CA2335246A1

CA2335246A1 - Detection of cancer using cellular autofluorescence

Info

Publication number: CA2335246A1
Application number: CA002335246A
Authority: CA
Inventors: Bhaskar Banerjee
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-06-16
Filing date: 1998-08-25
Publication date: 1999-12-23
Also published as: EP1087698A1; CN1301137A; AU9119498A; JP2002518664A; EP1087698A4; WO1999065394A1

Abstract

Apparatus and methods especially useful for detection of cancer using cellular autofluorescence are described. In one embodiment, an apparatus (10) includes a source (12) of white light which produces a beam of light transmitted to a tissue via one group (18) of optic fibers in a two-way fiber optic bundle (22). The two-way fiber optic bundle (22) may be passed through a conventional endoscope. The beam of light excites the tissue and results in an emission of cellular autofluorescence at a wavelength of about 330 nm. A light sample from the tissue is directed back through the two-way fiber optic bundle (22) and then passes through a photodetector (36). The photodetector (36) produces a signal, representative of the intensity of cellular autofluorescence, which can be passed to a monitor (38) as a wave form or meter response. The apparatus may further comprise a charge-coupled device and video imaging technology to produce real time video images of tissue being examined.

Description

WO 99165394 PCT/US98/i7597 DETECTION OF CANCEF~ USING
CELLULAR AUTOFLUORI~SCENCE
Field of the Invention This invention relates generally to detecticm of cancerous cells and more particularly, to detecting cancerous cells using cellular autofluorescence.
Background of the Invention The survival rate for cancer patients increases with early detection of cancer. Known methods of gaining early detection of cancer are limited to techniques such as surveillance endoscopy and random tissue biopsies, both of which are costly and inefficient. In addition, methods which employ relatively high levels of radiation which cause tissue damage generally are not preferred.
Autofluorescence has been used in attempts to detect cancerous tissue.
Particularly, fluorescence occurs when certain substances called fluorophores emit light of a longer wavelength after being excitedl by light of another, shorter wavelength. The fluorescence which occurs in human and animal tissues is commonly referred to as autofluorescence because; the fluorescence results from fluorophores occurring naturally in the tissues. The intensity of autofluorescence differs in normal and cancerous tissues, and autofluorescence can be used to detect cancerous tissue in different organs, including the colon, esophagus, breast, skin, and cervix.
In many medical and laboratory applicatioa~s, the use of autofluorescence often is preferred for detecting cancerous tissue because autofluorescence avoids the introduction of exogenous fluorophores or any other exogenous agent. The use of exogenous agents increases costs and results in time delays due to lag in incorporating the exogenous agents into the examined tissue. Exogenous agents also introduce the risk of adverse reaction.

