Spatially Selective Two-Photon Induction of Oxidative Damage in Fibroblasts Brett A. King and Dennis H. Oh Department of Dermatology University of California, San Francisco Dermatology Research Unit San Francisco VA Medical Center

Reactive Oxygen Species (ROS): Roles in Disease and Therapy Generated by endogenous processes and exogenous insults Damage nucleic acid, protein, and lipid Contribute to toxicity in skin from radiation and exogenous chemicals Factors in cellular senescence and death Mediators of photodynamic damage and therapy

Why Use Two-Photon Excitation? Permits generation of ROS with spatial selectivity Uses longer wavelengths to excite ultraviolet-absorbing chromophores Minimizes scatter to permit deeper tissue penetration Potentially permits greater chromophore specificity Allows for the assessment of the whole tissue response to damage targeted to specific cells Potential for applications in diagnostic imaging and photodynamic therapy

One- vs. Two-Photon Excitation At short wavelengths: depth of penetration is limited all chromophores in cone of light excited dose/effect is greatest at the surface At long wavelengths: depth of penetration is increased preferential chromophore excitation at focus dose/effect is greatest at the focus

One- and Two-Photon Excitation Differ in Dependence on Light Intensity Nabs µ sI Nabs µ dI2 (linear) (quadratic) Nabs = # of photons absorbed I = light intensity s = 1-photon constant d = 2-photon constant For two-photon excitation: A focused laser will produce maximal effect at the focal point Effect diminishes exponentially above and below focal plane

Assay for ROS in vivo using CM-H2DCFDA Chloromethyl-dihydro-dichlorofluorescein diacetate (CM-H2DCFDA) Rapidly loaded into and retained by intact cells Colorless prior to oxidation Oxidized by ROS to produce a derivative of DCF, a green fluorescent chromophore (see Spectra and Model below) Dichlorofluorescein (DCF) Reporter of ROS in cell A photosensitizer of H2DCF oxidation (Belanger et al., Free Radical Biology and Medicine, 2001) May be simultaneously exploited to generate and detect ROS (see Model below)

Spectra of CM-H2DCFDA, DCF, and Fluorescein absorption spectrum DCF absorption spectrum DCF fluorescence spectrum ROS Fluorescence Excitation Spectra of Fluorescein One-Photon (dashed line) Two-Photon (solid line) Xu et al., PNAS 1996

Simultaneous ROS Generation and Detection DCF both reflects and initiates ROS generation CM-H2DCFDA (non-fluorescent) DCF (excited state) photochemistry intracellular esterases and thiols 800 nm 2-photon abs 525 nm fluorescence ROS DCF H2DCF (non-fluorescent)

Two-Photon Induction of ROS in Fibroblasts 0 min 3 min 6 min 9 min 9 min 9 min 3 min 6 min

Two-Photon Excitation: Quadratic Dependence on Light Intensity Representative Contrast in Intensity Average of 3 paired cells 7.5 mW/cm2 15 mW/cm2

Two-Photon Excitation is Required to Generate ROS target 1-photon target 2-photon target 2-photon target Circles represent irradiated areas Two-photon excitation targeted to one subcellular area generates ROS throughout cell

Manipulating ROS Generation in Monolayers and 3-Dimensional Tissue Experiment Schematic Manipulating ROS Generation in Monolayers and 3-Dimensional Tissue A cell monolayer or dermal equivalent was incubated with CM-H2DCFDA Pulsed 800 nm radiation was scanned over a selected region of interest in the sample The visual field(s) was then imaged, detecting DCF fluorescence (ROS) monolayer or dermal equivalent coverslip stage microscope objective

Generation of ROS in Fibroblasts Embedded in a Collagen Matrix A dermal equivalent was incubated with CM-H2DCFDA Pulsed 800 nm radiation was scanned over the plane 100 m deep in the sample Fluorescence intensity (ROS) increases with increasing focus of the laser beam DCF Fluorescence Intensity Plane of Section of Dermal Equivalent (m)

Conclusions The commonly used reporter of ROS, DCF (dichlorofluorescein), is an efficient photosensitizer of ROS formation when excited by two-photon absorption. ROS generated focally within a cell rapidly diffuse throughout the whole cell. Two-photon excitation can be employed to generate ROS within both cellular monolayers and 3-dimensional tissues. In monolayers, ROS can be generated with 2-dimensional specificity in single cells. Within 3-dimensional dermal equivalents, ROS can be generated preferentially in a particular region. Supported by grants from the UCSF Academic Senate, NIAMS, and the Yale School of Medicine Office of Student Research (for partial support of Brett King)