Wavelength swept spectrally encoded confocal microscopy for biological and clinical applications

Abstract

Spectrally encoded confocal microscopy (SECM) is a technique that facilitates the incorporation of confocal microscopy into small, portable clinical instruments. This would allow in vivo evaluation of cellular and sub-cellular features in a non-destructive, minimally invasive manner. Prior studies have demonstrated the potential of the techniques as well as highlighted the need for faster acquisition rates and higher sensitivity. In this thesis, new laser sources, optical fiber arrangements and probe designs are explored to ultimately evaluate SECM's relevance as a clinical tool. Clinical imaging at cellular scales requires imaging rates on the order of tens of frames per second to reduce motion artifacts from unavoidable patient movements. Rapid SECM imaging was achieved through the development of a novel wavelength swept laser which simultaneously provided high output power (> 10mrW), narrow linewidth (10GHz), broad wavelength tuning (80 nm centered at 1310 nm) and fast repetition rates (up to 16,000 Hz), while being compact and environmentally stable. Imaging with a wavelength swept SECM system was characterized by coupling the laser to a tabletop imaging arm comprising a high density holographic grating, a galvanometer mounted mirror and a 0.9 NA water immersion microscope objective.

(cont.) Rapid SECM imaging is performed at a transverse resolution of 1.4 microns, axial resolution of 6 microns over a field of view of 440x440 microns and allows subcellular imaging ex vivo (excised specimens) and in vivo (human skin). A study on 40 excised head and neck specimens showed that SECM has the potential to perform tissue identification, but also revealed the presence of speckle noise due to the coherent nature of the illumination and collection schemes through a single mode optical fiber. A partially coherent system based on single mode fiber for illumination and multimode fiber for detection was simulated, implemented and tested to find adequate balance between attenuation of speckle noise and conservation of resolution. A coupling of 20 modes was found to reduce speckle by a factor 4.5 with a minimal sectioning penalty of 0.25, while allowing a signal increase of 8dB. This improvement in sensitivity allowed SECM table top system to be used for investigations in developmental biology where Dual clad fibers (DCF) were previously shown to allow partially coherent endoscopic imaging, using the single mode core for illumination and inner clad for multimodal collection.

(cont.) Commercially available DCF's which propagate thousands of modes are ill suited for confocal endoscopes as collecting such a number of modes would destroy the axial resolution. Based on results from the previous section and through modal analysis, a DCF was designed, drawn - via a collaboration with Boston University Photonics Center -, and tested for use with SECM. The prototype DCF yielded promising results (3 fold speckle attenuation, optical sectioning degradation of 0.85), and showed the need for implementation of better coupling mechanisms to take advantage of increased signal collection. Finally, a portable SECM system was built for in vivo evaluation of pediatric vocal fold. A preliminary study on porcine and cadaveric tissue showed that SECM can distinguish between epithelium, superior and intermediate layers of the lamina propria, which could help elucidate the development mechanism of the voice apparatus if performed in vivo. The handheld instrument comprises a custom grating scanner imaging the scanning pivot onto the back pupil of a high NA microscope objective. The imaging tube can easily be interchanged to accommodate geometrical constraints imposed by different age groups.

(cont.) The probe, currently under review by the biomedical engineering committee, revealed cellular and sub cellular details of human skin in vivo at depth and acquisition rates sufficient to capture blood cells flowing through capillaries. Through major improvements in acquisition speeds, sensitivity, and speckle appearance, this work established SECM as a potent clinical and biological imaging tool. Ultimate confirmation will be revealed through in vivo studies to come, but limitations are likely to be of engineering nature rather than from physical considerations. Future work should explore the possibility to combine SECM with other contrast mechanisms to provide imaging with increased specificity.