Hyperplasia
- increased production and growth of normal cells in a tissue or organ
- affected part becomes larger but retains its normal form
- e.g. breast growth during pregnancy
Dysplasia
- abnormal development of skin, bone or other tissues
- various stages: mild, moderate and severe
Carcinoma
- cancer that arises in epithelium, the tissue that lines the skin and internal organs of the body
- may occur in any tissue containing epithelial cells
- in many cases, the site of origin of the tumour may be identified by the naure of the cells it contains
- organs may exhibit more than one type of carcinoma; e.g. an adenocarcinoma and a squamous carcinoma may be found in the cervix (but not usually concurrently)
Carcinoma in situ (CIS)
- the earliest stage of cancer spread, in which the tumour is confined to the epithelium
- surgical removal of the growth should lead to cure
Invasive carcinoma
- from stages I - IV
Carcinomatosis
- carcinoma that has spread widely throughout the body
- spread of cancer cells occur via the lymphatic channels and bloodstream and across body cavities, for e.g. the peritoneal cavity
Morbidity
- the state of being diseased
Morbidity rate
- the number of cases of a disease found to occur in a stated number of the population
- usually given as cases per 100,000 or per million (number may be smaller for common diseases)
Mortality (mortality rate)
- the incidence of death in a population in a given period
Prognosis
- the probable outcome of the disease
Adenocarcinoma
- malignant tumour originating in glandular epithelium
Carcinosarcoma
- malignant tumour that is a mixture of carcinoma and sarcoma:
carcinoma: cancer of epithelial tissue, which is skin and tissues covering or lining the internal organs
sarcoma: cancer of connnective tissue, such as bone, cartilage and fat
Serous
- having to do with the serum, i.e. the clear liquid part of blood (i.e. plasma)
Pathogenesis
- The development of a disease. The origin of a disease and the chain of events leading to that disease.
Emesis
- vomiting
Adjuvant
- a pharmacological agent added to a drug to modify or increase or aid its effect. The Latin "adjuvans" means to help, particularly to reach a goal.
Monday, 19 March 2007
Thursday, 15 March 2007
Lam et al., Chest, Vol. 113, 1998
Localization of Bronchial Intraepithelial Neoplastic Lesions by Fluorescence Bronchoscopy
- Lung cancer patients have low survival rates because tumours are found at a late invasive stage, when the options for treatment are mostly palliative.
(Palliative treatment = treatment that concentrates on reducing the severity of disease symptoms or slowing the disease's progress, rather than providing a cure)
- Experience in other epithelial organs such as the cervix, esophagus and colon has shown that if the neoplastic lesions can be detected and treated in the intraepithelial stage, the cure rate can be significantly improved (2-4).
2. Anderson GH, Boyes DA, Benedet JL, et al. Organization and results of the cervical cytology screening program in British Columbia, 1955-85. BMJ 1988; 296:975-78
3. Winawer SJ, Zauber AG, Ho MN, et al. Prevention of colorectal cancer by colonoscopic polypectomy. N Engl J Med 1993; 329:1977-81
4. Jiang T, Yan S, Zhao L. Preventing effect of ‘liuwei dihuang decoction’ on esophageal carcinoma. Jpn J Cancer Chemother 1989; 16:1511-18
- Unfortunately in the tracheobronchial tree, identifying these leasions and localizing their exact sites remain problematic.
- During illumination of the bronchial (or any other tissue) surface, light can be:
- Tissue autofluorescence is not visible to the unaided eye because its intensity is very low and overwhelmed by the reflected and back-scattered light. It can only be visualized with suitable instrumentation.
- Fluorescence intensities differ significantly between normal and neoplastic tissues - this difference can be exploited to enhance our ability to localize areas of intraepithelial neoplasia in the tracheobronchial tree.
- Using Monte-Carlo simulation, one of the major causes for the loss of autofluorescence in areas of dysplasia or cancer was found to be a decrease in the extracellular matrix content (31, 32)
31 Qu J, MacAulay C, Lam S, et al. Optical properties of normal and carcinoma bronchial tissue. Appl Optics 1991; 11: 99-105
32 Qu J, MacAulay C, Lam S, et al. Laser induced fluorescence spectroscopy at endoscopy: tissue optics; Monte Carlo modeling and in vivo measurements. Optical Eng 1995; 34: 3334-43
- Only a proportion of the dysplastic lesions expressed matrix metalloproteinases that can degrade the extracellular matrix (30)
30 Bolon I, Brambilla E, Vandenbunder B, et al. Changes in the expression of matrix proteases and of the transcription factor c-Ets1 during progression of precancerous bronchial lesions. Lab Invest 1996; 75: 1-13
- Fluorescence bronchoscopy has made it possible to allow sampling of intraepithelial neoplastic lesions in vivo, especially serial sampling of the same area over time to study the pathogenesis of lung cancer and the natural history of these lesions.
