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Introduction to Biophotonics 生醫光子學導論 Chapter 3: Light-tissue interactions 孫家偉 Chia-Wei Sun 交通大學光電工程學系 Light-tissue interactions Light-tissue interaction, which is the basis for optically probing structure and function at cellular and tissue levels as well for the light-activated photodynamic therapy of cancer and other diseases; benefits from a molecular understanding of cellular and tissue structures and functions. The topics of biosensing, a hotly pursued area in view of possible threats of bioterrorism and constantly emerging new microbial infections, bioimaging, and multiple analyte detection using microarray technology, rely heavily on molecular recognition of biological species. Light-molecule interactions Light is an electromagnetic radiation consisting of oscillating electric and magnetic fields. Biological systems are molecular media. For such a medium the interaction with light can be described by the electronic polarization of a molecule subjected to an electric field. This approach is also referred to as the electric dipole (or simply dipole) approximation. Light-molecule interactions Light-molecule interactions Jablonski diagram Light-tissue interactions The variety of interaction mechanisms that may occur when applying light to biological manifold. Most important among optical tissue properties are the coefficients of reflection, absorption, and scattering. Together, they determine the total transmission of the tissue of the tissue at a certain wavelength. On the other hand, thermal tissue properties are decided by the parameters as well as wavelength, exposure time, applied energy, focal spot size, energy density, and power density. Light-cell interactions Biological cells span the size scale from submicron dimensions to over 20 µm. Therefore, they can be smaller than the wavelength of light or much larger. Interaction with light can lead to both scattering and absorption. Of particular interest in this regard is the Rayleigh scattering where even the subcellular components, organelles, can be a scattering center. Rayleigh scattering is dependent on three parameters: the size of the scattering centers (cells or organelles) the refractive index mismatch (difference) between a scattering center and the surrounding medium the wavelength of light Light-cell interactions The Rayleigh scattering is inversely proportional to the fourth power of wavelength. Therefore, a blue light (shorter wavelength) will be more scattered than a red light. On the basis of scattering alone as the optical loss mechanism (attenuation of light transmission), the longer the wavelength of light, the deeper it would penetrate in a biological specimen. However, an upper wavelength limit of transmission is set up by absorptive losses in IR due to water absorption and absorption by the -CH and the -OH vibrational overtone bands. Bulk scattering becomes more pronounced in tissues. Light-cell interactions Primary photoinduced cellular effects are produced by light absorption to induce transition between two electronic states (electronic or coupled electronic-vibrational transitions). Purely vibrational transitions (such as IR and Raman) are of significance only in structural identification and in conformational analysis. Light-cell interactions Light-absorbing components Endogeneous Exogeneous Constituents of cells and tissues Small molecules and molecular constituents of DNA, RNA, NADH: nucleotides, amino acids, water, protein-specific chromophores Photosensitizers added to absorb light and initiate physical and/or chemical changes in normal cellular components. Biopolymers Extracellular components Proteins present in tissues DNA Light absorption in proteins aliphatic 脂肪族的 aromatic 芳香的 benzene 苯 The basic constituents of proteins are amino acids, which can be aliphatic or aromatic (containing benzene or fused benzene type p-electron structures). The aliphatic amino acids absorb the UV light of wavelengths shorter than 240 nm. Colorless aromatic amino acids such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) absorb at wavelengths longer than 240 nm, but well below the visible. Light absorption in proteins However, the absorption by a protein is not completely defined by those of the constituent amino acid residues. Protein bonding involving the polypeptide bonds and disulfide linkage also absorb and contribute to the overall absorption of a protein. Furthermore, a protein may contain a chromophore such as the heme group (in hemoglobin) and cis-retinal (in case of retinal protein), which provide strong absorption bands. Light absorption in cellular components Hemoglobin has absorption peaks around 280 nm, 420 nm, 540 nm, and 580 nm. Melanin, the basic pigment of skin, has a broad absorption, covering the entire visible region, but decreasing in magnitude with the increase of wavelength. Light absorption in cellular components A cellular component, exhibiting absorption in the visible, is NADH, with absorption peaks at ~270 nm and 350 nm. Water does not have any bands from UV to near IR, but starts absorbing weakly above 1.3 µm, with more pronounced peaks at wavelengths ≥2.9 µm and very strong absorption at 10 µm, the wavelength of a CO2 laser beam. Therefore, most cells exhibit very good transparency between 800 nm and 1.3 µm. Light absorption in cellular components Light absorption in DNA The constituents of DNA and RNA are the nucleotides that contain carbohydrates and purine and pyrimide bases (A,C,T,G, and U). The absorption by carbohydrates is below 230 nm; the absorption by the carbohydrate groups generally does not produce any