Known uses of autofluorescence are, however, limited to reliance on the non-specific autofluorescence emitted from extra.cellular components of whole tissue. Specifically, several extracellular components of whole tissue exhibit autofluorescence, including blood, blood vessels, collagen and elastin. These extracellular components may change in non-specific ways from normal to _ cancerous tissue. More specifically, known uses of autofluorescence to detect cancerous tissue cannot distinguish between cellular changes and non-specific extracellular changes from normal to cancerous tissue. Therefore, the application of the known uses of autofluorescence to detect cancer rely on non-specific autofluorescence and therefore cannot track celhalar changes during the early stages of progression of cancer.
It would be desirable to provide apparatus and methods which facilitate the early detection of cancerous cells using autofl~uorescence. It also would be desirable to provide such autofluorescence apparatus and methods which exclude extracellular changes which are non-specific to cancer.
Summary of the Invention These and other objects may be attained by apparatus and methods for detecting the intensity of cellular autofluoresef;nce which enable the early detection of cancerous cells and exclude extracel',lular changes which are non-specific to cancer. In one embodiment, the apparatus includes a light source for producing a beam of light to excite a tissue to emit cellular autofluorescence.
The beam of light is first filtered through a narrow-band optical filter configured to pass light at a wavelength of about 200 - 3a9 nm, which is optimal for producing cellular autofluorescence. The beam of light is then transmitted to the 2S tissue via a two-way fiber optic bundle having a sampling end positioned at or near the tissue being examined. A lens-system is positioned between the sampling end of the two-way fiber optic bundle and the tissue, and the lens system is configured to collect a light sample from the tissue. The Light sample is transmitted back through the two-way fiber optic bundle and passes through a narrow-band optical filter configured to pass light at wavelengths of 320 -340.
A photodetector positioned at the output end of the two-way fiber optic bundle measures the intensity of cellular autofluorescenc~e emitted from the tissue.
In another aspect the present invention relates to a method for detecting pre-cancerous and cancerous cells in a tissue and un one embodiment, the method includes the steps of exciting the tissue with a beam of light delivered through a two-way fiber optic bundle, and measuring; the intensity of cellular autofluorescence emitted from the tissue. The two-way fiber optic bundle may be inserted through the biopsy channel of an endoscope or through a needle inserted into the tissue. The light beam has a wave;leng;th of about 200 - 329 nm, and the light sample is transmitted back through the two-way fiber optic bundle and through a narrow-band optical filter canfigurE:d to pass light at wavelengths of 320 - 340.
Measuring the intensity of the light sample: at an emission wavelength of about 330 nm enables detection of pre-cancerous and cancerous cells.
Specifically, the intensity of the light sample at 330 nm increases systematically with the progression of cancer from normal to cancerous tissue. In addition, at the wavelengths, identified above, extracellular changes which are non-specific to cancer are excluded and therefore, only the celluliar changes are detected. It is believed that the cell specific fluorescence originates from membranous structures in cells containing the amino acid Tryptophan.
Brief Description of the Drawings Figure 1 is a schematic illustration of an apparatus for detection of cancer using cellular autofluorescence in accordance with ~one embodiment of the present invention.

