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Spie Press Book

Optical Clearing of Tissues and Blood
Author(s): Valery V. Tuchin
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Book Description

This book describes an optical clearing method based on reversible reduction of tissue scattering due to refractive index matching of scatterers and ground matter. This technique, which has been of great interest for research and application in the last decade, is a promising technique for future developments in the fields of tissue imaging, spectroscopy, phototherapy, and laser surgery.

Book Details

Date Published: 7 November 2005
Pages: 256
ISBN: 9780819460066
Volume: PM154

Table of Contents
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Preface vii
Introduction: Brief Review ix
1 Tissue and Blood Optical Immersion by Exogenous Chemical Agents 1
1.1 Tissue Structure and Scattering Properties 1
1.2 Principles of Optical Immersion and Chemical Agent Diffusion 12
1.3 Thermodynamics of Water Transport Through a Semipermeable Membrane 18
1.4 Tissue Swelling and Hydration 19
2 Optical Clearing of Fibrous Tissues 23
2.1 Spectral Properties of Immersed Eye Sclera 23
2.1.1 Monte Carlo modeling 23
2.1.2 In vitro measurements 32
2.2 In vitro Frequency-Domain Measurements of Eye Sclera 52
2.3 In vivo Measurements of Eye Sclera 54
2.4 Dura Mater Immersion and Agent Diffusion Rate 58
2.5 Conclusions 60
3 Optical Clearing of Skin 61
3.1 Introduction 61
3.2 In vitro Speckle and Spectral Measurements 63
3.3 In vivo Spectral Reflectance Measurements 70
3.4 In vivo Frequency-Domain Measurements 75
3.5 OCT Imaging 78
4 Optical Clearing of Gastric Tissue 83
4.1 Spectral Measurements 83
4.2 OCT Imaging 84
5 Tissue Structural Properties Studies a tOptical Immersion 87
5.1 Polarization Measurements 87
5.2 Confocal Microscopy 94
5.3 Fluorescence Measurements 95
5.3.1 Transmittance of Fluorescent radiation 95
5.3.2 In vivo measurements for intact human skin 97
5.3.3 Two-photon scanning fluorescence microscopy 98
5.4 The Second Harmonic Generation 103
6 Optical Clearing of Blood, Cells, and Microorganisms 107
6.1 Blood Flow and Microvessel Network Imaging 107
6.2 OCT Measurements of Blood Optical Clearing 112
6.3 Theoretical Analysis of Blood Optical Clearing 125
6.4 Optical Immersion of Cells and Microorganisms 133
7 Chemical Agent Delivery 147
7.1 Introduction 147
7.2 Cosmetic Preparations and Skin Permeation 148
7.3 Diffusion of Macromolecules in Tissues 151
7.4 Enhanced Diffusion of Molecules Through Tissues 155
7.4.1 Occlusion 155
7.4.2 Chemical enhancers 156
7.4.3 Physical enhancers 157
7.5 Skin Reservoirs for Topically Applied Clearing Agents 162
8 Applications 167
8.1 Glucose Sensing 167
8.1.1 Introduction 167
8.1.2 Tissue and bloodscattering spectroscopy 167
8.1.3 OCT measurements 170
8.2 Speckle Technologies 175
8.2.1 Speckle topography and tomography 175
8.2.2 Diffusion-wave spectroscopy and functional imaging 186
8.3 Precision Tissue Photodisruption and Tattoo Removal 188
9 Other Methods of Tissue Optical Properties Control 193
9.1 Tissue Compression and Stretching 193
9.2 Temperature Effects and Tissue Coagulation 205
9.3 Tissue Whitening 211
Conclusion 213
References 217


This book describes an optical clearing method based on reversible reduction of tissue scattering due to refractive index matching of scatterers and ground matter, which was of great interest for research and application in the last decade and is a promising technique for future developments in the fields of tissue imaging, spectroscopy, phototherapy, and laser surgery.