- Lung cancer patients have low survival rates because tumours are found at a late invasive stage, when the options for treatment are mostly palliative.
(Palliative treatment = treatment that concentrates on reducing the severity of disease symptoms or slowing the disease's progress, rather than providing a cure)
- Experience in other epithelial organs such as the cervix, esophagus and colon has shown that if the neoplastic lesions can be detected and treated in the intraepithelial stage, the cure rate can be significantly improved (2-4).
2. Anderson GH, Boyes DA, Benedet JL, et al. Organization and results of the cervical cytology screening program in British Columbia, 1955-85. BMJ 1988; 296:975-78
3. Winawer SJ, Zauber AG, Ho MN, et al. Prevention of colorectal cancer by colonoscopic polypectomy. N Engl J Med 1993; 329:1977-81
4. Jiang T, Yan S, Zhao L. Preventing effect of ‘liuwei dihuang decoction’ on esophageal carcinoma. Jpn J Cancer Chemother 1989; 16:1511-18
- Unfortunately in the tracheobronchial tree, identifying these leasions and localizing their exact sites remain problematic.
- During illumination of the bronchial (or any other tissue) surface, light can be:
- reflected
- back-scattered
- absorbed
- induce tissue fluorescence
- Tissue autofluorescence is not visible to the unaided eye because its intensity is very low and overwhelmed by the reflected and back-scattered light. It can only be visualized with suitable instrumentation.
- Fluorescence intensities differ significantly between normal and neoplastic tissues - this difference can be exploited to enhance our ability to localize areas of intraepithelial neoplasia in the tracheobronchial tree.
- Using Monte-Carlo simulation, one of the major causes for the loss of autofluorescence in areas of dysplasia or cancer was found to be a decrease in the extracellular matrix content (31, 32)
31 Qu J, MacAulay C, Lam S, et al. Optical properties of normal and carcinoma bronchial tissue. Appl Optics 1991; 11: 99-105
32 Qu J, MacAulay C, Lam S, et al. Laser induced fluorescence spectroscopy at endoscopy: tissue optics; Monte Carlo modeling and in vivo measurements. Optical Eng 1995; 34: 3334-43
- Only a proportion of the dysplastic lesions expressed matrix metalloproteinases that can degrade the extracellular matrix (30)
30 Bolon I, Brambilla E, Vandenbunder B, et al. Changes in the expression of matrix proteases and of the transcription factor c-Ets1 during progression of precancerous bronchial lesions. Lab Invest 1996; 75: 1-13
- Fluorescence bronchoscopy has made it possible to allow sampling of intraepithelial neoplastic lesions in vivo, especially serial sampling of the same area over time to study the pathogenesis of lung cancer and the natural history of these lesions.
Lam et al., Chest, Vol 103, 1993
Detection and Localization of Early Lung Cancer by Imaging Techniques
- Fluorescence bronchoscopy can be employed to
- Lung cancer mortality may be reduced if it can be detected early at the in situ stage. This may prevent disease progression to the malignant stage.
- Yield of sputum cytology is low; improvements in the sensitivity of the method to detect cancer and better cytomorphology criteria are needed.
- Conventional white light bronchoscopy not accurate: Misses significant dysplasia or carcinoma in situ in about 10-20% of cases. Why?:
With drugs:
- Fluorescence bronchoscopy can be employed to
- localize the source of abnormal cells
- procuring premalignant tissue for molecular biology studies
- monitoring progress of patients in chemoprevention studies
- Lung cancer mortality may be reduced if it can be detected early at the in situ stage. This may prevent disease progression to the malignant stage.
- Yield of sputum cytology is low; improvements in the sensitivity of the method to detect cancer and better cytomorphology criteria are needed.