Figure 2 is a schematic illustration of an apparatus for detection of cancer using cellular autofluorescence in accordance wiith another embodiment of the present invention.
Figure 3 is a flow chart illustrating a method for detection of cancer using cellular autofluorescence in accordance with an embodiment of the present _ invention.
Figure 4 is a schematic illustration of an apparatus for detection of cancer using cellular autofluorescence in accordance with yet another embodiment of the present invention.
Detailed Description The present invention is directed to apparatus and methods for detecting cancer in vitro and in vivo using cellular autofluorescence. Although specific embodiments of the apparatus and methods are de,>cribed below, many variations and alternatives are possible. Also, the term tissue as used herein refers to both in vitro and in vivo tissues. In addition, the term tissue as used herein refers to tissue, organs (in vivo or in live animals or humans), as well as samples of cells, such as in cytology (examination of a film of cells on a glass slide).
Further, the cancer detection apparatus and methods can be used in connection with the detection of early cancer, or pre-cancer, or dysplasia.
Referring specifically to the drawings, Figure 1 is a schematic view of an apparatus 10 for detecting cancer in vitro or in vivo using cellular autofluorescence. Apparatus 10 includes a light source 12, such as a Xenon arc lamp or a laser, powered by a conventional power source. A first optical filter 14 with a narrow bandwidth of about 125 nm, configured to pass light at a wavelength in a range of about 200 - 329 nm is positioned in the path of the light beam produced by light source 12. In one embodiment, first optical filter 14 has a narrow bandwidth of about 35 nm and is configured to pass light at a wavelength in a range of about 280-315 nm. The Might beam emerging from first optical filter 14 passes through an optical chopper 16 which removes wavelengths of any background light. The light beam then passes through a two-way fiber optic bundle 22, sometimes referred to herein as a probe, which is positioned to catch the light beam as it emerges from optical chopper 16. The two-way fiber S optic bundle 22 has a sampling end 28, and comprises two groups of optic fibers. .
A first group of optic fibers 18 transmits light from source of white light 12 to a tissue T. A second group of optic fibers 32 transmits a light sample back from tissue T for analysis.
The two optical fiber groups of two-way fiber optic probe 22 are randomly intermeshed. Two-way fiber optic probe 22 is less than about 2.5 rnm in diameter and is Iong enough to pass through the biopsy channel of an endoscope, e.g., about 1 - 2 m in length. Specifically, probe 22 is configured to pass through the biopsy channel of a conventional endoscope 24, such as the endoscopes commonly used to examine the gastrointestinal tract or the lungs.
In an alternate embodiment, two-way fiber optic bindle 22 may be passed through a needle or trocar to obtain measurements of cellular autofluorescence intensity from solid masses or organs such as breast, liver or pancreas.
A lens system 30 is positioned between sannpling end 28 of two-way fiber optic bundle 22 and tissue T. Lens system 30 is provided to avoid direct contact between the tissue and probe 22. Light emerging from tissue T, including emissions of cellular autofluorescence and reflLected and scattered light, is collected by lens system 30 to form a light sample.
The light sample is directed to sampling end 28 of two-way fiber optic bundle 22. The light sample is then transmitted back through two-way fiber optic bundle 22, along second group of optic fibers 32, from sampling end 28 to a second optical filter 34. Second optical filter 34 has a narrow bandwidth of about 20 nm, configured to pass light at a wavelength ~of about 320 - 340 nm, and is positioned in the path ~of the light sample transmitted back from tissue T. A
photodetector 36 is positioned to collect the liglht sample as it emerges from second optical filter 34. Photodetector 36 is configured to measure the intensity of the light sample across wavelengths varying from about 320 nm to about 340 nm.
Photodetector 36 generates an electrical output signal a whose magnitude is proportional to the intensity of the light sample: at a wavelength of about nm. Electrical output signal a is amplified and displayed on a monitor 38 as a wave form or meter response. The intensity of cellular autofluorescence in tissue T may thus be noted and compared to the intensity of cellular autofluorescence at about 330 nm in a tissue whose condition is known, such as a cancerous, pre-cancerous or normal tissue. The presence of cancerous cells is indicated by an increase, relative to normal tissue, in intensity of cellular autofluorescence at an emission wavelength of about 330 nm. A ratiio of the intensity of cellular autofluorescence in the tissue F~ to the intensity oif cellular autofluorescence in a known normal sample Fn may be constructed. The: greater the value of F~/F", the more severe the degree of cancer or malignancy.
Figure 2 is a schematic view of an apparatus 100 for real time detection of cancer in vitro or in vivo using cellular autofluorescence and video imaging technology. Apparatus 100 includes a source of white light 102, such as a Xenon arc lamp or a laser, is powered by a conventional, power source and produces a beam of light. The light beam then passes throul;h a first group of optic fibers 104 of a two-way fiber optic bundle 108 which is positioned to catch the light beam as it emerges from white light source 102. The first group of optic fibers 104 transmits the light beam to a tissue T. Two-way optic fiber bundle 108 passes through a conventional endoscope 109. In alternate embodiments, the two-way fiber optic bundle may pass through a large-bore needle or trocar. . A
lens system 110 is part of the endoscope 109 and interposed between tissue T
and two-way fiber optic bundle 108. It is positioned t~o catch reflected and scattered light from tissue T, as well as emissions of cellular autofluorescence, to form a light sample from tissue T. A second group of optic fibers 106 in two-way fiber optic bundle 108 transmits the light sample back from tissue T.
The light sample transmitted along second group of optic fibers 106 of two-way fiber optic bundle 108 is directed into an image acquisition module by a lens 1 I2. Image acquisition module 114 uses a standard optical device such as a prism or dichromatic mirror to split the light sample into two beams of light b1 and b2, each comprising identical wavelengths,. Light beam bl is transmitted to a conventional video detector 116 which produces a video signal cl representative of the standard visual image obtained from tissue T with endoscope 109 and lens system 110. Light beam b2 is transmitted to an optical filter 118 with a bandwidth of about 20 nm at about 330 nm" Light beam b2 then impinges on an image intensifier 120, and then a charge-coupled device or CCD 122 which produces a second video signal c2. Video sigr~a.l c2 is representative of the intensity of cellular autofluorescence emitted from tissue T. Video signal c2 is 1S color-coded according to the intensity of cellular autofluorescence to visually represent different stages of malignancy of the lesian. Video signals cl and c2 are then directed via conventional cable means to a computerized image controller 124 which combines the two video signals cl and c2 into a single signal which represents the superimposition of the image represented by c2 onto the image represented by cl . The combined signal is then directed to a standard color video monitor 126 for display of the combined images.
Figure 3 is a flow chart illustrating a method 150 for utilizing autofluorescence to detect pre-cancer, early cancer, cancer, and dysplasia.
Method 150 includes exposing a first tissue to a Iig;ht beam 152 which excites the tissue and results in an emission of cellular autofluorescence at a wavelength of about 330 nm. In this embodiment, the first tissue is being examined for the detection of cancer. After exposure of the tissue to the beam of light, the intensity of cellular autofluorescence emitted from the tissue is measured, at a wavelength of about 330 nm, using a standard photodetector 154.

