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“Lighting the Way to Technology through Innovation”
The Institute for Lasers, Photonics and Biophotonics
University at Buffalo
Biophotonics
P.N.Prasad
www.biophotonics.buffalo.eduwww.biophotonics.buffalo.edu
PHOTOBIOLOGY
Various Molecular, Cellular and Tissue Components which Interact with Light
Various Light-Induced Cellular Processes
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
Photoaddition
Photofragmentation
PHOTOCHEMICAL PROCESSES
Photooxidation
Photoisomerization
(Retinal isomerization in the process of vision)
Retinal isomerization under light exposure
Rhodopsin (498 nm)
Lum irhodopsin (497 nm)
Bathorhodopsin (543 nm)
Photorhodopsin (570 nm)
M etarhodopsin I (478 nm )
M etarhodopsin II (380 nm) R*
M etarhodopsin III (465 nm)
All-trans-Retinal+opsin (387 nm)
Various intermediates formed after light absorption by Rhodopsin
Room temperature time-resolved resonance Raman spectra of rhodopsin and its intermediates. The rhodopsin spectrum is obtained using excitation at 458nm
HO
CH3
C CH2CH2CH2
CH3
H
C HH3C
CH3
HO
CH3
C CH2CH2CH2
CH3
H
C HH3C
CH3CH3 CH2h
Skin
7-Dehydrocholesterol Vitamine D3
(7)
Photorearrangement
(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
Photosensitized Oxidation
Photomedicine: Photodynamic Therapy
Photosensitization by Exogenous Molecules
Photodynamic Therapy
Porphyrin Porphyrin + O2 singlet
h O2
( Localizes and accumulatesat tumor sites )
Destroys Cancerous Cells
Mechanism of Photodynamic Photooxidation
PDT Drug (P)Light absorption
1P*
3P*
PDT drug in singlet state
PDT drug in triplet state
Type I process Type II process3P* + H20 HO. 3P* + 302 1P + 1O2*
Intersystem crossing
H2O2
Oxidation of cellular components
cytotoxicity
Light - Tissue Interactions
The four possible modes of interaction between light and tissue
The Various Light Scattering Processes in a Tissue
eI = I(z) z) + -(0
s
I
Penetration depths for commonly used laser wavelengths
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.
Light Induced Various Processes in Tissues
Thermal
Laser-Tissue Interaction
Photocoagulation:Absorption of visiblelight generating heat toproduce coagulation to seal leaky blood vessels or to repair a tear
Thermal keratoplasty:Absorption of IR beamproducing heat resultingin shrinkage
Photoablation:Photochemical ablation of tissues
Photodisruption:Mechanical disruptionby creation of plasma
PRK, LASIK Posteriorcapsulotomy
Various Laser-Tissue Mechanisms for Ophthalmic Applications
Various Types of Tissue Engineering using Lasers
L a se r B a s e d T is s u e E n g in e e rin g
T is su e c o n to u rin g a n dre s tru c tu r in g : U s e o f la se r s to a b la te , s h a p e o r c h a n g e p ig m e n ta tio n o f a ti s s u e
T is su e g e n e ra t io n :L a se r a c tiv a tio n o r in c is io n to s tim u la te n e w t is su e g e n e ra t io n
T is su e w e ld in g : L a se r in d u c e d w e ld in ga n d s o ld e rin g to fu s e tis s u e s , re p a ir a te a r, o rin h ib it v a s c u la r g ro w th
Tattoo removal using laser technology. Four treatments with Q-switched frequency doubled Nd:YAG laser (532nm green) removed the tattoo (Hogan, 2000).
