634 nm or near 634 nm.

8. The device of claim 7, wherein the light source is a white light source in combination with a filter.

9. The device of claims 7 or 8, wherein the light source is an LED.

10. The device of claims 7 or 8, wherein the light source is a laser diode.

11. The device of claims 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein the detector comprises a photodiode.

12. The device of claim 11 , further comprising a filter.

13. The device of claims 11 or 12, wherein the detector is a silicon photodarlington detector.

14. The device of claim 3, wherein the detector comprises a photodiode and a filter, and wherein the wavelength of the fluorescence detected is within the range of about 610 to about 650 nm.

15. The device of claim 7, wherein the detector comprises a photodiode and a filter, and wherein the wavelength of the fluorescence detected is within the range of about 690 to about 730 nm.

16. A method for detecting fluorescence in a biological sample, comprising providing a hand-held device comprising a light source, a detector, and circuit coupled to the detector to output an indication of the fluorescence; irradiating a biological sample with excitation light; detecting fluorescence emitted by the sample, the fluorescence resulting from irradiation of the sample with excitation light; and obtaining an indication of the detected fluorescence.

17. A method according to claim 16, wherein said biological sample comprises an added fluor or a precursor thereof.

18. A device for detecting fluorescence in abnormal tissue, comprising: an elongated housing having a tip end; a light source within the housing to irradiate tissue with excitation light from the tip end; a detector within the housing to detect fluorescence of the tissue, resulting from the irradiation with the excitation light, at the tip end; and a circuit coupled to the detector to output an indication of the fluorescence.

19. A device as set forth in claim 18, wherein the housing has a width of 1 inch or less.

20. A device as set forth in claim 18, wherein the housing has a width of YA of an inch or less.

21. A device as set forth in claim 18, wherein the housing has a width of Vz inch or less.

22. A device as set forth in claims 18, 19, 20 or 21 , wherein the housing has a length of 5 inches or less.

23. A device as set forth in claim 18, 19, 20 or 21 , wherein the housing has a length of 7 inches or less.

24. A device as set forth in claims 18, 19, 20 or 21 , wherein the housing has a length of 9 inches or less.

25. A device as set forth in claims 18, 19, 20 or 21 wherein the housing is generally cylindrical in shape.

26. A device as set forth in claims 18, 19, 20, 21 , 22, 23, 24 or 25 wherein the light source is a white light source and the device further comprises a filter in an optical path of the white light source to provide non-white excitation light.

27. A pen light device as shown and described in this application.

28. A pen light device having one or more of the components and/or features shown and/or described in this application.

29. A hand-held device for detecting fluorescence in a biological sample, including a light source to irradiate the sample with excitation light; a detector to detect fluorescence emitted by the sample, the fluorescence resulting from irradiation of the sample with excitation light; and an indicator coupled to the detector to output an indication of the fluorescence; and wherein said device is adapted to be hand-held.