In parallel, or in series, with steps 152 and 154, a second tissue whose condition is known as normal, pre-cancerous, or cancerous also is examined.
Particularly, the second tissue is exposed to a light beam 156 which excites the tissue and results in an emission of cellular autofluorescence at a wavelength of about 330 nm. After exposure of the tissue to the; beam of light, the intensity of cellular autofluorescence emitted from the tissue its measured, at a wavelength of about 330 nm, using a standard photodetector 158.
The intensity measurements from the first and second tissues are then compared 160. The intensity measurements obtained from the second tissue, which is of known condition, serves as a standard. Using the results of the comparison, the condition of the first tissue can be determined 162.
Method I50 may be practiced in vivo using a two-way fiber optic bundle passed through the biopsy channel of a conventional endoscope, as described above in connection with Figures 1 and 2. Alternatively, the first and second tissues may be collected tissue samples and metr~od 150 may be practiced in a laboratory. In addition, method 150 could be practiced in connection with the use of a charge-coupled device and video imaging equipment. With such devices.
and equipment, and at steps 154 and 158, the intensity of the autofluorescence could be visually represented in a real time video image. Real time video scanning of cellular autofluorescence would allow large areas of tissue to be scanned both in vitro and in vivo.
Figure 4 is a schematic illustration of an ;apparatus 200 for detection of cancer using cellular autofluorescence in accordance with yet another embodiment of the present invention: Apparatus 200 includes a light source 202 which may be a component of a conventional endoscopic illumination system. Light source may, for example, be a Xenon lamp or a source of laser energy. Source 202 is coupled to a lens system 204 by a optical fiber bundle 206. Lens system 204 is focused on a tissue T, such as a tissue, a tissue sample, an organ, or cells.
A lens system 208 is positioned to collect light from tissue T, and lens system 208 is coupled to an image acquisition module 210 by an optical fiber bundle 212. At image module 210, the light received from bundle 212 is split using a splitter such as a dichromatic mirror or a prism to produce two identical beams B 1 and Bz.
Light beam B1 is transmitted to a convenxionai video detector 214 which .
produces a video signal S1 representative of the standard visual image obtained from tissue T. Light beam B2 is transmitted to a~n optical filter 216 with a band width of about 125 nm which allows wavelenl;ths of about 290 nm to pass through. In one embodiment, optical filter 216 alllows wavelengths in the range of about 200 nm to about 329 nm to pass through. In an alternative embodiment, the band width of optical filter 216 is about 35 nrn which allows wavelengths in a range of about 280 - 315 nm to pass through. Liight beam B2 then impinges on an image intensifier 218, and then a charge-coupled device or CCD 220 which produces a second video signal S2. Video signal S2 is representative of the intensity of cellular autofluorescence emitted frorn tissue T.
Signals S1 and S2 are supplied to a computerized image controller 222 coupled to a display 224. The autofluorescence image from signal S2 could be color coded (i.e., different colors represent different grades of fluorescence intensities, and hence stages of malignancy) and superimposed on the standard endoscopic image from signal S 1. The intensity of cellular fluorescence would be stronger in malignant tissues than in normal tissue of the same organ, for example. The intensity of malignant areas also would be greater than that in dysplastic areas, which should be stronger than that in normal areas. If a laser source is used as light source 202, a gating rnE;chanism could be utilized to rapidly and alternately illuminate the sample with. white light (for routine video endoscopy) and the laser (for fluorescence imaging).
Using the above described methods and apparatus, fluorescence images can be obtained during endoscopy, from gastrointestinal organs, lungs, bladder, ureters, cervix, skin and bile ducts, and pancreatic ducts. Narrow caliber WO 99165394 PCT/US9$/17597 endoscopes can be passed through the biopsy ch~umels of larger endoscopes to obtain cellular fluorescence imaging from organs such as ureters, bile and pancreatic ducts, or may be passed through a large bore needle or trocar to examine solid organs such as the liver, pancreas, breast, prostrate, or other 5 masses.
Measuring the intensity of the light sample: at an emission wavelength of about 330 nm enables detection of pre-cancerous and cancerous cells.
Specifically, the intensity of the light sample at 3?~0 nm increases systematically with the progression of cancer from normal to cancerous tissue. In addition, at 10 the wavelengths identified above, extracellular changes which are non-specific to cancer are excluded and therefore, only the cellular changes are detected. It is believed that the cell specific fluorescence originates from membranous structures in cells containing the amino acid Tryptophan.
From the preceding description of various embodiments of the present invention, it is evident that the objects of the invention are attained.
Although the invention has been described and illustrated in detail, it is to be clearly understood that the same is intended by way of illustration and example only and is not to be taken by way of limitation. Accordingly, the spirit and scope of the invention are to be limited only by the terms of th.e appended claims.

