• ability to probe living tissue with subcellular resolution • enable the visualization of morphological details in tissue that can not be resolved by ultrasound or magnetic resonance imaging

• ability to probe living tissue with subcellular resolution • enable the visualization of morphological details in tissue that can not be resolved by ultrasound or magnetic resonance imaging

OOptical microscopy is unique methodptical microscopy is unique method

CARS makes use of two laser beams, a pump beam at frequency

p and a Stokes beam at frequency S

CARS makes use of two laser beams, a pump beam at frequency

p and a Stokes beam at frequency S

When the beat frequency (p - S) Matches the frequency of a particular Raman activemolecular vibration (vib), the resonantoscillators are coherently driven, resulting in a strong anti-Stokes

signal at as=2 p - S

Anti-Stokes Raman scattering microscopy

Schematic of the real-time CARS imaging microscope, based on a mode-locked Nd:vanadate picosecond laser and a tunable

synchronously pumped optical parametric oscillator (OPO)

Schematic of the real-time CARS imaging microscope, based on a mode-locked Nd:vanadate picosecond laser and a tunable

synchronously pumped optical parametric oscillator (OPO)

The CARS energy diagram with pump (p) and Stokes (s) excitation

frequencies, the blue-shifted anti-Stokes (as) signal frequency, and the vibrational

frequency (vib)

The CARS energy diagram with pump (p) and Stokes (s) excitation

frequencies, the blue-shifted anti-Stokes (as) signal frequency, and the vibrational

frequency (vib)

Anti-Stokes Raman scattering microscopy

CARS Tissue ImagingImages of a hairless mouse earImages of a hairless mouse ear

(A) Stratum corneum with bright signals from the lamellar lipid intercellular space that surrounds the polygonal corneocytes.

Bright punctuated dots are ducts of sebaceous glands.

(B) Sebaceous glands at ~30 m from skin surface.

(C) Individual cells of the gland compartment can berecognized, with nuclei visible as dark holes (arrow).

(D) Adipocytes of the dermis at ~60 m

from skin surface.

(E) Adipocytes of the subcutaneous layer at a

depth of ~100 m.

(F) 2D projection of 60 depth-resolved slices separated by 2 m. Panels to the

right and under F show the yz and xz cross

sections taken at the white lines, respectively.

CARS Offers Chemical SelectivityA combined sequential CARS and two-photon fluorescence tissue imageA combined sequential CARS and two-photon fluorescence tissue image

The CARS signal is colored blue, and the two-photon fluorescenceis colored red. The Raman shift is set to 2,845 cm-1, with the 816.7-nm pump

Beam driving two-photon fluorescence excitation of the injected DiD dye.The sebaceousglands can be seen within the branched and looped capillary network

CARS Offers Chemical SelectivitySpectral differences between sebaceous glands Spectral differences between sebaceous glands

and dermal adipocytesand dermal adipocytes

(A) In vivo CARS spectrum of sebaceous gland (black) and adipocyte (red) (B) obtained by point-by-point wavelength scanning of thepumpbeam.

(B) Ex vivo Raman spectrum of individual sebaceous glands (black) and adipocytes (red)recorded from 10 m thick microtomed tissue sections.

CARS Offers Chemical SelectivityDiffusion of mineral oil through mouse epidermisDiffusion of mineral oil through mouse epidermis

(A) Externally applied mineral oil penetrates the stratum corneum through the lipid clefts between

corneocytes. Image was taken 20 m below the surface 15 min after application of oil

(B) The same area is shown 5 min later. Brighter signalindicates a higher oil concentration caused by time-dependent diffusion,

which can be clearly seen during the 5-min time window.