Lund Reports on Atomic Physics Laser-Induced Fluorescence for Medical Diagnostics Stefan Andersson-Engels LRAP-108 , ISSN 0281-2762 ISBN 91-7900-891-7 Laser-Induced Fluorescence for Medical Diagnostics Stefan Andersson-Engels Lund Reports on Atomic Physics LRAP-108, December 1989 Contents Abstract 3 List of papers 4 I. Introduction 7 II. The interaction of light with tissue 9 Introduction 9 Theoretical models for light propagation in tissue 14 A theoretical model for thermal propagation in tissue 21 Experimental investigations to measure optical coefficients in tissue 21 Applications 26 Photothermal interactions 27 Photo-ablation 28 Photochemical interactions 31 Tissue transillumination 33 Blood and tissue oxygenation 34 III. Photosensitizers and photodynamic therapy (PDT) 36 Porphyrins 36 Photophysical properties of porphyrins 37 Photodynamic therapy 40 Characteristics of PDT 47 Photosensitizers under investigation for PDT 48 IV. Tissue fluorescence 52 Introduction 52 Theory 56 In vivo Applications 62 Malignant tumour detection and investigation 62 Identification of atherosclerotic plaque 63 Instrumentation 64 '' Brief summary of the papers 67 acknowledgements 69 References 71 Japers Abstract Laser-induced fluorescence as a tool for tissue diagnostics is dis- cussed. Both spectrally and time-resolved fluorescence signals are studied to optimize the demarcation of diseased lesions from normal tissue. The presentation is focused on two fields of application: the identification of malignant tumours and atherosclerotic plaques. Tissue autofluorescence as well as fluorescence from administered drugs have been utilized in diseased tissue diagnosis. The fluorescence criterion for tissue diagnosis is, as far as possible, chosen to be independent of unknown fluorescence parameters, which are not correlated to the type of tissue investigated. Both a dependence on biological parameters, such as light absorption in blood, and instrumental characteristics, such as excitation pulse fluctuations and detection geometry, can be minimized. Several chemical compounds have been studied in animal experiments after intraveneous injection to verify their capacity as malignant tumour marking drugs under laser excitation and fluorescence detection. Another objective of these studies was to improve our understanding of the mechanism and chemistry behind the retention of the various drugs in tissue. The properties of a chemical which maximize its selective reten- tion in tumours are discussed. In order to utilize this diagnostic moda- lity, three different clinically adapted sets of instrumentation have been developed and are presented. Two of the systems are nitrogen-laser- based fluorosensors; one is a point-monitoring system with full spectral resolution and the other one is an imaging system with up to four simul- taneously recorded images in different spectral bands. The third system is a low-cost point-monitoring mercury-lamp-based fluorosensors with the potential of recording fluorescence excitation, fluorescence emission as well as reflection characteristics of tissue. - 3 - List of papers This thesis is based on sixteen papers co-authored by the author of this thesis. During the course of this work, in connection with his marriage, the author's name was changed from PS Andersson to S Andersson-Engels. The papers included in the thesis are: I. Andersson PS, Montån S, Svanberg S. Remote sample characterization based on fluorescence monitoring. Appl Phys B 1987, 844:19-28 II. Andersson-Engels S, Johansson J, Svanberg S, The use of time- resolved fluorescence for diagnosis of atherosclerotic plaque and malignant tumours. Submitted to Spectrochim Acta (1989) III. Andersson PS, Gustafson A, Stenram U, Svanberg K, Svanberg S. Diagnosis of arterial atherosclerosis using laser-induced fluore- scence. Lasers Med Sci 1987, 2:261-6 IV. Andersson-Engels S, Gustafson A, Johansson J, Stenram U, Svanberg K, Svanberg S. Laser-induced fluorescence used in localizing atherosclerotic lesions. Lasers Med Sci 1989, 4:171-81 V. Andersson-Engels S, Johansson J, Stenram U, Svanberg K, Svanberg S. Time-resolved laser-induced fluorescence spectroscopy for enhanced demarcation of human atherosclerotic plaques. J Photochem PhotobiohB 1989 (in press) VI. Andersson-Engels S, Johansson J, Stenram U, Svanberg K, Svanberg S. Malignant tumor and atherosclerotic plaque diagnostics using laser-induced fluorescence. Invited paper for IEEE J Quantum Electr special issue on Lasers in Medicine, March, 1990 (to appear) VII. Andersson PS, Kjellén E, Montån S, Svanberg K, Svanberg S. Auto- fluorescence of various rodent tissues and human skin tumour samples. Lasers Med Sci 1987, 2:41-9 VIII. Andersson-Engels S, Brun A, Kjellén E, Salford LG, Strömblad L-G, Svanberg K, Svanberg S. Identification of brain tumours in rats using laser-induced fluorescence and haematoporphyrin derivative. Lasers Med Sci 1989, 4 (in press) IX. Andersson-Engels S, Ankerst J, Montån S, Svanberg K, Svanberg &. Aspects of tumour demarcation in rats by means of laser-induced fluorescence and haematoporphyrin derivatives. Laser Med Sci 1988, 3:239-48 X. Andersson-Engels S, Ankerst J, Johansson J, Svanberg K, Svanberg S. Tumour marking properties of different haematoporphyrins and tetrasulfonated phthalocyanine • a comparison. Lasers Med Sci 1989, 4:115-23 - 4 - XI. Andersson-Engels S, Johansson J, Killander D, Kjellén E, Svaasand