Transcript Lighting the Way to Technology through Innovation”

“Lighting the Way to
Technology through Innovation”
The Institute for Lasers, Photonics and
Biophotonics
University at Buffalo
Biophotonics
P.N.Prasad
www.biophotonics.buffalo.edu
PHOTOBIOLOGY
Various Molecular, Cellular and Tissue Components which
Interact with Light
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
Proteins
DNA
Extracellular components
present in tissues
Various Light-Induced Cellular Processes
Light Absorption by Cellular Components
Radiative Process
Non-radiative Process
Fluorescence
(Also called autofluorescence or
Endogeneous fluorescence)
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
Photochemical
Excited State Chemistry
Water vaporization at stronger
Heating, leading to disruption
of cellular structure
Photoaddition Photofragmentation Photo-oxidation Photohydration Cis-trans Isomerization photorearrangement
The absorption spectra of some important cellular constituents
The absorption spectra of
important cellular constituents
The absorption (left) and the fluorescence (right) spectra of
important tissue flourophores. The Y-axes represent the
absorbance (left) and florescence intensity (right) on a
relative scale
PHOTOCHEMICAL PROCESSES
Photoaddition
O
O
H
2
CH 3
N
O
H
h
N
H
N
H
O
O
NH2
CH3
N
HS CH2 C CH2COOH
+
H
h
H
N
H
N
O
H
O
N
O
N
H
CH3 H3C
O
H
N
(1)
O
NH2
S CH2 C CH2COOH
H
CH3
(2)
N
H
Photofragmentation
O
H3C
H3C
N
N
O
N
N
H
h
O
CH2CHOHCHOHCHOHCH
H3C
H3C
2OH
+
N
N
H
N
N
H
(3)
O
CH3CCHOHCHOHCH 2OH
O
Photooxidation
O
O
h
HN
O
N
H
Uracil
H2O
H
HN
O
H
N
H
OH
H
6-Hydroxy-5-Hydrouracil
Photoisomerization
(Retinal isomerization in the process of vision)
(5)
Retinal isomerization under light exposure
Various intermediates formed after light absorption by Rhodopsin
R h o d o p s in ( 4 9 8 n m )
P h o to r h o d o p s in ( 5 7 0 n m )
B a th o r h o d o p s in ( 5 4 3 n m )
L u m ir h o d o p s in ( 4 9 7 n m )
M e ta r h o d o p s in I ( 4 7 8 n m )
Room temperature time-resolved resonance Raman spectra
of rhodopsin and its intermediates. The rhodopsin spectrum
is obtained using excitation at 458nm
Photorearrangement
CH3
CH3
H C
CH3
H C
CH3
CH2CH2CH2
H3C
CH3
C
H
CH3
h
CH2
CH2CH2CH2
H3C
C
H
CH3
(7)
Skin
HO
HO
7-Dehydrocholesterol
Vitamine D3
Photosensitized Oxidation
(i)
S0 (photosensitizer)  hv Si (photosensitizer)  T1 (photosensitizer)
(ii)
T1 (photosensitizer) + T0 (oxygen)  S0 (photosensitizer) + S1 (oxygen)
(iii)
S1 (oxygen) + A cellular component  Photooxidation of the cellular
component
Photomedicine: Photodynamic Therapy
Photosensitization by Exogenous Molecules
Photodynamic Therapy
Porphyrin
h
Porphyrin
+
O2
O2 singlet
( Localizes and
accumulates
at tumor sites )
Destroys
Cancerous Cells
Mechanism of Photodynamic Photooxidation
PDT Drug (P)
Light absorption
1P*
PDT drug in singlet state
Intersystem crossing
3P*
Type I process
3P*
+ H 20
PDT drug in triplet state
Type II process
3P* + 30
1P + 1O *
2
2
HO.
H 2O2
Oxidation of cellular
components
cytotoxicity
Light - Tissue Interactions
The four possible modes of interaction between light and tissue
Refraction
Reflection
Absorption
Scattering
The Various Light Scattering Processes in a Tissue
Light Scattering
Elastic Scattering
Inelastic Scattering
Incident and Scattered Photons
are of the Same Frequency
Incident and Scattered Photons
are of Different Frequencies
Rayleigh Scattering
•Scattering by particles of size
smaller than the wavelength
of light.
