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Spatially Selective Two-Photon
Induction of Oxidative Damage
in Fibroblasts
Brett A. King and Dennis H. Oh
Department of D...
Reactive Oxygen Species (ROS):
Roles in Disease and Therapy
•Generated by endogenous processes and exogenous insults
•Dama...
Why Use Two-Photon Excitation?
• Permits generation of ROS with spatial selectivity
• Uses longer wavelengths to excite ul...
One- vs. Two-Photon Excitation
At short wavelengths:
•depth of penetration is limited
•all chromophores in cone of light e...
1-photon
absorption
ground state
excited state
2-photon
absorption
energy
Nabs ∝ σI Nabs ∝ δI2
(linear) (quadratic)
Nabs =...
Assay for ROS in vivo using CM-H2DCFDA
Chloromethyl-dihydro-dichlorofluorescein diacetate (CM-H2DCFDA)
• Rapidly loaded in...
Xu et al., PNAS 1996
CM-H2DCFDA
absorption
spectrum
DCF
absorption
spectrum
DCF
fluorescence
spectrum
Fluorescence
Excitat...
CM-H2DCFDA
(non-fluorescent)
DCF
(excited state)
ROS
800 nm
2-photon abs
O
Cl
OCH3C
O
O C
O
CH3
COOH
Cl
ClH2C
O
Cl
-
O O-
...
0 min 3 min 6 min
9 min 9 min
3 min 6 min
9 min
Two-Photon Induction of ROS in Fibroblasts
7.5 mW/cm2
15 mW/cm2
Two-Photon Excitation:
Quadratic Dependence on Light Intensity
Average of 3 paired cellsRepresentativ...
2-photon
target
1-photon
target
2-photon
target
1-photon
target
Two-Photon Excitation is Required to
Generate ROS
• Circle...
coverslip
Experiment Schematic
Manipulating ROS Generation in Monolayers and 3-Dimensional Tissue
• A cell monolayer or de...
0
0.05
0.1
0.15
0 10 20 30 40 50 60 70 80
ROSsignal(increaseovert=0)
level (microns)
Generation of ROS in Fibroblasts
Embe...
Conclusions
• The commonly used reporter of ROS, DCF (dichlorofluorescein), is an efficient
photosensitizer of ROS formati...
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  1. 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. 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. 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. 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 near-infrared laser beam maximum intensity at target DEJ diminished intensity at target DEJ ultraviolet radiation O ne -photon activation Tw o-photon activation
  5. 5. 1-photon absorption ground state excited state 2-photon absorption energy Nabs ∝ σI Nabs ∝ δI2 (linear) (quadratic) Nabs = # of photons absorbed I = light intensity σ = 1-photon constant δ = 2-photon constant One- and Two-Photon Excitation Differ in Dependence on Light Intensity For two-photon excitation: • A focused laser will produce maximal effect at the focal point • Effect diminishes exponentially above and below focal plane
  6. 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. 7. Xu et al., PNAS 1996 CM-H2DCFDA absorption spectrum DCF absorption spectrum DCF fluorescence spectrum Fluorescence Excitation Spectra of Fluorescein One-Photon (dashed line) Two-Photon (solid line) ROS Spectra of CM-H2DCFDA, DCF, and Fluorescein
  8. 8. CM-H2DCFDA (non-fluorescent) DCF (excited state) ROS 800 nm 2-photon abs O Cl OCH3C O O C O CH3 COOH Cl ClH2C O Cl - O O- COO- Cl RSCH2 O Cl - O O COO- Cl RSCH2 intracellular esterases and thiols O Cl - O O COO- Cl RSCH2 DCF Simultaneous ROS Generation and Detection 525 nm fluorescence photochemistry H2DCF (non- fluorescent) DCF both reflects and initiates ROS generation
  9. 9. 0 min 3 min 6 min 9 min 9 min 3 min 6 min 9 min Two-Photon Induction of ROS in Fibroblasts
  10. 10. 7.5 mW/cm2 15 mW/cm2 Two-Photon Excitation: Quadratic Dependence on Light Intensity Average of 3 paired cellsRepresentative Contrast in Intensity 0 1 2 3 4 5 6 7 7.5 15 relativemeanfluorescentintensity incident laser intensity (mW/cm2 )
  11. 11. 2-photon target 1-photon target 2-photon target 1-photon target Two-Photon Excitation is Required to Generate ROS • Circles represent irradiated areas • Two-photon excitation targeted to one subcellular area generates ROS throughout cell
  12. 12. coverslip 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) stage microscope objective monolayer or dermal equivalent
  13. 13. 0 0.05 0.1 0.15 0 10 20 30 40 50 60 70 80 ROSsignal(increaseovert=0) level (microns) 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 µm deep in the sample • Fluorescence intensity (ROS) increases with increasing focus of the laser beam DCFFluorescenceIntensity Plane of Section of Dermal Equivalent (µm)
  14. 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)

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