Spatially Selective Two-Photon Induction of Oxidative Damage

in Fibroblasts

Brett A. King and Dennis H. OhDepartment of Dermatology

University of California, San Francisco

Dermatology Research UnitSan 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

near-infrared laser beam

maximum intensity at target

DEJdiminished intensity

at target

DEJ

ultraviolet radiation

One-photon activation Two-photon activation

1-photon absorption

ground state

excited state

2-photon absorption

en

erg

y

Nabs I Nabs I2

(linear) (quadratic)Nabs = # of photons absorbedI = light intensity

= 1-photon constant= 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

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)

Xu et al., PNAS 1996

CM-H2DCFDA absorptionspectrum

DCFabsorptionspectrum

DCFfluorescence

spectrum

FluorescenceExcitation Spectra

of FluoresceinOne-Photon (dashed line)

Two-Photon (solid line)

ROS

Spectra of CM-H2DCFDA, DCF, and Fluorescein

CM-H2DCFDA(non-fluorescent)

DCF(excited state)

ROS

800 nm2-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 nmfluorescence

photochemistry

H2DCF(non-fluorescent)

DCF both reflects and initiates ROS generation

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 cellsRepresentative Contrast in Intensity

0

1

2

3

4

5

6

7

7.5 15

rela

tive

mea

n flu

ores

cent

inte

nsity

incident laser intensity (mW/cm 2)

2-photontarget

1-photontarget

2-photontarget

1-photontarget

Two-Photon Excitation is Required to Generate ROS

• Circles represent irradiated areas• Two-photon excitation targeted to one subcellular area generates ROS throughout cell

coverslip

Experiment SchematicManipulating 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

microscopeobjective

monolayer ordermal equivalent

0

0.05

0.1

0.15

0 10 20 30 40 50 60 70 80

RO

S s

igna

l (in

crea

se o

ver

t=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 m deep in the sample • Fluorescence intensity (ROS) increases with increasing focus of the laser beam

DC

F F

luor

esce

nce

Inte

nsity

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 YaleSchool of Medicine Office of Student Research (for partial support of Brett King)