The basic principles, recent results, advantages, limitations, and future of the optical immersion method applied to clearing of the naturally turbid biological tissues and blood are overviewed in the book. The refractive index matching concept for enhancement of in- depth light and laser beam penetration into tissues and blood is discussed on the basis of in vitro and in vivo studies using optical spectroscopy, polarization, and coherence domain techniques. The optical properties of tissues with basic multiple scattering, which are transformed to a low scattering mode, are analyzed. It is shown that light reflection, transmission, scattering, and polarization can be effectively controlled. The diagnostic abilities of the method based on contrasting of abnormalities, on in-depth profiling of tissue and blood, and on monitoring of endogenous and exogenous matter diffusion within tissue are demonstrated.

The author is grateful to Ms. Sharon Streams, who has supported the idea to publish the book, for her assistance in editing and production.

The author is very thankful to attendees of the short courses �Coherence, Light Scattering, and Polarization Methods and Instruments for Medical Diagnosis,� �Tissue Optics and Spectroscopy,� and �Tissue Optics and Controlling of Tissue Optical Properties,� which the author has given during the SPIE Photonics West Symposia, SPIE/OSA European Conferences on Biomedical Optics, and OSA CLEO/QELS Conferences over last ten years, for their stimulating questions, fruitful discussions, and critical evaluations of the presented materials. Their responses were very valuable for the preparation of this edition.

The original part of this work was supported within both Russian and international research programs by grant N25.2003.2 of the President of Russian Federation �Supporting of Scientific Schools,� grant N2.11.03 �Leading Research-Educational Teams,� contract No. �Biophotonics� of the Ministry of Industry, Science and Technologies of RF, grant REC-006 of CRDF (U.S. Civilian Research and Development Foundation for the Independent States of the Former Soviet Union) and the Russian Ministry of Education, the Royal Society grant for a joint project between Cranfield University (UK) and Saratov State University, grants of National Nature Science Foundation of China (NSFC), and by Palomar Medical Technologies Inc.

The author greatly appreciates the cooperation, contribution, and support of all his colleagues from the Optics and Biomedical Physics Division of the Physics Department and Research-Educational Institute of Optics and Biophotonics at Saratov State University, especially of Profs. Yu.P. Sinichkin, D.A. Zimnyakov, V.I. Kochubey, I.L. Maksimova, and E.I. Galanzha, and Drs. A.N. Bashkatov, E.A. Genina, A.B. Pravdin, G.V. Simonenko, K.V. Larin, I.V. Meglinsky, S.P. Chernova, and I.V. Yaroslavsky.

The author would like to thank all of his numerous colleagues and friends all over the world for collaboration. He is especially grateful to Dr. B. Chance and Dr. A. Yodh from the University of Pennsylvania (USA) for collaboration in the framework of the CRDF Research Program, to Dr. R.K. Wang and Dr. X. Xu from Cranfield University (UK), Dr. Q. Luo from HUST University (China), and Stoyan Tanev from VITESSE Re-Skilling Canada Inc. (Canada).

The author expresses gratitude to his wife, Nataliya, and to all of his family, especially to his daughter Nastya and grandchildren Dasha, Zhenya, and Stepa, for their indispensable support, understanding, and patience during the writing of this book.

Valery Tuchin
April 2005

Introduction: Brief Review

Reflection, absorption, scattering, and fluorescence in living tissues and blood can be effectively controlled by various methods. Staining (sensitization) of biological materials is extensively used to study mechanisms of interaction between their constituent components and light, and also for diagnostic purposes and selective photodestruction of individual components of living tissues. This approach underlies the diagnosis and photodynamic therapy (PDT) of malignant neoplasm, UV-A photochemotherapy of psoriasis and other proliferative disorders, angiography in ophthalmology, and many other applications in medicine.

