Shau Poh Chong

Hyperspectral optical coherence tomographic imaging of the retina using the visible-near-infrared wavelength range improves the use of spectroscopic information for biomarkers of retinal diseases without compromising the achievable axial resolution. Previous efforts to develop an optical coherence tomograph that covers both visible and near-infrared wavelengths were hampered by the challenges in designing a broadband spectrometer with adequate performance. In this work, we describe an ultrabroadband OCT system we designed that advances the state-of-the art in two major aspects: the design and implementation of a broadband spectrometer employing a 4k-pixel line camera achieving a spectral range of greater than 450nm with a central wavelength of 680nm; and an achromatised imaging system that minimises aberrations in the visible-near-infrared wavelength range for imaging mouse retina. The system provides an axial imaging range greater than 0.67mm within the retina with a larger than 40 ^∘ field angle. With this, we demonstrated ultrabroadband mouse retinal structural imaging in vivo and simultaneous visible and NIR waveband visualization of axon fiber bundle within the retinal nerve fiber layer for the first time. Our system opens new avenues for enhanced functional imaging of the mouse retina.

Nonlinear microscopy, including two-photon microscopy, requires pulsed lasers as light source. Typically, when choosing the appropriate pulsed laser for two-photon microscopy, the pulse repetition rate, pulse width, total power output, and output beam diameter are among the critical parameters which are often emphasised. Here, we demonstrate that the pulse shape, often overlooked, can have significant impact on the two-photon microscopy excitation efficiency and the effective signal brightness. We provide metrics to ease practical selection of pulsed laser sources for two-photon microscopy.

Nonlinear microscopy, including two-photon microscopy, requires pulsed lasers as light source. Typically, when choosing the appropriate pulsed laser for two-photon microscopy, the pulse repetition rate, pulse width, total power output, and output beam diameter are among the critical parameters which are often emphasised. Here, we demonstrate that the pulse shape, often overlooked, can have significant impact on the two-photon microscopy excitation efficiency and the effective signal brightness. We provide metrics to ease practical selection of pulsed laser sources for two-photon microscopy.

We have developed a multi-functional laser speckle imaging system, which can be operated in both the surface illumination laser speckle contrast imaging (SI-LSCI) mode and the line scan laser speckle contrast imaging (LS-LSCI) mode. The system has been applied to imaging the chicken embryos to visualize both the blood flow and morphological details of the vasculature. The experimental results demonstrated that LS-LSCI is capable of detecting and quantifying blood flow in blood vessels smaller and deeper than those detectable by conventional SI-LSCI. Furthermore, the line scan mode is also capable of producing depth-resolved absorption-based morphological images of tissue, augmenting flow-based functional images.

Recent development of visible light Optical Coherence Microscopy (i.e. Optical Coherence Tomography employing high numerical aperture Objective) has pushed the axial/transverse resolution limit to < 2 μm within tissues. Here, a fiber-based visible light Fourier domain (FD) Optical Coherence Microscopy system is presented, employing a 10x water immersion objective to achieve higher transverse resolution and reduced specular reflection when imaging human skin in vivo, using ~ 1-2 mW of incident power. Though with limited penetration depth, we demonstrated its application for in vivo imaging of human skin, and further measured the thicknesses of epidermis and dermo-epidermal junction. Furthermore, the total attenuation coefficient of the skin tissues was also estimated across epidermis and dermis.

In biological tissue, longer near-infrared wavelengths generally experience less scattering and more water absorption. Here we demonstrate an optical coherence tomography (OCT) system centered at 2.1 microns, whose bandwidth falls in the 2.2 micron water absorption optical window, for in vivo imaging of the rodent brain. We show in vivo that at 2.1 microns, the OCT signal is actually attenuated less in cranial bone than at 1.3 microns, and is also less susceptible to multiple scattering tails. We also show that the 2.2 micron window enables direct spectroscopic OCT assessment of tissue water content. We conclude that with further optimization, 2.2 micron OCT will have advantages in low-water-content tissue such as bone, as well as applications where extensive averaging is possible to compensate absorption losses.

Most flying-spot Optical Coherence Tomography (OCT) and Optical Coherence Microscopy (OCM) systems use a symmetric confocal geometry, where the detection path retraces the illumination path starting from and ending with the spatial mode of a single mode optical fiber. Here, we describe a visible light OCM instrument that breaks this symmetry to improve transverse resolution without sacrificing collection efficiency in scattering tissue. This was achieved by overfilling a 0.3 numerical aperture (NA) water immersion objective on the illumination path, while maintaining a conventional Gaussian mode detection path (1/e2 intensity diameter ~0.82 Airy disks), enabling ~1.1 μm full-width at half-maximum (FWHM) transverse resolution. At the same time, a ~0.9 μm FWHM axial resolution in tissue, achieved by a broadband visible light source, enabled femtoliter volume resolution. We characterized this instrument according to paraxial coherent microscopy theory, and then used it to image the meningeal layers, intravascular red blood cell-free layer, and myelinated axons in the mouse neocortex in vivo through the thinned skull. Finally, by introducing a 0.8 NA water immersion objective, we improved the lateral resolution to 0.44 μm FWHM, which provided a volumetric resolution of ~0.2 fL, revealing cell bodies in cortical layer I of the mouse brain with OCM for the first time.

Chromatic aberrations are an important design consideration in high resolution, high bandwidth, refractive imaging systems that use visible light. Here, we present a fiber-based spectral/Fourier domain, visible light OCT ophthalmoscope corrected for the average longitudinal chromatic aberration (LCA) of the human eye. Analysis of complex speckles from in vivo retinal images showed that achromatization resulted in a speckle autocorrelation function that was ~20% narrower in the axial direction, but unchanged in the transverse direction. In images from the improved, achromatized system, the separation between Bruch’s membrane (BM), the retinal pigment epithelium (RPE), and the outer segment tips clearly emerged across the entire 6.5 mm field-of-view, enabling segmentation and morphometry of BM and the RPE in a human subject. Finally, cross-sectional images depicted distinct inner retinal layers with high resolution. Thus, with chromatic aberration compensation, visible light OCT can achieve volume resolutions and retinal image quality that matches or exceeds ultrahigh resolution near-infrared OCT systems with no monochromatic aberration compensation.