significant photophysical or photochemical effect. The purine and pyrimidine bases absorb light of wavelengths in the range of 230–300 nm. This absorption is mainly responsible for DNA damage. Light-induced cellular processes Light absorption by cellular components Radiative processes Nonradiative processes Autofluorescence Thermal effect (i) Light converted into local heating by IC and/or ISC and vibrational relaxation (ii) Thermal conduction to other areas Protein denaturation due to conformational changes Loss of enzymatic activity Water vaporization at stronger heating, leading to disruption of cellular structure Photochemical Excited-state chemistry Photoaddition Photofragmentation Photo-oxidation Photohydration Cis–trans Isomerization Photorearrangement Autofluorescence A number of cellular constituents fluoresce when excited directly or excited by energy transfer from another constituent. This fluorescence is called endogenous fluorescence or autofluorescence, and the emitting constituent is called an endogenous fluorophore (also called fluorochrome). Fluorescence originates from an excited singlet state and has typical lifetimes in the range of 1– 10 nsec. Phosphorescence, which is emission from an excited triplet, is generally not observed from cellular components. Some of the fluorophores native to cells are NADH, flavins and aromatic amino acid constituents of proteins. Autofluorescence In addition, some important endogenous fluorophores are present in the extracellular structures of tissues. For example, collagen and elastin, present in the extracellular matrix (ECM), fluoresce as a result of cross-linking between amino acids. Endogenous fluorophores absorption fluorescence GFP An important fluorescing protein that has received considerable attention during recent years for fluorescence-tagging of proteins is the green fluorescent protein (GFP) derived from jellyfish. In its native form, it absorbs at 395 nm and 475 nm with emission maximum in green, around 508. Intensive mutagenesis of the primary sequence has produced a wide variety of GFPs with broad spectral and biochemical properties. The GFP and its variants have been utilized as multicolor fluorescent markers to be used as subcellular probes. Photoaddition An important photoaddition process responsible for UVinduced molecular lesions in DNA is the photodimerization of thymine. Another important photoaddition is that of cysteine (in protein) to thymine (in DNA), which can lead to photochemical cross-linking of DNA to protein. Photofragmentation In a photofragmentation reaction the original molecule, when photoexcited, decomposes into smaller chemical fragments by the cleavage of a chemical bond. This type of reaction is very common in biomolecules when exposed to short wavelength UV light. An example is the photofragmentation of riboflavin: Photooxidation Here the molecule, when excited, adds an oxygen molecule from the surroundings (a chemical process called oxidation). An example is the photooxidation of cholesterol: Photohydration This type of reaction is also responsible for creating lesions in DNA. In this reaction, an excited molecule adds a water molecule to produce a new product, as illustrated for uracil. Photoisomerization Photoisomerization here specifically refers to the change in geometry or conformation of stereoisomers. An important photoisomerization process responsible for retinal vision is that of 11cis-retinal which upon excitation rotates by 180° around a double bond to produce a geometric isomer, the all-trans-retinal. isomerization 同質異構化 Photorearrangement In this photoinduced process the chemical formula of the molecule does not change, only a rearrangement of bonds occurs as illustrated for 7-dehydrocholesterol in skin which upon UV exposure produces vitamin D3: UV damage In the case of DNA, UV irradiation can also lead to the breaking of either one or both DNA strand and intra- and intermolecular DNA cross-linking. The photochemistry of RNA is very similar to that of DNA. UV damage The draft ISO standard on determining solar irradiances (ISO-DIS-21348) describes the following ranges: Ultraviolet A, long wave, or black light UVA 400 nm – 320 nm Ultraviolet B or medium wave UVB 320 nm – 280 nm Ultraviolet C, short wave, or germicidal UVC 280 nm – 100 nm UV damage The UV-visible transmission of the Caucasian stratum corneum and epidermis is affected by tryptophan, tyrosine, and other aromatic chrophores that absorb near 280 nm. Nucleic acids and urocanic acid also contribute to the 280 nm absorption band. While the degree of absorbance of the stratum corneum and epidermis below 250 nm is largely due to peptide bonds. UV damage It is thus a critical protection mechanism by melanogenesis and epidermal hyperplasia. Melanin absorption steadily increases from 250 to 1200 nm. Beyond 1100 nm, both transmittance and remittance are unaffected by melanin. UV damage The prevalence of sunburn, abnormal photosensitivity, skin cancer, and cutaneous “aging” decreases with increasing melanin concentration. The transmission of dermis is affected by the existence of collagen, which has the same order of the wavelength of light and causes scattering effect. Longer wavelengths exhibit both greater and more forwarddirected transmission. UV damage Harmful effects An overexposure to UVB radiation can cause sunburn and some forms of skin cancer. In humans, prolonged exposure to solar UV radiation may result in acute and chronic health effects on the skin, eye, and immune system. However the most deadly form - malignant melanoma - is mostly caused by the indirect DNA damage (free radicals and oxidative stress). This