Optical methods for imaging and focusing are advantageous in many scenarios as optics can provide exquisite spatial resolution, has multiple sources of contrast, and does not impart ionizing radiation. However, optical scattering remains a fundamental challenge which limits the depth at which we can perform imaging with good spatial resolution. This challenge motivated our investigations into methods that could make use of the scattered light in order to extend the depth of imaging through or within scattering media. In particular, we focus on answering: (1) Can one ‘unscramble’ the scattered light in order to recover information about the otherwise hidden object?; and (2) Can we preferentially detect the more forward scattered photons in an efficient manner in order to allow deeper penetration with modest resolution? These two questions are explored in the first two projects of the thesis:
Optical methods offer the benefit of visualizing samples that would otherwise appear transparent. Using light, one is able to visualize and measure the thickness of transparent films and coatings in a non-contact manner. The third project in my thesis focuses on using light to non-destructively visualize and characterize the evenness of the silicone oil layer that typically coats the inner surface of prefilled syringes. Characterizing the evenness of this silicone oil layer is important as it impacts the functionality of the prefilled syringe and may correlate with particle formation, which is undesirable as the number of particles in a syringe is regulated due to potential health concerns.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/4hkq-dz43, author = {Xu, Jian}, title = {Optical Light Manipulation and Imaging Through Scattering Media}, school = {California Institute of Technology}, year = {2021}, doi = {10.7907/4hkq-dz43}, url = {https://resolver.caltech.edu/CaltechTHESIS:09092020-162015646}, abstract = {Typical optical systems are designed to be implemented in free space or clean media. However, the presence of optical scattering media scrambles light waves and becomes a problem in light field control, optical imaging, and sensing.
To address the problem caused by optical scattering media, we discuss two types of solutions in this thesis. One type of solution is active control, where active modulators are used to modulate the light wave to compensate the wave distortion caused by optical scattering. The other type of solution is computational optics, where physical and mathematical models are built to computationally reconstruct the information from the measured distorted wavefront.
In the part of active control, we first demonstrate coherent light focusing through scattering media by transmission matrix inversion. The transmission matrix inversion approach can realize coherent light control through scattering media with higher fidelity compared to conventional transmission matrix approaches. Then, by combining the pre-designed scattering metasurface with wavefront shaping, we demonstrate a beam steering system with large angular and high angular resolution. Next, we present optical-channel-based intensity streaming (OCIS), which uses only intensity information of light fields to realize light control through scattering media. This solution can be used to control spatially incoherent light propagating through scattering media. In the part of computational optics, we first demonstrate the idea of interferometric speckle visibility spectroscopy (ISVS) to measure the information cerebral blood flow. In ISVS, a camera records the speckle frames of diffused light from the human subject interferometrically, and the speckle statistics is used to calculate the speckle decorrelation time and consequently the blood flow index. Then, we compare the two methods of decorrelation time measurements - temporal sampling methods and spatial ensemble methods - and derive unified mathematical expressions for them in terms of measurement accuracy. Based on current technology of camera sensors and single detectors, our results indicate that spatial ensemble methods can have higher decorrelation time measurement accuracy compared to commonly used temporal sampling methods.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/8W3A-HE02, author = {Chung, Jaebum}, title = {Computational Imaging: a Quest for the Perfect Image}, school = {California Institute of Technology}, year = {2019}, doi = {10.7907/8W3A-HE02}, url = {https://resolver.caltech.edu/CaltechTHESIS:05202019-151055724}, abstract = {A physical lens is limited in its ability to capture an image that is both high- resolution and wide-field due to aberrations even with a sophisticated lens design. This thesis explores computational methods that expand on the recently developed Fourier ptychographic microscopy (FPM) to overcome the physical limitations. New algorithms and imaging methods extend the computational aberration correction to more general imaging modalities including fluorescence microscopy and incoherent bright-field imaging so as to allow even a crude lens to perform like an ideal lens. This paradigm shift from the lens design to computational algorithms democratizes high-resolution imaging by making it easier to use and less complicated to build.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/0PP8-2E39, author = {Brake, Joshua Harris}, title = {Seeing Through the Fog: Using Scattered Light to Peer Deeper into Biological Tissue}, school = {California Institute of Technology}, year = {2019}, doi = {10.7907/0PP8-2E39}, url = {https://resolver.caltech.edu/CaltechTHESIS:05282019-104728085}, abstract = {Optical scattering is a fundamental problem in biomedical optics and limits most optical techniques to shallow operating depths less than 1 millimeter. However, although the scattering behavior of tissue scrambles the information it contains, it does not destroy it. Therefore, if you can unscramble the scattered light, it increases the accessible imaging depths up the absorption limit of light (several centimeters deep).
