Supraja Murali

Gabor-based fusion technique for Optical Coherence Microscopy

We recently reported on an Optical Coherence Microscopy technique, whose innovation intrinsically builds on a recently reported - 2 microm invariant lateral resolution by design throughout a 2 mm cubic full-field of view - liquid-lens-based dynamic focusing optical probe [Murali et al., Optics Letters 34, 145-147, 2009]. We shall report in this paper on the image acquisition enabled by this optical probe when combined with an automatic data fusion method developed and described here to produce an in-focus high resolution image throughout the imaging depth of the sample. An African frog tadpole (Xenopus laevis) was imaged with the novel probe and the Gabor-based fusion technique, demonstrating subcellular resolution in a 0.5 mm (lateral) x 0.5 mm (axial) without the need, for the first time, for x-y translation stages, depth scanning, high-cost adaptive optics, or manual intervention. In vivo images of human skin are also presented.

Three-dimensional adaptive microscopy using embedded liquid lens.

We report on the compact optical design of a high-resolution 3D scanning microscope with adaptive optics capability for refocusing with no moving parts designed for clinical research. The optical aberrations arising from refocusing are compensated for as part of the multiconfiguration optical design process. The lateral scanning is provided by a scanning mirror, and the depth scan is provided by an adaptive liquid lens embedded within the microscope as an integrated component of a custom optical design. The microscope achieves a performance of 250 lp/mm-a tenfold increase in performance over a liquid lens used as a stand-alone optical element. Results show that the optical design provides invariant modular transfer function over a 2 mm x 2 mm x 2 mm imaging volume, fully compensating (i.e., diffraction limited) for dynamic aberrations contributed by the scanning, the variation in the shape of the liquid lens, and the change in spherical aberration with depth in a slab of average index of refraction of skin. This design can find applications in biomedical imaging, white light interferometry for surface roughness measurements, and other 3D imaging systems.

Invariant resolution dynamic focus OCM based on liquid crystal lens.

A primary limitation of optical coherence microscopy is the lack of sufficient lateral resolution over a usable imaging volume for diagnostic applications, even with high-numerical aperture imaging optics. In this paper, we first motivate the benefit of refocusing at multiple depths in a highly scattering biological sample using optical coherence microscopy, which experimentally shows invariant 2.5 mum axial and 6.5 mum lateral resolution throughout the sample. We then present the optical system design of a hand-held probe with the advanced capability to dynamically focus with no moving parts to a depth of 2 mm in skin-equivalent tissue at 3 mum resolution throughout an 8 cubic millimeter imaging volume. The built-in dynamic focusing ability is investigated with an addressable liquid crystal lens embedded in a custom-designed optics optimized for a Ti:Sa pulsed broadband laser source of bandwidth 100nm centered at 800nm. The design was developed not only to account for refocusing into the tissue but also to minimize and compensate for the varying on-axis and off-axis optical aberrations that would be introduced throughout a 2 mm thick and 2 mm wide skin imaging volume. The MTF contrast functions and distortion plots at three different skin depths are presented.

Assessment of a liquid lens enabled in vivo optical coherence microscope.

The optical aberrations induced by imaging through skin can be predicted using formulas for Seidel aberrations of a plane-parallel plate. Knowledge of these aberrations helps to guide the choice of numerical aperture (NA) of the optics we can use in an implementation of Gabor domain optical coherence microscopy (GD-OCM), where the focus is the only aberration adjustment made through depth. On this basis, a custom-designed, liquid-lens enabled dynamic focusing optical coherence microscope operating at 0.2 NA is analyzed and validated experimentally. As part of the analysis, we show that the full width at half-maximum metric, as a characteristic descriptor for the point spread function, while commonly used, is not a useful metric for quantifying resolution in non-diffraction-limited systems. Modulation transfer function (MTF) measurements quantify that the liquid lens performance is as predicted by design, even when accounting for the effect of gravity. MTF measurements in a skinlike scattering medium also quantify the performance of the microscope in its potential applications. To guide the fusion of images across the various focus positions of the microscope, as required in GD-OCM, we present depth of focus measurements that can be used to determine the effective number of focusing zones required for a given goal resolution. Subcellular resolution in an onion sample, and high-definition in vivo imaging in human skin are demonstrated with the custom-designed and built microscope.

