Live tissue imaging (cornea)

Optical imaging of tissue cells

Histology and Electron Microscopy can image ECM.•Requires sectioning and staining.

Stroma Extracellular Matrixcollagen is ubiquitous

Light scattering Cellular fluorescence

nonlinear microscopy endogenous nonlinear optical signals

High Resolution Imaging

Cornea (Rabbit)

Epithelium

Stroma

Endothelium

100 m

14 m

Corneal Stroma

30 m

xy z

y

x

z

xyz

28 x 32 x 33 m

Inte

nsit

y (a

.u.)

Wavelength (nm)

750 nm

Inte

nsit

y (a

.u.) 800 nm

8 m

0

350 400 450 500 550 600

0

Corneal imaging signals

Spectral filtering of imaging signals

520 nm 400 nm

8 m

keratocytes 30 m

TPEF + SHG TPEF SHG

SHG in Collagen

Collagen fiberquaternarystructure

tertiarystructure

~

Polarization dependence of SHG

Ex(t)

Ey(t)

P(t)

xy

z Collagen fiber

Fundamental SHG

SHG Polarization Dependence

Incident laserpolarization

8 m

Optical characterization of tissue

Polarization dependence of SHG in collagen

Endogenous nonlinear optical signals to characterize tissue

Linear (circular) polarization

Spectroscopy of nonlinear optical signals to enhance contrast

constituent specific imaging

sensitive to long range order of collagen molecules

Cornea(diagnosis window to the

eye)

Wilmer Eye Institute, Johns Hopkins University

U.S. 3 million cases of glaucomaU.S. 1.5 million diagnosed OHT and treated to reduce IOP

Applanation Tonometry

Corneal tissue properties affectIOP measurement

central cornea thickness

low tensionglaucoma

OHT56%

NLOM: characterize tissue material properties dependence on IOP