Raman Signal Enhancement and CARS Microscopy

Abstract

Raman biosensors are appealing for many biomedical applications, due to their accuracy and speed. In addition, Raman microscopy is a non-labeled imaging technique that offers chemical contrast based on Raman vibrational frequencies. However, the weak Raman signal represents a significant obstacle to using Raman in biological applications. The objective of my PhD research, presented in this thesis, is to enhance the Raman signal, thereby enabling it to be used in a wide variety of biomedical applications. More specifically, the research focuses on two different Raman signal enhancement techniques. The first is to improve the Raman signal using hollow-core photonic crystal fibers; this enhanced the Raman signal of ethanol 40 times. The second approach is by generating a coherent anti-Stokes Raman scattering (CARS) signal. We demonstrated CARS microscopy of myelin (lipid-rich) structures using a single femtosecond Ti:sapphire laser, and a photonic crystal fiber (PCF) with two closely lying zero dispersion wavelengths (ZDWs). Generating low noise supercontinuum (Stokes beam) out of two closely lying ZDW PCFs, enabled us to perform fast data acquisition (84 μs per pixel) CARS imaging using a homebuilt microscope. However, the application of this fiber is often limited to CARS imaging of molecular species with vibrations at wavenumbers ≥ 2000 cm−1 Raman shift. In addition, as it is not a polarization maintaining fiber, it cannot be used for polarization CARS microscopy. A polarization-maintaining PCF with two far-lying zero dispersion wavelengths offers important advantages for polarization CARS microscopy, and for CARS imaging in the fingerprint region. This PCF, though commercially available, has had limited use for CARS microscopy in the C-H bond region. The main problem is that the supercontinuum from this fiber is typically noisier than that from a standard PCF with two closely-lying zero dispersion wavelengths. To overcome this, we determined the optimum operating conditions for generating a low-noise supercontinuum out of a PCF with two far-lying zero dispersion wavelengths, in terms of the input parameters of the excitation pulse. We measured the relative intensity noise (RIN) of the Stokes and the corresponding CARS signal, as a function of the input laser parameters in this fiber. We demonstrated that the results of CARS imaging using this alternate fiber are comparable to those achieved using the standard fiber for input laser pulse conditions of low average power, narrow pulse width with a slightly positive chirp, and polarization direction parallel to the slow axis of the selected fiber. Finally, we demonstrated a novel fiber-delivered, portable, multimodal CARS exoscope, for minimally invasive in-vivo imaging of tissues. The device was based on a micro-electromechanical system-scanning mirror and miniaturized optics, and light delivery by photonic crystal fibre. A single Ti:sapphire femtosecond laser approach is used to produce CARS and two photon excitation fluorescent and second harmonic generation images of different samples using the new setup. The high resolution and distortion-free images achieved with various samples, particularly in the reverse direction (epi), successfully demonstrate proof of concept, and paves the way to minimally or non-invasive in vivo imaging. Moreover, combining this novel endoscope with a portable femtosecond fiber laser will accelerate delivering multimodal nonlinear imaging endoscopy/microscopy to clinical bed-side applications.