Fiber delivered probe for efficient CARS imaging of tissues

Abstract

We demonstrate a fiber-based probe for maximum collection of the coherent anti-Stokes Raman scattering (CARS) signal in biological tissues. We discuss the design challenges including capturing the backscattered forward generated CARS signal in the sample and the effects of fiber nonlinearities on the propagating pulses. Three different single mode fibers (fused silica fiber, photonic crystal fiber and double-clad photonic crystal fiber) were tested for the probe design. We investigated self-phase modulation, stimulated Raman scattering (SRS) and four-wave-mixing (FWM) generation in the fiber: nonlinear processes expected to occur in a two-beam excitation based probe. While SPM and SRS induced spectral broadening was negligible, a strong non phase-matched FWM contribution was found to be present in all the tested fibers for excitation conditions relevant to CARS microscopy of tissues. To spectrally suppress this strong contribution, the pro design incorporates separate fibers for excitation light delivery and for signal detection, in combination with dichroic optics. CARS images of the samples were recorded by collecting the back-scattered forward generated CARS signal in the sample through a multi-mode fiber. Different biological tissues were imaged ex vivo in order to assess the performance of our fiber-delivered probe for CARS imaging, a tool which we consider an important advance towards label-free, in vivo probing of superficial tissues.

Fig. 1

Schematic diagram of the fiber-delivered probe for CARS tissue imaging: M-mirrors; D1- 1000 nm longpass dichroic mirror; D2- 760 nm longpass dichroic mirror, L-lens; Obj-objective; S-sample; F- 670 nm bandpass filter. Fiber 1 is used for delivery of the excitation pulses and fiber 2 is used for detecting the CARS radiation. The dimensions of the probe are indicated in cm.

Fig. 2

Intensity spectra of the pump beam measured before and after the (a) DCPCF16 fiber (FWHM=9.7 nm before the fiber; FWHM=9.7 nm after the fiber) and (b) the LMA-20 PCF (FWHM=10 nm before the fiber; FWHM=11 nm after the fiber)

Fig. 3

Spectrally-resolved anti-Stokes four-wave-mixing signal measured at the output of (a) the LMA20 fiber (FWHM=7.9 nm) output and (b) the silica SMF (FWHM=8.6 nm).

Fig. 4

CARS signal intensity from the DMSO sample and FWM from the fiber as a function of time delay between the pump and the Stokes beam for the DCPCF16 fiber (a) and the LMA-20 fiber (b).

Fig. 5

CARS images of thick tissue samples ex vivo at 2842 cm⁻¹.a) Small adipocytes of mouse ear skin. b) Adipocytes of subcutaneous layer of rabbit skin tissue. C) Meibomian gland in mouse eyelid. Images were acquired in 2s. Scale bar is 50 µm.