can be seen from the absence of a UV-signature mutation in 92% of all melanoma. UV damage UVC rays are the highest energy, most dangerous type of ultraviolet light. Little attention has been given to UVC rays in the past since they are filtered out by the atmosphere. However, their use in equipment such as pond sterilization units may pose an exposure risk, if the lamp is switched on outside of its enclosed pond sterilization unit. Light-tissue interactions A tissue is a self-supporting bulk medium. In other words, unlike cells, which have to be supported in a medium (in an aqueous phase as in vitro specimen or in a tissue either as an ex vivo or an in vivo specimen), tissues do not need a medium. Tissues, therefore, behave like any bulk medium in which light propagation produces absorption, scattering, refraction, and reflection. Modes of light-tissue interaction Light-tissue interactions Absorption Scattering Fluorescence Photothermal interaction Photochemical interaction Photoacoustic effect Two-photon absorption Light-tissue interactions The absorption of light under weak illumination is a linear absorption described by Beer-Lambert’s law. The absorption is due to various intracellular as well as extracellular constituents of the tissue. However, the most pronounced effect in a tissue is scattering. A tissue is a highly scattering turbid medium. The turbidity or apparent nontransparency of a tissue is caused by multiple scattering from a very heterogeneous structure consisting of macromolecules, cell organelles, and a pool of water. Light-absorbing components in tissues Light scattering processes in tissues Light scattering Elastic scattering Rayleigh scattering Mie scattering Inelastic scattering Brillouin scattering Raman scattering Light scattering processes in tissues Elastic scattering (no change in frequency of scattered light) Rayleigh scattering: Particles are much smaller than the wavelength of the radiation. Scattering intensity falls off as 1/λ4, hence significantly more for blue compared to red light. Forward and backward scattering is the same. Mie scattering: Particles are comparable in size to the wavelength of the radiation. Mie scattering shows a weaker dependence on wavelength (~λ-x with 0.5≥x≥0.4) and preferably takes place in the forward direction. Light scattering processes in tissues Light scattering processes in tissues Both the Rayleigh and the Mie theories, which are based on the Maxwell equations, model the scattering of a plane monochromatic optical wave by a single particle. The Rayleigh theory is applicable only to particles that are much smaller than the optical wavelength, whereas the Mie theory is valid for homogeneous isotropic sphere of any size. The Mie Theory reduces to the Rayleigh theory when the particle is much smaller than the wavelength. Light scattering processes in tissues Inelastic scattering (small change in frequency) Raman scattering: Light is frequency-shifted with respect to the excitation frequency, but the magnitude of the shift is independent of the excitation frequency. This "Raman shift" is therefore an intrinsic property of the sample. The difference in energy generates a vibrational excitation in the molecule. Brillouin scattering: It arises from acoustic waves propagating through a medium, thereby inducing inhomogeneities of the refractive index. Brillouin scattering of light to higher (lower) frequencies occurs, because scatters are moving toward (away from) the light source. It can thus be regarded as an optical Doppler effect. The difference in energy generates acoustic phonons Penetration depths for laser wavelengths increasing particle size (g) Scattering-induced polarization effect unscattered light present scattered light depolarized unscattered light present no unscattered light Scattered light scattered light retains depolarized polarization no unscattered light but scattered light retains polarization increasing concentration Jablonski energy diagram Light-induced processes in tissues Radiative Tissue autofluorescence Photochemical Excited state reaction. Occurs even at low optical power density. Tissue-light interaction Photoablation Direct breaking of cellular structure. Performed by highenergy UV radiation. Thermal Light absorption converted to heat. Can produce coagulation, vaporization, carbonization, and melting. Nonradiative Photodisruption Shockwave generation at high pulse intensity. Fragmentation and cutting of the tissue by mechanical force of shockwave. Plasma-Induced Ablation Induced by high-intensity short pulse. Dielectric breakdown creates ionized plasma that interacts with light to produce ablation. Light-induced processes in tissues Photodisruption Power density (w/cm2) 1015 109 103 10-3 Plasmainduced ablation Photoablation 10 00 J/c 1J m2 /cm 2 Thermal interaction Photochemical interaction 10-15 10-9 10-3 Exposure time (s) 103 Thermal effects inside tissue The thermal effects result from the conversion of the energy of light, absorbed in tissues, to heat through a combination of nonradiative processes such as internal conversion (IC), intersystem crossing (ISC), and vibrational relaxations. Thermal effects can be induced both by lamp as well as by CW and pulse laser sources and they are nonspecific; that is, they do not show a wavelength dependence, implying that no specific excited state need to be populated to create these effects. Thermal effects inside tissue The heating of an area in a tissue can produce four effects: coagulation vaporization carbonization melting. Thermal effects inside tissue Laser beam Carbonization Coagulation Tissue Vaporization Hyperthermia Thermal