“Lighting the Way to Technology through Innovation” The Institute for Lasers, Photonics and Biophotonics University at Buffalo Biophotonics P.N.Prasad.

“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)