In the visible and NIR spectrums, tissues and bioliquids are low absorbing, but highly scattering media. Scattering defines spectral and angular characteristics of light interacting with living objects, as well as its penetration depth; thus, optical properties of tissues and blood may be effectively controlled by changes of scattering properties. The living tissue allows one to control its optical (scattering) properties using various physical and chemical actions such as compression, stretching, dehydration, coagulation, UV irradiation, exposure to low temperature, and impregnation by chemical solutions, gels, and oils. All these phenomena can be understood if we consider tissue as a scattering medium that shows all optical effects that are characteristic to turbid physical systems. It is well known that the turbidity of a dispersive physical system can be effectively controlled by providing matching of refractive indices of the scatterers and the ground material. This is a so-called optical immersion technique. Another possibility for controlling the optical properties of a disperse system is to change its packing parameter and/or scattered sizing.

Control of optical properties tissues in vivo is very important for many medical applications. A number of laser surgery, therapy, and diagnostic technologies include tissue compression and stretching used for better transportation of the laser beam to underlying layers of tissue. The human eye compression technique allows one to perform transscleral laser coagulation of the ciliary body and retina/choroid. The possibility of selective translucence of the upper tissue layers should be very useful for developing of the eye globe imaging techniques and for detecting local inhomogeneities hidden by a highly scattering medium in functional tomography. Results on the control of human sclera optical properties by tissue impregnation with osmotically active chemicals such as Trazograph (X-ray contrast), glucose, and polyethylene glycol (PEG), as well as hypaque-60 (X-ray contrast), were reported.

In general, the scattering coefficient ?s and scattering anisotropy factor g of a tissue is dependent on the refractive index mismatch between cellular tissue components: cell membrane, cytoplasma, cell nucleus, cell organelles, melanin granules, and the extracellular fluid. For fibrous (connective) tissue (eye scleral stroma, corneal stroma, skin dermis, cerebral membrane, muscle, vessel wall noncellular matrix, female breast fibrous component, cartilage, tendon, etc.), index mismatch of the interstitial medium and long strands of scleroprotein (collagen-, elastin-, or reticulin-forming fibers) is important. The refractive index matching is manifested in the reduction of the scattering coefficient (?s?0) and increase of single scattering directness (g?1). For skin dermis and eye sclera ?s, reduction can be very high. For hematous tissue such as the liver, its impregnation by solutes with different osmolarity also leads to refractive index matching and reduction of the scattering coefficient, but the effect is not so pronounced as for skin and sclera, due to cells changing size as a result of osmotic stress.

It is possible to achieve a marked impairment of scattering by means of the intratissue administration of appropriate chemical agents. Conspicuous experimental optical clearing in human and animal sclera; human, animal, and artificial skin; human gastrointestinal tissues; and human and animal cartilage and tendon in the visible and NIR wavelength ranges induced by administration of X-ray contrast agents (Verografin, Trazograph, and Hypaque-60), glucose, propylene glycol, polypropylene glycol-based polymers (PPG), polyethylene glycol (PEG), PEG-based polymers, glycerol, and other solutions.

Coordination between refractive indices in multicomponent transparent tissues showing polarization anisotropy (e.g., cornea) leads to its decrease. In contrast, for a highly scattering tissue with a hidden linear birefringence or optical activity, its impregnation by immersion agents may significantly improve the detection ability of polarization anisotropy due to reduction of the background scattering.

Concentration-dependent variations in scattering and transmission profiles in -crystalline suspensions isolated from calf lenses are believed to be related to osmotic phenomenon. Osmotic and diffusive processes that occur in tissues treated with Verografin, Trazograph, glucose, glycerol, and other solutions are also important. Osmotic phenomena appear to be involved when optical properties of biological materials (cells and tissues) are modulated by sugar, alcohol, and electrolyte solutions. This may interfere with the evaluation of hemoglobin saturation with oxygen or identification of such absorbers as cytochrome oxidase in tissues by optical methods.

Experimental studies on optical clearing of normal and pathological skin and its components (epidermis and dermis) and the management of reflectance and transmittance spectra using water, glycerol, glycerol-water solutions, glucose, sunscreen creams, cosmetic lotions, gels, and pharmaceutical products were carried out in Refs. The control of skin optical properties was related to the immersion of refractive indices of scatterers (keratinocyte components in epidermis and collagen, and elastic fibers in dermis) and ground matter, and/or reversible collagen dissociation. In addition, some of the observed effects appear to have been due to the introduction of additional scatterers or absorbers into the tissue or, conversely, to their washing-out.