- Conventional white light bronchoscopy not accurate: Misses significant dysplasia or carcinoma in situ in about 10-20% of cases. Why?:
- carcinomas are only a few cell laters thick (0.2 to 1 mm) and a few millimeters in surface diameter
- since they are small and thin, they usually do not produce any visible abnoramlity on conventional white-light bronochoscopy
With drugs:
- these drugs are preferentially retained in tumours
- bulk of research focused on hematoporphyrin derivative (HpD) or its partially purified form, dihematoporphyrin ether/ester (photofrin):
- HpD or photofrin emits red fluorescence when excited by violet light
- Since the concentration of HpD or photofrin is higher in malignant tumours than most nonmalignant tissue, tumours can thus be detected by their more intense fluorescence
- Autofluorescence approach
- use of in vivo spectroscopy with a helium-cadmium laser (442 nm) for illumination showed a significant decrease in autofluorescence intensity, predominantly in the green region of the visible spectrum in areas with dysplasia or carcinoma in situ, compared to normal bronchial tissue.
- comprises of a helium-cadmium laser, two image-intensified CDD cameras with green and red band-pass filters, a computer with imaging board and a color video monitor
- real time imaging
- even precancerous lesions (moderate and severe dysplasia) can be detected by this means
- sensitivity: 72.5%, specificity: 94% (white-light sensitivity: 48.4%, specificity same)
- detecting and localizing early lung cancer
- staging the extent of endobronchial spread of bronchial cancers
- detecting and localizing premalignant lesions (moderate/severe dysplasia in chemoprevention studies
- providing a vehicle to educate bronchoscopists in recognizing subtle changes during routine bronchoscopy
Venmans et al., Chest, Vol. 117, 2000
Outcome of Bronchial Carcinoma In Situ
- Squamous cell carcinoma of the bronchus arises after a serios of progressive histological changes in the epithelium. These are in the order of increasing severity:
- Squamous cell carcinoma of the bronchus arises after a serios of progressive histological changes in the epithelium. These are in the order of increasing severity:
- basal cell metaplasia
- metaplasia
- dysplasia:
- mild
- moderate
- severe
- carcinoma in situ
Monday, 12 March 2007
Cothren et al., Gastrointestinal Endoscopy Vol 44, No. 2, 1996
Detection of dysplasia at colonoscopy using laser-induced fluorescence: a blinded study
- Methods: Fluorescence spectra were collected from normal mucosa and colonic polyps during colonoscopy using 370 nm excitation. Tissue was classified as normal, hyperplastic, or adenomatous by histologic examination. Preliminary data was used to devise an algorithm to differentiate tissue type based on probability distributions of the fluorescence intensity at 460 nm and the ratio of the intensity at 680 nm to that at 600 nm. The algorithm was then tested in a blinded fashion.
- Previously published studies have employed a variety of wavelengths in the ultraviolet range. Kapadia et al. distinguished excised adenomatous polyps from normal mucosa using 325 nm excitation light produced by a helium-cadmium (HeCd) laser and step-wise multivariate linear regression analysis. Marchesini et al. used 410 nm excitation light from a conventional spectrofluorimeter to discriminate dysplastic from nondysplastic colonic biopsy specimens.
(Kapadia CR, Cutruzzola FW, O'Brien KM, Stetz ML, Enriquez R, Deckelbaum LI. Laser-induced fluorescence spectroscopy of
human colonic mucosa: detection of adenomateus transformation. Gastroenterology 1990;99:150-7.)
(Marchesini R, Brambilla M, Pignoli E, et al. Light-induced fluorescence spectroscopy of adenomas, adenocarcinomas and nonneoplastic mucosa in human colon. I. In vitro measurements. J Photochem Photobiol B 1992;14:219-30.)
- We have systematically studied the fluorescence spectra of excised colon tissue excited by ultraviolet and visible wavelengths of light using a spectrofluorimeter to generate fluorescence excitation-emission matrices. The results of these preliminary observations identified excitation light of wavelengths near 330 nm, 370 nm, and 430 nm as optimal for the discrimination of normal and dysplastic colon tissue.
- Fluorescence spectra from normal mucosa, a hyperplastic polyp, and an adenomatous polyp from a typical patient are shown in Figure 2A. Each fluorescence spectrum exhibits a strong emission peak near 460 nm and only weak fluorescence at wavelengths greater than 600 nm. Generally, the peak fluorescence intensity of normal mucosa at 460 nm was greater than that of adenomatous polyps, with hyperplastic polyps intermediate in intensity.
