Vibrational phase contrast CARS microscopy

Abstract

This thesis describes a new technique that improves specificity, selectivity and sensitivity in coherent anti-Stokes Raman scattering (CARS) microscopy. CARS microscopy is a nonlinear optical technique that utilizes specific bonds of molecules, sometimes referred to as the `fingerprint' of a molecule, to obtain chemically specific imaging of a sample. A single `unique' vibrational resonance within a molecule can be addressed and the appearance of this resonance within a sample can be imaged. This nonlinear vibrational microscopy technique provides a noninvasive method to identify structures of a specific chemical composition. CARS features high collection efficiency and absence of interference from one-photon autofluorescence. However, CARS suffers from a frequency-independent background signal, the `non-resonant background'. In mixtures, the resonant CARS signal of less abundant constituents may be overwhelmed by the non-resonant background, preventing detection of the resonant molecules. A cascaded phase preserving chain is used for heterodyne detection and yields amplitude and phase. The vibrational phase of the oscillators in the focal volume can be obtained by the subtraction of the local excitation phase from the heterodyne phase. We call this technique ``Vibrational Phase Contrast CARS'' and can be regarded as a vibrational extension of the linear (refractive index) phase contrast microscopy introduced by Zernike in 1933. The phase detection allows for rejection of the non-resonant background signal, without reduction of the resonant signal. CARS microscopy is applied to biology, pharmacology and food, where chemical selectivity and label-free detection are essential. Other nonlinear signals, generated simultaneously, can be used to obtain more information from the sample; CARS, second harmonic generation microscopy and spontaneous Raman spectroscopy are used side-by-side. Two in-depth studies are presented on the dissolution behavior of a drug and on early bone growth in cells.