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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):

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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 long wavelengths:

  • depth of penetration is increased

  • preferential chromophore excitation at focus

  • dose/effect is greatest at the focus

  • At short wavelengths:

  • depth of penetration is limited

  • all chromophores in cone of light excited

  • dose/effect is greatest at the surface


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 Intensityin 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-H Intensity2DCFDA, DCF, and Fluorescein

DCF

absorption

spectrum

CM-H2DCFDA

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 Intensity

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 Intensity

0 min

3 min

6 min

9 min

9 min

9 min

3 min

6 min


Two-Photon Excitation: Intensity

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 Intensity

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 Intensity

  • 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 Intensity

  • 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 Intensity

  • 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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