- Previous studies by our group demonstrated that excitation wavelengths throughout the near-ultraviolet range are potentially suitable for the detection of colonic dysplasia. However, our excitation and emission spectral data indicated that 370 nm excitation light provides more specific information than shorter ultraviolet excitation wavelengths. In particular, shorter wavelengths were found to be less likely to excite fluorescence observed at 680 nm.
- Methods: Fluorescence spectra were collected from normal mucosa and colonic polyps during colonoscopy using 370 nm excitation. Tissue was classified as normal, hyperplastic, or adenomatous by histologic examination. Preliminary data was used to devise an algorithm to differentiate tissue type based on probability distributions of the fluorescence intensity at 460 nm and the ratio of the intensity at 680 nm to that at 600 nm. The algorithm was then tested in a blinded fashion.
- Previously published studies have employed a variety of wavelengths in the ultraviolet range. Kapadia et al. distinguished excised adenomatous polyps from normal mucosa using 325 nm excitation light produced by a helium-cadmium (HeCd) laser and step-wise multivariate linear regression analysis. Marchesini et al. used 410 nm excitation light from a conventional spectrofluorimeter to discriminate dysplastic from nondysplastic colonic biopsy specimens.
(Kapadia CR, Cutruzzola FW, O'Brien KM, Stetz ML, Enriquez R, Deckelbaum LI. Laser-induced fluorescence spectroscopy of
human colonic mucosa: detection of adenomateus transformation. Gastroenterology 1990;99:150-7.)
(Marchesini R, Brambilla M, Pignoli E, et al. Light-induced fluorescence spectroscopy of adenomas, adenocarcinomas and nonneoplastic mucosa in human colon. I. In vitro measurements. J Photochem Photobiol B 1992;14:219-30.)
- We have systematically studied the fluorescence spectra of excised colon tissue excited by ultraviolet and visible wavelengths of light using a spectrofluorimeter to generate fluorescence excitation-emission matrices. The results of these preliminary observations identified excitation light of wavelengths near 330 nm, 370 nm, and 430 nm as optimal for the discrimination of normal and dysplastic colon tissue.
- Fluorescence spectra from normal mucosa, a hyperplastic polyp, and an adenomatous polyp from a typical patient are shown in Figure 2A. Each fluorescence spectrum exhibits a strong emission peak near 460 nm and only weak fluorescence at wavelengths greater than 600 nm. Generally, the peak fluorescence intensity of normal mucosa at 460 nm was greater than that of adenomatous polyps, with hyperplastic polyps intermediate in intensity.
- Previous studies by our group demonstrated that excitation wavelengths throughout the near-ultraviolet range are potentially suitable for the detection of colonic dysplasia. However, our excitation and emission spectral data indicated that 370 nm excitation light provides more specific information than shorter ultraviolet excitation wavelengths. In particular, shorter wavelengths were found to be less likely to excite fluorescence observed at 680 nm.
Panjehpour et al., Gastrointestinal Endoscopy Vol. 41 (6), 1995
Spectroscopic diagnosis of esophageal cancer: new classification model, improved measurement system.
- A nitrogen-pumped dye-laser (model LC300C, Laser Photonics, Inc., Orlando, Fla.) was used to deliver 5-ns pulses of excitation light. The dye-laser was tuned at 410 nm.
- Each fluorescence lineshape spectrum was sampled at 15-nm intervals from 430 to 716 nm, excluding 430 nm. This resulted in intensities at 19 wavelengths being used in the statistical analysis.
- The fluorescence lineshapes are clearly different in normal and malignant tissue. The two spectra intersect at about 540 nm. Below 540 nm, the normal tissue fluorescence is stronger than that of malignant tissue. Above 540 nm, the normal tissue
fluorescence is weaker than that of malignant tissue.
- The stepwise discriminate analysis selected only five wavelengths: 490 nm, 580 nm, 670 nm, 685 nm, and 715 nm.
- In this study, the excitation wavelength of 410 nm proved to be suitable for diagnosis of esophageal malignancies. This is in close agreement with data from Richards-Kortum et al. reporting excitation wavelength of 430 + 10 nm for classification of colonic lesions.
(10. Richards-Kortum R, Rava RP, Petras RE, Fitzmaurice M, Sivak M, Feld MS. Spectroscopic diagnosis of colonic dysplasia.
Photochem Photobiol 1991;53:777-86.)