T iss u e B o n d in g
D ire c t W e ld in g o f T is su e s :
L o c a l h e a tin g to ~ 6 0 ºC - 8 0 ºCB y la se r e n e rg y a b so rp tio n(p h o to th e rm o ly s is ) to d e n a tu rec o lla g e n , u n c o ilin g th e ir n a tiv etr ip le h e lic a l s tru c tu re a n d p ro d u c in g c o lla g e n b o n d in g
L a se r S o ld e rin g :
U s e o f p ro te in e o u sS o ld e r a t th e s u rfa c e s to b ejo in e d fo llo w e d b y a p p lic a tio no f la s e r l ig h t to se le c tiv e lyh e a t th e s o ld e r a n d se a l i t toth e su r ro u n d in g t is s u e
D y e -e n h a n c e d L a s e r S o ld e r in g :
A d y e a b so rb in g a t th e la s e rw a v e le n g th o f s o ld e r in g a d d e dto th e s o ld e r to e n h a n c e se le c tiv ea b s o rp tio n a n d s u b se q u e n t h e a tin go f th e so ld e r a n d n o t o f th en o n ta rg e t tis su e
The Approaches for Tissue Bonding
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.html).
FemtoLaser Surgery
Schematics of various optical interactions with a tissue used for optical biopsyAlfano et al., 1996
Fluorescence spectra of the normal breast tissue (BN) and the tumor breast tissue (BT) excited at 488 nm
In vivo spectroscopy
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
Electron Microscopy
Nearfield Microscopy,FRET technique
Confocal Microscopy, Multiphoton Microscopy,
Coherence Tomography etc.
Simple microscope,Whole body imaging tools
Bio
Imag
ing
Tas
ks :
M
ole
cula
r le
vel t
o W
ho
le b
od
y im
agin
g
Optical Imaging
Confocal Microscopy (CSLM)
Multi-photon Microscopy
Nearfield Microscopy
Optical Coherence Tomography
Total Internal Reflection Imaging (TIR)
TOOLS
Fluorescence Microscopy
Raman Imaging ( e.g. CARS)
Interference Imaging (e.g. OCT)
Techniques
Whole body imaging
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.
Applications
Propagation of a laser pulse through a turbid medium
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
3 > c
c : critical angle
1 32
Principle of total internal reflection
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
Polarized Fluorescence Imaging :
Fluorescence Resonance Energy Transfer ( FRET )
Fluorescence Recovery After Photobleaching (FRAP)
Fluorescence Life time imaging ( FLIM)
Molecular diffusion and Mobility measurements in living cells
( e.g. Protein mobility and interactions )
Molecular diffusion and Mobility measurements in living cells
( e.g. Protein mobility and interactions )
Molecular interactions and conformational changes in living cells
( e.g. Protein interactions and conformational changes )
•Environmental changes inside cells
•Complements FRET technique
Fluorescence Imaging Techniques
Nonlinear Optical Techniques
• Second harmonics Imaging - membrane dynamics - excitation at , signal at 2
• CARS Imaging - vibrational imaging - excitation at p and s, signal at 2p –s with Raman resonance at p –s
Schematics of a synchronized mode-locked picosecond Ti-Sapphire laser system for backward detection CARS microscopy. Millenia is the diode pumped Nd Laser. Tsunami is the Ti-Sapphire Laser.
Bioimaging Applications
Fluorescence labels:
• Near IR dyes• Two-photon emitters• Green fluorescent proteins• Quantum Dots• Rare-earth up-convertors
NCH CH CH CH CH
(CH2)4SO3
CH CH
NNaO3S(CH2)4
H3C CH3 H3CCH3
OCl
O
CH3 CH3
ClO4
O
CH CH CH CH CH
O
CH3 CH3
ClO4
Some new Near-IR and IR dyes
Commercially available Indocyanine Green, Absorption λmax: 780nm (water), Fluorescence λmax: 805
nm (water)
New IR dye*, absorption λmax: 1127 nm
(dichloroethane), Emission λmax: 1195nm
(dichloroethane)
New IR dye*, absorption λmax 1056 nm (dichloroethane), Emission λmax: 1140nm (dichloroethane)
*Developed at ILBP
N
S
H 3 C O H
( C H 2 ) 6 O H O O
N
S
H 3 C O H
( C H 2 ) 6 O N a O O
N
S
H 3 C S H
( C H 2 ) 6 O H O O
A P S S W a t e r - s o l u b l e A P S S
A P S S - S H C 6 2 5
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
O
NCH3
X N
D
R'
X
1 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=1300nm emmax=553nm
pump
He et al., Nature 415, 767 (2002)