Transcript No Slide Title

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
One-photon activation
Two-photon activation
ultraviolet radiation
diminished intensity
at t arget
near-infrared laser beam
DEJ
At short wavelengths:
• depth of penetration is limited
• all chromophores in cone of light excited
• dose/effect is greatest at the surface
maximum intensity
at target
DEJ
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
energy
excited state
ground state
1-photon
absorption
Nabs I
(linear)
2-photon
absorption
Nabs I2
(quadratic)
Nabs = # of photons absorbed
I = light intensity
= 1-photon constant
= 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
CM-H2DCFDA
absorption
spectrum
ROS
Fluorescence
Excitation Spectra
of Fluorescein
One-Photon (dashed line)
Two-Photon (solid line)
DCF
absorption
spectrum
DCF
fluorescence
spectrum
Xu et al., PNAS 1996
Simultaneous ROS Generation and Detection
DCF both reflects and initiates ROS generation
O
O
-
H3 C
C
O
O
O
Cl
C
O
CH3
O
O
Cl
Cl
Cl
COO -
COOH
RSCH 2
ClH2C
CM-H2DCFDA
(non-fluorescent)
DCF
(excited state)
photochemistry
intracellular
esterases and thiols
-
O
800 nm
2-photon abs
-
O
O-
O
Cl
Cl
525 nm
fluorescence
ROS
O
Cl
Cl
COO -
COO -
RSCH 2
H2DCF
(non-fluorescent)
O
RSCH 2
DCF
Two-Photon Induction of ROS in Fibroblasts
0 min
9 min
3 min
6 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
relative mean fluorescent intensity
7
6
5
4
3
2
1
0
7.5
15
incident laser intensity (mW/cm2 )
Two-Photon Excitation is Required to
Generate ROS
1-photon
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
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
ROS
signal (increase Intensity
over t=0)
Fluorescence
DCF
0.15
0.1
0.05
0
0
10
20
30
40
50
60
70
80
Plane of Section level
of Dermal
(microns) 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)