“lighting the way to technology through innovation” the institute for lasers, photonics and...
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![Page 1: “Lighting the Way to Technology through Innovation” The Institute for Lasers, Photonics and Biophotonics University at Buffalo Biophotonics P.N.Prasad](https://reader030.vdocuments.site/reader030/viewer/2022032800/56649d4d5503460f94a2ba76/html5/thumbnails/1.jpg)
“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
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PHOTOBIOLOGY
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Various Molecular, Cellular and Tissue Components which Interact with Light
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Various Light-Induced Cellular Processes
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The absorption spectra of some important cellular constituents
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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
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Photoaddition
Photofragmentation
PHOTOCHEMICAL PROCESSES
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Photooxidation
Photoisomerization
(Retinal isomerization in the process of vision)
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Retinal isomerization under light exposure
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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
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Room temperature time-resolved resonance Raman spectra of rhodopsin and its intermediates. The rhodopsin spectrum is obtained using excitation at 458nm
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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
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Photomedicine: Photodynamic Therapy
Photosensitization by Exogenous Molecules
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Photodynamic Therapy
Porphyrin Porphyrin + O2 singlet
h O2
( Localizes and accumulatesat tumor sites )
Destroys Cancerous Cells
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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
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Light - Tissue Interactions
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The four possible modes of interaction between light and tissue
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The Various Light Scattering Processes in a Tissue
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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.
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Light Induced Various Processes in Tissues
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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
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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
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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).
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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
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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
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Schematics of various optical interactions with a tissue used for optical biopsyAlfano et al., 1996
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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
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Raman spectra from normal, benign and malignant breast tumors
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Bioimaging: Principles and Techniques
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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
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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
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Propagation of a laser pulse through a turbid medium
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Confocal and multiphoton imaging. The bottom panel demonstrates the vertical cross-section of the photo-bleached area in a sample.
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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.
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A table top OCT design using a SLD light source.
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A fiber based OCT design
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1 < c
2 = c
3 > c
c : critical angle
1 32
Principle of total internal reflection
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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.
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Different modes of Near field microscopy
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Schematics of experimental arrangement for obtaining fluorescence spectra from a specific biological site (e.g. organelle) using a CCD coupled spectrograph.
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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
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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
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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.
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Bioimaging Applications
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Fluorescence labels:
• Near IR dyes• Two-photon emitters• Green fluorescent proteins• Quantum Dots• Rare-earth up-convertors
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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
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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)
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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.
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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
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Three-Photon Excited Amplified Emission
pump=1300nm emmax=553nm
pump
He et al., Nature 415, 767 (2002)