slide1
Download
Skip this Video
Download Presentation
Spatially Selective Two-Photon Induction of Oxidative Damage in Fibroblasts

Loading in 2 Seconds...

play fullscreen
1 / 14

Spatially Selective Two-Photon Induction of Oxidative Damage in Fibroblasts - PowerPoint PPT Presentation


  • 60 Views
  • Uploaded on

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):

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Spatially Selective Two-Photon Induction of Oxidative Damage in Fibroblasts' - russell-odom


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

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

slide2

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
slide3

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
slide4

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
slide5

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
slide6

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)
slide7

Spectra of CM-H2DCFDA, 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

slide8

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)

slide9

Two-Photon Induction of ROS in Fibroblasts

0 min

3 min

6 min

9 min

9 min

9 min

3 min

6 min

slide10

Two-Photon Excitation:

Quadratic Dependence on Light Intensity

Representative Contrast

in Intensity

Average of 3 paired cells

7.5 mW/cm2

15 mW/cm2

slide11

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
slide12

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

slide13

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)

slide14
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)

ad