1. 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

2. 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

3. 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

4. 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

5. 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

6. 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)

7. 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

8. 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)

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

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

11. 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

12. 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

13. 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)

14. 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)