Claims

Claims:

1. An apparatus for detection of cancerous tissue, said apparatus comprising:
a source of white light producing a light beam;
a first optical filter configured to optimally pass wavelengths of the light beam which optimize an emission of cellular autofluorescence in a tissue;
a two-way fiber optic bundle having a sampling end, said two-way fiber optic bundle configured to transmit the light beam to the tissue and transmit a light sample back from the tissue;
a lens system positioned between the sampling end of the two-way fiber optic bundle and the tissue;
a second optical filter configured to optionally pass wavelengths of the light sample which comprise the emission of cellular autofluorescence; and a photodetector configured to receive the light sample from the tissue.

2. An apparatus in accordance with Claim 1 wherein said first optical filter comprises a filter configured to optimally pass wavelengths of light about 290 nm.

3. An apparatus in accordance with Claim 1 wherein said second optical filter comprises a filter configured to optimally pass wavelengths of light about 320 - 340 nm.

4. An apparatus in accordance with Claim 1 further comprising a charge-coupled device configured to provide a visual representation of said emission of cellular autofluorescence.

5. An apparatus in accordance with Claim 1 further comprising an endoscope having a biopsy channel configured to allow insertion of said two-way fiber optic bundle therethrough.

6. A method comprising the steps of:
exposing a first tissue to a beam of light; and measuring an intensity of cellular autofluorescence emitted in the first tissue.

7. A method in accordance with Claim 6 wherein said step of exposing the first tissue to a beam of light comprises the step of exposing the first tissue to a beam of light having a wavelength in a range of about 200 nm to about 329 nm.

8. A method in accordance with Claim 7 wherein said step of exposing the first tissue to a beam of light comprises the step of exposing the first tissue to a beam of light having a wavelength in a range of about 280 nm to about 315 nm.

9. A method in accordance with Claim 6 wherein measuring the intensity of cellular autofluorescence comprises the step of obtaining intensity measurements at an emission wavelength of about 330 nm.

10. A method in accordance with Claim 6 wherein measuring the intensity of cellular autofluorescence comprises the step of visually representing the cellular autofluorescence using a charge-coupled device.

11. A method in accordance with Claim 6 wherein exposing the first tissue to a beam of ultraviolet light comprises the step of delivering the beam of light to the first tissue using a two-way fiber optic bundle.

12. A method in accordance with Claim 11 wherein delivering the beam of light to the tissue using a two-way fiber optic bundle comprises the step of passing the two-way fiber optic bundle through a biopsy channel of an endoscope.

13. A method comprising the steps of:
exposing a first tissue to a beam of ultraviolet light;
measuring an intensity of cellular autofluorescence emitted in the first tissue;
exposing a second tissue whose condition is known to a beam of ultraviolet light;
measuring an intensity of cellular autofluorescence emitted in the second tissue; and comparing the intensity measurements of the first tissue sample to the intensity measurements for the second tissue sample whose condition is known to determine if the first tissue is cancerous.

14. A method in accordance with Claim 13 wherein comparing the intensity measurements of the first tissue with the intensity measurements of the second tissue further comprises the step of comparing the intensity measurements at an emission wavelength of 330 nm.

15. A method in accordance with Claim 13 further comprising the step of producing a signal representative of the difference between the intensity measurements of the first tissue and the intensity measurements of the second tissue.