Extended Focus Optical Coherence Microscopy : Theory and Application

Optical coherence tomography (OCT) is an interferometric imaging technique that can provide depth resolved cross-sectional views of biological tissue. OCT employs light with low temporal and yet high spatial coherence. The sample is illuminated point by point and raster scanned in the lateral directions. Part of the incident light is back-scattered by differences in the tissue refractive index and combined with a strong reference signal. The detection in the Fourier, or frequency domain (FDOCT), separates the interference signal spectrally and records the resulting pattern with a line camera. Processing of this pattern extracts the sample structure along the axial direction but without axial scanning and in parallel with high resolution of 2-3 µm. The interferometric detection conveys a very high sensitivity that allows imaging several hundred micrometers deep into the tissue. A single two dimensional scan thus can extract the three dimensional structure of the object, resulting in very high volume acquisition rates and offering an exceptional speed advantage over other optical imaging methods. Combined with its ability to measure an intrinsic sample property without the need of adding extrinsic labels or contrasting agents, FDOCT has become a confirmed tool for minimally invasive in vivo measurements and comprehensive volumetric imaging. While the axial resolution in OCT is defined by the coherence length of the employed light source, it is the optical focusing that specifies the resolution in the lateral direction, which reaches in general only approximately 10 µm. To improve the resolution, classical optical coherence microscopy (OCM) uses higher numerical apertures. However, this implies a reduction of the lateral dimension of the focal spot which leads to a dramatic decrease in the depth of field (DOF). This imposes a severe limitation on FDOCT's parallel depth extraction. Therefore, the motivation behind this thesis work was to circumvent the compromise between the lateral resolution and the depth of field by engineering an extended focal volume (xf). Combining FDOCT's assets of speed and sensitivity with the high lateral resolution of microscopy provided a very promising tool for rapid in vivo imaging at close to cellular resolution. To study the mechanisms limiting the DOF and to investigate what impact they have on the tomogram reconstruction process, we developed a model for image formation in FDOCT. Generally, the tomogram is reconstructed from the two dimensional interference patterns in the individual spectral channels of the detection. By making use of the coherent transfer functions (CTF), the spatial frequency content made accessible by each channel, was analyzed. This provided a novel perspective on the entire tomogram formation. We were able to show that the out-of-focus structures suffer from two signal degrading mechanisms. First, a de-focusing effect, induced by additional phase arguments in the spatial frequency domain provo