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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.