- Schomacker et al. used a nitrogen laser (337 nm) in vivo and in vitro to excite fluorescence of colonic tissue. Using a multivariate linear regression analysis, they distinguished neoplastic tissue from tissue that was not neoplastic with sensitivity and specificity of 80 % and 92 %, respectively.
(3. Schomacker KT, Frisoli JK, Compton CC, et al. Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential. Lasers Surg Med 1992;12:63~78.)
- Cothren et al. used 370-nm excitation light to examine in vivo fluorescence of colonic tissue. Using a two-dimensional scatter plot of fluorescence intensities at 460 nm versus 680 nm, they defined a straight line representing a decision surface, which minimized the number of misclassified samples. Using this technique, adenomas could be distinguished from nonadenomatous tissue in approximately 97 % of cases.
(4. Cothren RM, Richards-Kortum R, Sivak MV Jr, et al. Gastrointestinal tissue diagnosis by laser-induced fluorescence spectroscopy at endoscopy. Gastrointest Endosc 1990;36:105-11.)
- A nitrogen-pumped dye-laser (model LC300C, Laser Photonics, Inc., Orlando, Fla.) was used to deliver 5-ns pulses of excitation light. The dye-laser was tuned at 410 nm.
- Each fluorescence lineshape spectrum was sampled at 15-nm intervals from 430 to 716 nm, excluding 430 nm. This resulted in intensities at 19 wavelengths being used in the statistical analysis.
- The fluorescence lineshapes are clearly different in normal and malignant tissue. The two spectra intersect at about 540 nm. Below 540 nm, the normal tissue fluorescence is stronger than that of malignant tissue. Above 540 nm, the normal tissue
fluorescence is weaker than that of malignant tissue.
- The stepwise discriminate analysis selected only five wavelengths: 490 nm, 580 nm, 670 nm, 685 nm, and 715 nm.
- In this study, the excitation wavelength of 410 nm proved to be suitable for diagnosis of esophageal malignancies. This is in close agreement with data from Richards-Kortum et al. reporting excitation wavelength of 430 + 10 nm for classification of colonic lesions.
(10. Richards-Kortum R, Rava RP, Petras RE, Fitzmaurice M, Sivak M, Feld MS. Spectroscopic diagnosis of colonic dysplasia.
Photochem Photobiol 1991;53:777-86.)
- Schomacker et al. used a nitrogen laser (337 nm) in vivo and in vitro to excite fluorescence of colonic tissue. Using a multivariate linear regression analysis, they distinguished neoplastic tissue from tissue that was not neoplastic with sensitivity and specificity of 80 % and 92 %, respectively.
(3. Schomacker KT, Frisoli JK, Compton CC, et al. Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential. Lasers Surg Med 1992;12:63~78.)
- Cothren et al. used 370-nm excitation light to examine in vivo fluorescence of colonic tissue. Using a two-dimensional scatter plot of fluorescence intensities at 460 nm versus 680 nm, they defined a straight line representing a decision surface, which minimized the number of misclassified samples. Using this technique, adenomas could be distinguished from nonadenomatous tissue in approximately 97 % of cases.
(4. Cothren RM, Richards-Kortum R, Sivak MV Jr, et al. Gastrointestinal tissue diagnosis by laser-induced fluorescence spectroscopy at endoscopy. Gastrointest Endosc 1990;36:105-11.)
Saturday, 10 March 2007
Wang et al., Applied Spectroscopy Vol. 45 (3), 1991
A Two-Dimensional Fluorescence Lifetime Imaging System Using a Gated Image Intensifier
A simple fluorescence lifetime imaging system using a gated microchannel plate (MCP) image intensifier coupled to a CCD camera has been developed. Nanosecond-level time-resolved fluorescence images of a sample under a pulsed light excitation can be detected directly. With a rapid lifetime determination method for multigate detection, fluorescence lifetime imaging can be promptly performed. In the present system, laser excitation of sample and shutter action of an image intensifier are fully synchronized by means of an optical fiber delay line. In order to compensate for fluctuations in the excitation source, a simple intensity monitor circuit was developed. Details of the instrumental system and verification measurements on two component samples are presented.
Index Headings: Spectroscopic techniques; Time-resolved spectroscopy;
Fluorescence lifetime imaging; Gated image intensifier; Instrumentation.
