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

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

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

Photorearrangement (i)S 0 (photosensitizer)  hv  S i (photosensitizer)  T 1 (photosensitizer) (ii)T 1 (photosensitizer) + T 0 (oxygen)  S 0 (photosensitizer) + S 1 (oxygen) (iii)S 1 (oxygen) + A cellular component  Photooxidation of the cellular component Photosensitized Oxidation

Photomedicine: Photodynamic Therapy Photosensitization by Exogenous Molecules

Photodynamic Therapy Porphyrin Porphyrin + O 2 singlet h O2O2 ( Localizes and accumulates at tumor sites ) Destroys Cancerous Cells

Mechanism of Photodynamic Photooxidation PDT Drug (P) Light absorption 1 P* 3 P* PDT drug in singlet state PDT drug in triplet state Type I processType II process 3 P* + H 2 0 HO. 3 P* P + 1 O 2 * Intersystem crossing H2O2H2O2 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

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; I 0 is the intensity when it enters the tissue; α = absorption coefficient and α s = scattering coefficient. Therefore, α + α s is the total optical loss.

Light Induced Various Processes in Tissues

Thermal Laser-Tissue Interaction 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 Laser-Tissue Mechanisms for Ophthalmic Applications

Various Types of Tissue Engineering using Lasers

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

Laser tissue ablation using lasers of two different pulse widths. Top: pulse width of 200ps; bottom: pulse width of 80fs (Source: ml). FemtoLaser Surgery

Schematics of various optical interactions with a tissue used for optical biopsy Alfano 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:

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 Imaging Tasks : Molecular level to Whole body imaging

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1 33 22 Principle of total internal reflection

Evanescent wave extending beyond the guiding region and decaying exponentially. For waveguiding n 1 > n 2, n 2 = refractive index of surrounding medium. n 1 = 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 2 p – 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

Some new Near-IR and IR dyes Commercially available Indocyanine Green, Absorption λ max : 780nm (water), Fluorescence λ max : 805 nm (water) New IR dye *, absorption λ max : 1127 nm (dichloroethane), Emission λ max : 1195nm (dichloroethane) New IR dye *, absorption λ max 1056 nm (dichloroethane), Emission λ max : 1140nm (dichloroethane) *Developed at ILBP

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)

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 em max =553nm pump He et al., Nature 415, 767 (2002)