Super-resolution imaging combining the design of an optical coherence microscope objective with liquid-lens based dynamic focusing capability and computational methods

In this paper, we present the design of a 0.2 NA microscope objective operating across a 120nm broadband spectral range that requires only two doublets and an embedded liquid lens to achieve 3 μm invariant lateral resolution throughout a large 8 cubic millimeter imaging sample. Achieving invariant lateral resolution comes with some sacrifice in imaging speed, yet in the approach proposed, high speed in vivo imaging is maintained up to a resolution of 3 μm for a 2x2 mm sample size. Thus, in anticipation to ultimately aim for a resolution of 0.5 to 1 μm, we are investigating the possibility to further gain in resolution using super-resolution methods so both hardware solutions and image processing methods together can provide the best trade-off in overall resolution and speed of imaging. As a starting point to investigate super-resolution methods, we evaluate in this paper three well-known super-resolution algorithms used to reconstruct a high resolution image from down-sampled low resolution images of an African frog tadpole acquired en face using our OCM set-up. To establish ground truth necessary for assessment of the methods, low resolution images were simulated from a high resolution OCM image. The specification and design performance of the custom designed microscope will be presented as well as our first results of super-resolution imaging. The performance of each algorithm was analyzed and all performances compared using two different metrics. Early results indicate that super-resolution may play a significant role in the optimization of high invariant resolution OCM systems.

Dynamic-Focusing Microscope Objective for Optical Coherence Tomography

Optical Coherence Tomography (OCT) is a novel optical imaging technique that has assumed significant importance in bio-medical imaging in the last two decades because it is non-invasive and provides accurate, high resolution images of three dimensional cross-sections of body tissue, exceeding the capabilities of the current predominant imaging technique - ultrasound. In this paper, the application of high resolution OCT, known as optical coherence microscopy (OCM) is investigated for in vivo detection of abnormal skin pathology for the early diagnosis of cancer. A main challenge in OCM is maintaining invariant resolution throughout the sample. The technology presented is based on a dynamic focusing microscope imaging probe conceived for skin imaging and the detection of abnormalities in the epithelium. A novel method for dynamic focusing in the biological sample is presented using variable-focus lens technology to obtain three dimensional images with invariant resolution throughout the cross-section and depth of the sample is presented and discussed. A low coherence broadband source centered at near IR wavelengths is used to illuminate the sample. The design, analysis and predicted performance of the dynamic focusing microscope objective designed for dynamic three dimensional imaging at 5μm resolution for the chosen broadband spectrum is presented.

Dynamic focus catheter design for endoscopic optical coherence tomography

To maintain high lateral resolution throughout depth scanning, a dynamically focusing catheter with a lateral resolution < 10 mum and a depth scanning range > 4 mm was designed without a mechanically re-focusing system.

Invariant high resolution optical skin imaging

Optical Coherence Microscopy (OCM) is a bio-medical low coherence interferometric imaging technique that has become a topic of active research because of its ability to provide accurate, non-invasive cross-sectional images of biological tissue with much greater resolution than the current common technique ultrasound. OCM is a derivative of Optical Coherence Tomography (OCT) that enables greater resolution imposed by the implementation of an optical confocal design involving high numerical aperture (NA) focusing in the sample. The primary setback of OCM, however is the depth dependence of the lateral resolution obtained that arises from the smaller depth of focus of the high NA beam. We propose to overcome this limitation using a dynamic focusing lens design that can achieve quasi-invariant lateral resolution up to 1.5mm depth of skin tissue.

Performance of a Liquid Lens Enabled Optical Coherence Microscope with Gabor Fusion

A custom microscope with an integrated Varioptic liquid lens has been fabricated and shown to provide subcellular resolution using Gabor image fusion. MTF testing and final fused images are shown.

Sub-cellular resolution imaging with Gabor domain optical coherence microscopy

Optical Coherence Microscopy (OCM) utilizes a high NA microscope objective in the sample arm to achieve an axially and laterally high resolution OCT image. An increase in NA, however, leads to a dramatically decreased depth of focus (DOF), and hence shortens the imaging depth range so that high lateral resolution is maintained only within a small depth region around the focal plane. One solution to increase the depth of imaging while keeping a high lateral resolution is dynamic-focusing. Utilizing the voltage controlled refocus capability of a liquid lens, we have recently presented a solution for invariant high resolution imaging using the liquid lens embedded within a fixed optics hand-held custom microscope designed specifically for optical imaging systems using a broadband light source at 800 nm center wavelength. Subsequently, we have developed a Gabor-Domain Optical Coherence Microscopy (GD-OCM) that utilizes the high speed imaging of spectral domain OCT, the high lateral resolution of OCM, and the ability of real time refocusing of our custom design variable focus objective. In this paper we demonstrate in detail how portions of the infocus cross-sectional images can be extracted and fused to form an invariant lateral resolution image with multiple crosssectional images acquired corresponding to a discrete refocusing step along depth enabled by the varifocal probe. We demonstrate sub-cellular resolution imaging of an African frog tadpole (Xenopus Laevis) taken from a 500 μm x 500 μm cross-section.