A simple fluorescence lifetime imaging system using a gated microchannel plate (MCP) image intensifier coupled to a CCD camera has been developed. Nanosecond-level time-resolved fluorescence images of a sample under a pulsed light excitation can be detected directly. With a rapid lifetime determination method for multigate detection, fluorescence lifetime imaging can be promptly performed. In the present system, laser excitation of sample and shutter action of an image intensifier are fully synchronized by means of an optical fiber delay line. In order to compensate for fluctuations in the excitation source, a simple intensity monitor circuit was developed. Details of the instrumental system and verification measurements on two component samples are presented.
Index Headings: Spectroscopic techniques; Time-resolved spectroscopy;
Fluorescence lifetime imaging; Gated image intensifier; Instrumentation.
Cubeddu et al., IEEE Journal of Quantum Electronics. Vol. 26, No. 12, Dec 1990
Time-Resolved Fluorescence Spectroscopy of the Retinal Pigment Epithelium: Age-Related Studies
- Lipofuscin granules contain fluorophores which emit yellow-orange light upon UV illumination.
- An Ar-ion or a Kr-ion laser working in mode-locking (M-L) regime are used as the principal excitation source. The two lasers can be tuned to all their emission source. The two lasers can be tuned to all their emission lines (except for the 488 nm line of the Ar-ion laser), thus providing excitation pulses at wavelengths ranging from 364 to 700 nm at intervals of = 50 nm.
- Melanin samples exhibited fluorescence spectra peaking in the blue (440 nm, corresponding excitation peak at 350 nm.
- Lipofuscin samples exhibited a broad emission peak around 590-600 nm, with a corresponding almost flat excitation spectrum from 350 to 500 nm. A secondary peak around 650 nm appeared in samples from older age groups.
- Lipofuscin granules contain fluorophores which emit yellow-orange light upon UV illumination.
- An Ar-ion or a Kr-ion laser working in mode-locking (M-L) regime are used as the principal excitation source. The two lasers can be tuned to all their emission source. The two lasers can be tuned to all their emission lines (except for the 488 nm line of the Ar-ion laser), thus providing excitation pulses at wavelengths ranging from 364 to 700 nm at intervals of = 50 nm.
- Melanin samples exhibited fluorescence spectra peaking in the blue (440 nm, corresponding excitation peak at 350 nm.
- Lipofuscin samples exhibited a broad emission peak around 590-600 nm, with a corresponding almost flat excitation spectrum from 350 to 500 nm. A secondary peak around 650 nm appeared in samples from older age groups.
Thursday, 8 March 2007
Colon Tissue Phantoms
- Purchased from Buy-A-Mag Co. (Murukeshan and Sujatha, Opt.
- Rat colon tissue (animal cancer model - rats treated with azoxymethane (AOM), a colon-specific carcinogen. Carcinogenisis will occur in the distal part of the colon.)
- Purchased from Phantom Lab,
- Cadaveric colon phantom
- Lucite cyclinder into which fabricated polyps were inserted (Dijkers et. al.)
- Pig colon phantom
Wagnieres et al., Photochem. Photobil. 68(5), 1998
In Vivo Fluorescence Spectroscopy and Imaging for Oncological Applications
Introduction
Historical background
Clinical context
Fluorophores
Fluorophore precursors
Physical Principles and Instrumentation
Clinical applications in the GI tract
Introduction
- Critical status report on the detection and characterisation of premalignant or malignant lesions using in vivo fluorescence spectroscopy and imaging
- Also considered is use of these techniques to guide tissue biopsy and surgical resection
- Approaches to tissue characterisation by light-induced fluorescence spectroscopy (LIFS) and imaging classified according to fluorophore type investigated and principle of instrumentation used. 3 main categories:
- Endogenous fluorophores (causes autofluorescence)
- Fluorophores synthesised in tissue after external administration of precursor molecule:
- protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (
- Fluorophores administered as exogenous drugs:
- fluorescein
- indocyanin green (ICG)
- photosensitizers used for photodynamic therapy (PDT), eg. hematoporphyrin derivatives (HpD) and tetra(m-hydroxyphenyl)chlorin (mTHPC)
Historical background
- History: First study concerning fluorescence in tumours under illumination with UV/violet light (Policard, 1924). The fluorescence was attributed to endogenous porphyrins
Clinical context
- Often, early tumours arise in a region of transformed mucosa on the surface of hollow organs (e.g. bronchi, GI tract, bladder and female reproductive tract) and are radiologically and endoscopically occult, i.e. not detectable upon gross examination)
- In the GI tract, conventional white-light endoscopy has a low sensitivity for detection of dysplastic lesions.