LO, Svanberg K, Svanberg S. Photodynamic therapy and simultaneous near-infrared light-induced hyperthermia in human malignant tumours. A methological case study. LI A ICALEO 1987, 60:67-74 XII. Andersson-Engels S, Johansson J, Killander D, Kjellén E, Olivo M, Svaasand LO, Svanberg K, Svanberg S. Photodynamic therapy alone and in conjunction with near-infrared light-induced hypertliermia in human malignant tumors: a methodological case study. SP1E, Bellingham Wa USA 1988, 908:116-25 XIII. Andersson-Engels S, Elner Å, Johansson J, Karlsson S-E, Salford LG, Strömblad L-G, Svanberg K, Svanberg S. Clinical recording of laser-induced fluorescence spectra for evaluation of tumour demar- cation feasibility in selected clinical specialities. Lasers Med Sci 1989 (in press) XIV. Andersson PS, Montån S, Persson T, Svanberg S, Tapper S. Fluore- scence endoscopy instrumentation for improved tissue characteri- zation. Med Phys 1987, 14:633-6 XV. Andersson PS, Montån S, Svanberg S. Multispectral system for medi- cal fluorescence imaging. IEEE J Quantum Electr 1987, QE-23:1798-805 XVI. Andersson-Engels S, Johansson J, Svanberg S, Multicolor fluore- scence imaging system for tissue diagnostics. Manuscript for SPIE, Bellingham, 1990, 1205 (to appear) - 5 - Additional material not included in this thesis, using similar techni- ques, is presented in the following papers: A. Andersson PS, Montån S, Svanberg S. Oil slick characterization using an airborne laser fluorosensor - construction considerations. Lund Reports on Atomic Physics LRAP-45, 1985 B. Andersson PS, Montån S, Svanberg S. Flashlamps for remote fluore- scence characterization of oil slicks. Lund Reports on Atomic Physics LRAP-57, 1986 C. Andersson PS, Montån S, Svanberg S. Fluorosensor for remote cha- racterization of marine oil-slicks. In: Reuter R, Gillot RH (eds) Remote Sensing of Pollution of the Sea, Proc of the International Colloquium, Oldenburg FRG, March 31 - April 3, 1987, pp 223-36 Reviews of the work are also described in: a. Andersson S, Ankerst J, Kjellén E, Montån S, Sjöholm E, Svanberg K. Svanberg S. Tumour localization by means of laser-induced fluore- scence in hematoporphyrin derivative (HPD) - bearing tissue. In: Hänsch TW, Shen YR (eds) Laser Spectroscopy VII, Springer Series in Optical Sciences Vol 49, Springer Heidelberg 1985, pp 401-6 (invi- ted paper) b. Andersson-Engels S, Ankerst J, Brun A, Elner Å, Gustafson A, Johansson J, Karlsson S-E, Killander D, Kjellén E, Lindstedt E, Montån S, Salford LG, Simonsson B, Stenram U, Strömblad L-G, Svanberg K, Svanberg S. Tissue diagnostics using laser-induced fluorescence. Ber Bunsenges Phys Chem 1989, 93:335-42 (invited paper) c. Andersson-Engels S, Berg R, Johansson J, Svanberg K, Svanberg S. Medical applications of laser spectroscopy. In: Feld M (ed) Laser Spectroscopy IX, Academic Press, New York 1989 (invited paper) d. Andersson-Engels S, Johansson J, Svanberg K, Svanberg S. Fluore- scence diagnostics and photochemical treatment of diseased tissue using lasers. Part I. Accepted for publication in Analytical Chemistry, scheduled for the December Issue 1989 (invited paper) e. Andersson-Engels S, Johansson J, Svanberg K, Svanberg S. Fluore- scence diagnostics and photochemical treatment of diseased tissue using lasers. Part II. Accepted for publication in Analytical Chemistry, scheduled for January Issue 1990 (invited paper) f. Andersson-Engels S, Johansson J, Svanberg K, Svanberg S, Tissue diagnostics using laser-induced fluorescence. SPIE, Bellingham 1990, 1203 contribution no 9 (invited paper) (to appear) 6 - I. Introduction To meet medical demands for alternative and improved methods of diagno- sis as well as therapy, medical laser techniques are developing rapidly. The interaction of ionizing radiation with tissue has iong been used clinically to diagnose and treat various disorders. It has also been known for some time that non-ionizing electromagnetic radiation hus u potential for these purposes. A wide field of applications has been developed for ultraviolet radiation, visible light and infrared light, mainly following the availability of new laser sources and fibre-optical delivery systems. The most commonly used interaction mechanism between light and tissue has so far been the heating of tissue following the absorption of light, in, for example, laser surgery. Ophthalmology is often the clinical specialty to first use new laser techniques. Two factors make the eye well suited to laser therapy: the eye is a good optical system, making it easy to deliver the light to the lesiorT of interest; the relatively small tissue volume involved requiring only relatively small light energies and powers. Much more powerful systems are necessary for larger organs, which has delayed the development of these fields somewhat. The interaction between non-ionizing radiation with tissue is very different from that of X-rays or r-radiation. In tissue non-ionizing radiation interacts mainly with the outer electrons in the molecules. Upon absorption of a photon, a molecule is excited to a higher electro- nic state. This means that the molecule gains internal energy, but it is still intact with all electrons bound to the molecule. Tissue is there- fore not destroyed by photon absorption. The excess energy absorbed in the molecule can, however, be transformed into heat or may cause chemi- cal reactions which can damage the tissue. Another possibility is