•Scattering depends on λ-4
hence significantly more for
blue compared to red light.
•Forward and backward
scattering is the same.
Mie Scattering
•Scattering of particles of
size comparable to λ
•Weaker wavelength
dependence
-X
λ with 0.4 < X < 0.5
Preferably forward
scattering.
Brillouin Scattering
The difference in energy
generates acoustic
phonons.
Raman Scattering
The difference in energy
generates a vibrational
excitation in the molecule.
-( +  s ) z
I(z) = I0 e
The total intensity attenuation in a tissue can be described as In this equation I(z) is the
intensity at a depth z in the tissue; I0 is the intensity when it enters the tissue; α =
absorption coefficient and αs = scattering coefficient. Therefore, α + αs is the total
optical loss.
Penetration depths for commonly used laser wavelengths
Laser:
CO2
WaveLength: 10.6 m
Holmium
2.9 m
Argon
488, 514 nm
Nd:YAG
1064 nm
I
Tissue Surface
0.1-0.2 mm
0.4-0.6 mm
0.5-2 mm
2-6 mm
Light Induced Various Processes in Tissues
Tissue-Light Interaction
Radiative
Non-radiative
Tissue Autofluorescence
Fluorescence from
various constituents
of the tissue
Photochemical
•Excited State Reaction
•Occurs even at low
optical power density
Photoablation
•Direct breaking of
cellular structure
•Performed by highenergy uv radiation
Thermal
•Light absorption
converted to heat
•Can produce coagulation,
vaporization, carbonization
and melting
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
create ionized plasma which
interact with light to produce ablation
Various Laser-Tissue Mechanisms for Ophthalmic Applications
Laser-Tissue Interaction
Thermal
Photocoagulation:
Absorption of visible
light generating heat to
produce coagulation to
seal leaky blood vessels
or to repair a tear
Thermal keratoplasty:
Absorption of IR beam
producing heat resulting
in shrinkage
Photoablation:
Photochemical
ablation of tissues
Photodisruption:
Mechanical disruption
by creation of plasma
PRK, LASIK
Posterior
capsulotomy
Various Types of Tissue Engineering using Lasers
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Tattoo removal using laser technology. Four treatments
with Q-switched frequency doubled Nd:YAG laser
(532nm green) removed the tattoo (Hogan, 2000).
The Approaches for Tissue Bonding
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FemtoLaser Surgery
Laser tissue ablation using lasers of two different pulse widths.
Top: pulse width of 200ps; bottom: pulse width of 80fs (Source:
http://www.eecs.umich.edu/CUOS/Medical/Photodisruption.ht
ml).
Schematics of various optical interactions with a tissue used for optical biopsy
Alfano et al., 1996
In vivo spectroscopy
Fluorescence spectra of the normal breast tissue (BN) and the tumor breast tissue (BT)
excited at 488 nm
Alfano, R.R. et al., J. Opt. Soc. Am. B. 6:1015-1023 1989
Raman spectra from normal,
benign and malignant breast
tumors
Bioimaging: Principles and Techniques
Bio Imaging Tasks :
Molecular level to Whole body imaging
Simple microscope,
Whole body imaging tools
Confocal Microscopy,
Multiphoton Microscopy,
Coherence Tomography etc.
Nearfield Microscopy,
FRET technique
Electron Microscopy
Techniques
Fluorescence
Microscopy
Optical Imaging
Raman Imaging
( e.g. CARS)
TOOLS
Interference Imaging
(e.g. OCT)
Confocal Microscopy (CSLM)
Multi-photon Microscopy
Applications
Nearfield Microscopy
Whole body imaging
Optical Coherence Tomography
Total Internal
Reflection Imaging
(TIR)
Drug distribution/ Interaction in cells,
Organelles or tissue
Bio-molecular (e.g. Proteins) activity and
organization in cells
Identification of Structural changes in cells,
organelles, tissues etc.