- Patients at high risk require frequent surveillance, including mutiple biopsies, over a long period to increase the chances of detecting dysplastic or early-stage neoplastic lesions.
- Additional potential applications of fluorescent techniques:
- provide guidance in locating optimum sites for biopsy (7)
- define the surgical margins for tumor resection (73, 105, 106)
- optimisation and monitoring of PDT treatments (21, 94, 107, 108)
- Optical biopsy: A controversial but recurring concept; envisaged that diagnosis based on in situ optical measurements could be performed without tissue removal for histopathological examination
- This paper thinks it is probably an unachievable goal, and detracts from the valuable and realistic uses of fluorescence and other optical techniques.
- Potential clinical advantages:
- high signal sensitivity, esp. if point measurements are used
- particularly suited for examination of tissue surfaces
- flexibility in anatomical sites tt can be investigated, esp. using small diameter optical fibre probes
- reduction in tissue biopsies
- ease of use by clinician
- potential for reduced healthcare costs as a consequence of minimally-invasive nature
- speed of technique - fast!
- improved patient outcome
Fluorophores
- Endogenous fluorophores:
- Most associated with structural matrix of tissues (most impt of which are collagen and elastin); fluorescence is as a result of cross-linking between amino acids
- Or they can also be involved in cellular metabolic processes (NADH, flavins)
- Others include aromatic amino acids (tryptophan, tyrosine, phenylalanine), various porphyrins and lipopigments (ceroids, lipofuscin) that are end-products of lipid metabolism
- Each fluorophore has distinct a excitation/emission spectrum
- Any tissue contains a mixture of many fluorophores of different concentrations.
- Fluorophores are not uniformly distributed in tissue and vary with depth below the tissue surface. E.g. GI tract has a distinct layered structure (mucosa, submucosa, muscularis), each of which has a different fluorophore composition.
- Early detection of premalignant lesions/cancer using autofluorescence is dependent on the following fields:
- Fluorophore concentration/Spatial distribution
- Metabolic status (NADH fluorescent when reduced)
- Biochemical/Biophysical microenvironment
- Tissue architecture (mucosal thickening/loss of layer)
- Wavelength-dependent light attentuation
- In vivo fluorescence signal is dependent on the excitation/emission wavelengths used. E.g.:
- Fluorescence from aromatic amino acids observed only with UV excitation
- Depth of penetration of excitation light increases with wavelength
- Exogenous fluorophores developed primarily as PDT (photodynamic therapy) sensitizers
Fluorophore precursors
- 5-Aminolevulinic acid (ALA) - rate limiting precursor in heme biosynthesis. Penultimate step of the synthesis process produces the fluorophore protoporphyrin IX (PpIX)
- PpIX excited in the Soret band around 400 nm, or around 635 nm at the highest Q-band. Typical red emission in 625-725 nm region.
- Impt for tumour detection by exogenous fluorescence: Why do certain drugs appear to localize preferentially in tumours?
- tumour pH lower than that or normal tissue
- prescence of plasma lipoproteins, in particular LDL
- tumour vasculature
Physical Principles and Instrumentation
- General principles
- Light sources: arc lamps and CW, intensity-modulated or pulsed lasers. Lasers allow use with standard endoscopes!
- Illumination and detection optics: 2 ways - fibre is places in direct contact with tissue (might be afected by pressure at point of contact; or a larger tissue surface area is illuminated (spectral distortion may arise)
- For spectroscopic measurements: fluorescence emission collected via same fibre as used for light delivery
- Detectors: Intensified charge-coupled device (CCD) camera, nonintensified cameras, high-sensitivity photographic film or direct viewing by eye
- Point systems
- Most in vivo instruments employ optical fibres to guide the fluorescence excitation and emission light
- Basic instrument structure: light source, fibre light guides, detector.
- Light attenuation: determines how fast the light intensity decreases with distance from objects.
Clinical applications in the GI tract
- Strong rationale for developing fluorescence diagnostics in the GI tract:
- Detection of dysplastic and early malignant lesions
- Differentiating hyperplastic, metaplastic and dysplastic pathologies
- Challenges faced when performing fluorescence spect. or imaging in GI tract:
- prescence of pre-exsiting benign disease (ulcerative colitis, Barrett's esophagus) that may also have altered fluorescence compared with normal tissue - might interfere/affect results of procedure
- contamination due to food residues, stoll and mucus
- the large and motile luminal surface
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