de- excitation through the emission of a photon - fluorescence. Different forms of laser therapy started to develop immediately after the invention of the laser in 1960. The field of diagnostics using lasers started later but has also gained interest during the last de- cade. Diffusely reflected or fluorescence light from tissue has been employed to measure blood perfusion and blood oxygenation as well as to demarcate diseased lesions. Fluorescence diagnostics of tissue has been applied by utilizing intrinsic tissue autofluorescence, or the fluore- scence from an administered chromophore. This thesis is divided into two parts, the first presenting certain aspects of the interaction between light and tissue, mainly focusing on laser-induced fluorescence, and the second containing the papers on which this thesis is based. The first part is subdivided into three main sections treating three different fields of interest for fluorescence diagnostics of tissue. The first section deals with the transport of light in tissue. Various theoretical models for light distribution are presented, as are the tissue parameters necessary for each model. The different mechanisms for tissue response during and after light irradia- tion are also discussed. Most medical applications of these responses are outlined. The next section discusses research into new chemicals which can be used in photochemotherapy and as tumour marking drugs for fluorescence diagnostics. This is presently a field of intense research. - 7 - The discussion is focused on the spectroscopical and photophysical characteristics of a number of the proposed drugs. The last section includes an overview of the field of tissue diagnostics using laser- induced fluorescence. Spectroscopical criteria to optimize the contrast between diseased and surrounding normal tissue with respect of being as independent as possible to other unknown parameters are discussed. Such uncertainties may be instrumental (measurement geometry, excitation pulse fluctuation and detection efficiency variation) or biological (effects of chromophores in the tissue that are of no interest in the diagnostics). Time-integrated, as well as time-resolved spectral charac- teristics are discussed. As a consequence of these studies three diffe- rent fluorosensors adapted for tissue diagnostics have been developed. Presentations of these systems are included in this thesis. - 8 - II. The interaction of light with tissue In this thesis the term light is used to denote electromagnetic radiation in the UV, visible and near- and mid-infrared spectral regions from 190 nm (the limit for light transmission in the atmosphere) to 10 urn. The interaction between tissue and light used for medical diagnostics and therapies will be discussed. Introduction We are all surrounded by light, most of it emanating from the sun which is propelled by nuclear fusion reactions. Light is utilized in several ways in our bodies - most obvious are the reactions in the photochemical receptors in the eye enabling us to see. In the skin, light triggers important photochemical and photobiological reactions. However, most of the body tissue will never see any light. The properties of tissue do not permit visible light to penetrate very deeply. The pigmentation in the skin shields the rest of the body from light. The optical properties and the way light is transported in tissue are not only of relevance for photo-induced reactions in the body, but they also determine the appearance of a person as seen by an observer. The colour and intensity of the light reflected or diffusely scattered from the tissue depend on the optical properties of the tissue. Light was found to be useful for medical purposes quite long ago. A few examples from modern time can be mentioned: diaphanography or light scanning, i.e. diagnostics using tissue transillumination with light. has been used for breast cancer tumour detection since 1929 [lj; pathological changes in the skin, such as psoriasis, were first treated with UV light at the beginning of this century [2,3]; and fluorescent drugs in combination with light irradiation were tested for tumour therapy starting around 1900 [4]. An interesting historical review of cutaneous photobiology is given in Ref. [5]. Other forms of photo- therapy have been suggested, among them wound healing, pain relief and light illumination as a step in psycho therapy. Medical examination and treatment methods involving light were developed and gained popularity after the invention of the first laser in 1960. Only the photothermal effects on the tissue during laser radiation have been explored in any depth - in laser surgery [6,7]. The increase in the tissue temperature is due to a single photon absorption process in the tissue chromophores. The absorbed energy is transformed into heat. Due to the ideal optical properties of the eye, ophthalmological lasers were among the first to be utilized [8]. Dermatological applications of lasers came naturally also early and proved to be straightforward. In order to optimize the cutting capability and healing and to be able to predict the tissue response of the laser irradiation, light dosimetry models for tissue have been developed and are being constantly improved. Also, the various mechanisms involved in the interaction between light and tissue are being studied, giving a deeper understanding of the processes involved. - 9 -