A marked clearing effect through the hamster and the human skin, the human and rabbit sclera, and rabbit dura matter was occurred for an in vivo tissue within a few minutes of topical application (eye, dura matter, skin) or intratissue injection (skin) of glycerol, glucose, propylene glycol, Trazograph, and PEG and PPG polymers.

Albumin, a useful protein for index matching in phase contrast microscopy experiments, can be used as the immersion medium for tissue study and imaging. Proteins smaller than albumin may offer a potential alternative because of relatively high scattering of albumin. Sometimes medical diagnosis or contrasting of a lesion image can be provided by the enhancement of a tissue�s scattering properties by applying, for instance, acetic acid, which has been successfully used as a contrast agent in optical diagnostics of cervical tissue. It has been suggested that the acetowhitening effect seen in cervical tissue is due to coagulation of nuclear proteins. Therefore, an acetic acid probe may also prove extremely significant in quantitative optical diagnosis of precancerous conditions because of its ability to selectively enhance nuclear scatter.

Evidently, the loss of water by tissue seriously influences its optical properties. One of the major reasons for tissue dehydration in vivo is the action of endogenous or exogenous osmotic liquids. In in vitro conditions, spontaneous water evaporation from tissue, tissue sample heating at a noncoagulating temperature, or its freezing in a refrigerator push tissue to loose water. Typically in the visible and NIR spectrums, far from water absorption bands, the absorption coefficient increases by a few dozen percent, and the scattering coefficient by a few percent due to closer packing of tissue components caused by its shrinkage. However, the overall optical transmittance of a tissue sample increases due to the decrease of its thickness at dehydration. Specifically, in the vicinity of the strong water absorption bands, the tissue absorption coefficient decreases due to less concentration of water in spite of higher density of tissue at its dehydration.

It is possible to significantly increase transmission through a soft tissue by its squeezing (compressing) or stretching it. The optical clarity of living tissue is due to its optical homogeneity, which is achieved through the removal of blood and interstitial liquor (water) from the compressed site. This results in a higher refractive index of the ground matter, whose value becomes close to that of scatterers (cell membrane, muscle, or collagen fibers). Closer packing of tissue components at compression makes the tissue a less chaotic and more organized system�what may give less scattering due to cooperative (interference) effects. Indeed, the absence of blood in the compressed area also contributes to altered tissue absorption and refraction properties. Certain mechanisms underlying the effects of optical clearing and changing of light reflection by tissues at compression and stretching were proposed in Refs.

Long-pulsed laser heating induces reversible and irreversible changes in the optical properties of tissue. In general, the total transmittance decreases and the diffuse reflectance increases, showing nonlinear behavior during pulsed laser heating. Many types of tissues slowly coagulated (from 10 min to 2 hrs) in a hot water or saline bath (70�85 C) exhibit an increase of their scattering and absorption coefficients.

UV irradiation causes erythema (skin reddening), stimulates melanin synthesis, and can induce edema and tissue proliferation if the radiation dose is sufficiently large. All these photobiological effects may be responsible for variations in the optical properties of skin, and need to be taken into consideration when prescribing phototherapy. Also, UV treatment is known to cause color development in the human lens.

Natural physiological changes in cells and tissues are also responsible for their altered optical properties, which may be detectable and, thus, used as a measure of these changes. For example, measurements of the scattering coefficient allow one to monitor glucose or edema in the human body, as well as blood parameters. Many papers report optical characteristics of blood as functions of hemoglobin saturation with oxygen. The alterations of the optical properties of blood caused by changes of the hematocrit value, temperature, and flow parameters.

As a particle system, whole blood shows pronounced clearing effects that may be accompanied by induced or spontaneous aggregation and disaggregation processes, as well as RBC swelling or shrinkage at application of biocompatible clearing agents with certain osmotic properties.

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