effects inside tissue For coagulation, the local temperature of a tissue has to reach at least 60°C, where the coagulated tissue becomes necrotic. Both CW (e.g., Nd:YAG) and pulse (e.g., Er:YAG) lasers have been used for different tissues. For a vaporization effect to manifest, the local temperature of a tissue has to reach 100°C, where water starts converting into steam, producing photothermal ablation of the tissue. This ablation is a purely thermomechanical effect produced by the pressure buildup due to steam formation and is thus different from photoablation. In this process the tissue is torn open by the expansion of steam, leaving behind an ablation crater with lateral tissue damage. Thermal effects inside tissue In a living tissue, the blood vessels can transport heat away from the ablation site, creating damage at other sites and thus the spread of the lateral damage. If one wishes to reduce the lateral thermal damage from thermal diffusion, one must ablate the tissue with a short pulse laser. Based on the thermal diffusion characteristics of biological tissues, it can be assumed that if the energy is deposited in the tissue in tens of microseconds, the thermal ablation remains primarily localized around the focal spot of the beam and the lateral thermal damage is minimized. However, the use of ultrashort and high-intensity pulses can lead to other complications such as nonlinear optical Thermal effects inside tissue Carbonization occurs when the tissue temperature reaches above 150°C, at which tissue chars, converting its organic constituents into carbon. This process has to be avoided because it is of no benefit and leads to irreparable damage of a tissue. At sufficiently high power density from a pulse laser (generally in microseconds to nanoseconds), the local temperature of a tissue may reach above its melting point. This type of process can be used for tissue welding. Thermal effects inside tissue Laser & optical issue parameters Thermal tissue parameters Type of tissue Heat generation Heat transport Heat effects Tissue damage Photoablation This is a process whereby the various cellular and extracellular components are photochemically decomposed by the action of an intense UV laser pulse. The result is the release of photofragmented species from a tissue, causing etching (or ablation). This ablation is localized within the beam spot and is thus very clean. Typical power densities are 107–1010 W/ cm2. A convenient UV laser source is an excimer laser that provides a number of lasing wavelengths in the range of 193–351 nm. The pulses from these lasers are typically 10–20 nsecs. This method is very useful for tissue contouring, such as in refractive corneal surgery. Photoablation Plasma-Induced Ablation When exposed to a power density of 1011 W/cm2, the tissue experiences an electric field of 107 V/ cm associated with the light. This field is considerably larger than the average coulombic attraction between the electrons and the nuclei and causes a dielectric breakdown of the tissue to create a very large free electron density (plasma) of ~1018 cm3 in the focal volume of the laser beam in an extremely short period (less than hundreds of picoseconds). This high-density plasma strongly absorbs UV, visible, and IR light, which is called optical breakdown and leads to ablation. Plasma generation Photodisruption This effect occurs in soft tissues or fluids under high intensity irradiation that produces plasma formation. At higher plasma energies, shock waves are generated in the tissue which disrupt the tissue structure by a mechanical impact. When a laser beam is focused below the tissue, cavitation occurs in soft tissues and fluids produced by cavitation bubbles that consist of gaseous vapors such as water vapor and CO2. In contrast to a plasma-induced ablation, which remains spatially localized to the breakdown region, photodisruption involving shockwaves and cavitation effects spreads into adjacent tissues. For nanosecond pulses, the shockwave formation and its effects dominate over plasma-induced ablation. However, for shorter pulses both plasmainduced ablation and photodisruption may occur and it is not easy to distinguish between these two processes. Photoacoustic effects The time-dependent heat generated in a tissue via interaction with pulsed or intensity-modulated optical radiation is know as optothermal effect. Such interaction also induces a number of thermoelastic effects; in a tissue in particular, it causes generation of acoustic waves. Detection of acoustic waves is the basis for optoacoustic or photoacoustic methods. Photoacoustic effects Two main modes can be used for excitation of tissue thermal response: A pulse of light excites the sample and the signal is detected in the time domain with a fast detector attached to a wide-band amplifier. An intensity-modulated light source, a lowfrequency transducer, and phase-sensitive detection for noise suppression are provided. Photoacoustic effects Laser beam Photoacoustic effects Two-photon absorption The understanding of the occurrence of two-photon absorption requires quantum mechanics, particularly the uncertainty principle. Because of the energy discrepancy in each step from energy conservation, the two-photon transition must occur in a very short time. For instance, if ΔE=1 eV, according to the uncertainty principle ΔEΔt ≥ ħ/2, the limit of Δt should be in the range of a few fsec. Two-photon absorption excited state virtual state ground state Two-photon absorption Two-photon excitation exhibits localized excitation, the inherent advantage which accounts for the improved resolution available with this method.