Propagation of a laser pulse through a turbid medium
Optical Imaging
Transmission
(Transillumination)
Spatial Filtering
Confocal Microscopy
Polarization Gating
Time Gating
Frequency
Domain
Methods
Reflection
(Back Scattering)
Spatial Filtering
Confocal Microscopy
Interferometric
Optical Coherence Tomography
Fluorescence
Spatial Filtering
Confocal
Microscopy
Spatially
resolved
Localized
spectroscopy
Polarization
Resolved
Time resolved
Fluorescence
Lifetime Imaging
(FLIM)
Fluorescence Resonance
Energy Transfer
(FRET)
Confocal and multiphoton imaging. The bottom panel demonstrates the vertical
cross-section of the photo-bleached area in a sample.
Low coherence interferometer. The interference signal as a function of the
reference mirror displacement in case of a coherent source (e.g. laser) and a
low-coherence source (e.g., SLD) are shown here.
A table top OCT design using a SLD light source.
A fiber based OCT design
1 < c
2 = c
1
3 > c
2
c : critical angle
Principle of total internal reflection
3
Evanescent wave extending beyond the guiding region and decaying
exponentially. For waveguiding n1 > n2 , n2 = refractive index of
surrounding medium. n1 = refractive index of guiding region.
Different modes of Near field microscopy
Schematics of experimental arrangement for obtaining fluorescence spectra from a
specific biological site (e.g. organelle) using a CCD coupled spectrograph.
Fluorescence Imaging Techniques
•Environmental changes inside cells
Molecular diffusion and Mobility
measurements in living cells
( e.g. Protein mobility and interactions )
•Complements FRET technique
Fluorescence
Life time imaging
( FLIM)
Polarized
Fluorescence
Imaging :
Fluorescence
Fluorescence Recovery
After Photobleaching (FRAP)
Fluorescence Resonance
Energy Transfer
( FRET )
Molecular interactions and
conformational changes in living cells
( e.g. Protein interactions and
conformational changes )
Molecular diffusion and Mobility
measurements in living cells
( e.g. Protein mobility and interactions )
Nonlinear Optical Techniques
• Second harmonics Imaging
- membrane dynamics
- excitation at , signal at 2
• CARS Imaging
- vibrational imaging
- excitation at p and s, signal at 2p –s with
Raman resonance at p –s
Schematics of a synchronized mode-locked picosecond TiSapphire laser system for backward detection CARS microscopy.
Millenia is the diode pumped Nd Laser. Tsunami is the TiSapphire Laser.
Bioimaging Applications
Fluorescence labels:
• Near IR dyes
• Two-photon emitters
• Green fluorescent proteins
• Quantum Dots
• Rare-earth up-convertors
Some new Near-IR and IR dyes
H3C
CH3
CH3
H3C
CH CH CH CH CH CH CH
N
Commercially available Indocyanine Green, Absorption
λmax: 780nm (water), Fluorescence λmax: 805 nm (water)
N
NaO3S(CH2)4
(CH2)4SO3
CH3
O
CH3
Cl
New IR dye*, absorption λmax: 1127 nm
(dichloroethane), Emission λmax: 1195nm
(dichloroethane)
O
ClO 4
CH3
CH3
New IR dye*, absorption λmax 1056 nm
(dichloroethane), Emission λmax: 1140nm
(dichloroethane)
*Developed at ILBP
O
ClO 4
O
CH CH CH CH CH
H3C
OH
N
O
S
O
( C 2H)6O H
APSS
H3 C
OH
N
O
S
O
(CH
2)6 O N a
Water-soluble
APSS
H3 C
SH
N
O
S
O
(CH
2)6 O H
APSS- SH
C625
Lists a chromophore, APSS, and its various derivatives
developed at our Institute which can very efficiently be
excited at 800 nm and emit in the green ( 520 nm peak)
O
O
O
O
O
X
D
O
X
N
1
N R'
CH 3
2
Examples of highly efficient two-photon
active ionic dyes developed at the Institute
for Lasers, Photonics and Biophotonics.
Excitation and emission spectra of wild type
fluorescent protein (FP) as well as the enhanced
variants of GFP (eCFP, eGFP, eYFP and eRFP)
C = cyan, G = green, y = yellow, R = red
Three-Photon Excited Amplified Emission
pump
lpump=1300nm
lemmax=553nm
He et al., Nature 415, 767 (2002)