One such way to beat optical scattering is using wavefront shaping. Borrowing ideas from adaptive optics in astronomy and phased arrays in radar and ultrasonic imaging, the basic concept of wavefront shaping is to control the phase and amplitude of the light field in order to harness scattered light. Using wavefront shaping techniques, scattered light can be used to form focal spots or transmit information through or inside optically scattering media. Furthermore, even without correcting for scattering directly by shaping the input light field, the properties of the scattered light can be analyzed to recover information about the structure and dynamic properties of a sample using methods from diffuse optics.
The main contributions of this thesis are along these two lines of research: moving wavefront shaping toward more practical applications and developing new techniques to recover useful physiological information from scattered light. This is developed through three main projects: (1) an investigation of how dynamic samples impact the scattering process and the practical implications of these dynamics on wavefront shaping systems, (2) the development of a wavefront shaping system combining light and ultrasound to focus light inside acute brain slices to improve light delivery for optogenetics, (3) a novel method to sensitively detect the dynamics of scattered light and use it to tease out information about the flow of blood within the tissue sample of interest.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/Z9H9937R, author = {Kim, Jinho}, title = {Compact Microscope System for Biomedical Applications}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z9H9937R}, url = {https://resolver.caltech.edu/CaltechTHESIS:04262017-114441886}, abstract = {Demands for an imaging system which has high space-bandwidth product (SBP) are increasing in modern biomedical research as the amount of information to be dealt with is increasing. However, conventional microscopy has a limited SBP of about 10 mega pixels, and as such if a user wants an image in high resolution, the field of view (FOV) of the image is reduced, or if a wide FOV is necessary, the user needs to give up the resolution of image. A common way of overcoming this SBP limit in the conventional microscopy is to use mechanical moving stages and scan through wide sample area, however, it is time consuming to image large area using a high numerical aperture (NA) objective lens. This thesis presents compact imaging systems based on Fourier ptychographic microscopy for biomedical applications which are able to increase SBP without having any mechanical moving parts: one imaging system for an incubator embedded imaging system to be used in in-vitro cell culture monitoring, and the other for a high throughput 96 well plate imaging system for fast drug screening.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/Z9TX3CD1, author = {Zhou, Edward Haojiang}, title = {Optical Focusing and Imaging through Scattering Media}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z9TX3CD1}, url = {https://resolver.caltech.edu/CaltechTHESIS:05172017-103505376}, abstract = {Optical techniques, which have been widely used in various fields including bio-medicine, remote sensing, astronomy, and industrial production, play an important role in modern life. Optical focusing and imaging, which correspond to the basic methods of utilizing light, are key to the implementation of optical techniques. In free space or a nearly transparent medium, optical imaging and focusing can be easily realized by using conventional optical elements, such as lenses and mirrors, due to the ballistic propagation of light in these media. However, in scattering media like biological tissue and fog, refractive index inhomogeneities cause diffusive propagation of light that increases with depth, which restricts the use of optical methods in thick, scattering media. Generally speaking, scattering media poses three challenges to optical focusing and imaging: wavefront aberrations, glare, and decorrelation. Wavefront aberrations can randomize light traveling through a scattering medium, disrupt the formation of focus, and break the conjugate relation in imaging. Glare caused by backscattering will largely impair the visibility of imaging, and decorrelation in dynamic media requires systems that counter the effect of scattering to operate faster than the decorrelation time. In this thesis, we explored solutions to the problem of scattering from different aspects. We presented Time Reversal by Analysis of Changing wavefronts from Kinetic targets (TRACK) technique to realize noninvasive optical focusing through a scattering medium. We showed that by taking the difference between time-varying scattering fields caused by a moving object and applying optical phase conjugation, light can be focused back to the location previously occupied by the object. To tackle the decorrelation of living tissue, we built up a fast digital optical phase conjugation (DOPC) system based on FPGA and DMD, which has a response time of 5.3 ms and was the fastest DOPC system in the world before 2017. We demonstrated that the system is fast enough to focus light through 2.3mm-thick living mouse skin. As for glare, inspired by noise canceling headphones, we invented an optical analogue termed coherence gated negation (CGN) technique. CGN can optically cancel out the glare in an active illumination imaging scenario to realize imaging through scattering media, like fog. In the experiment, we suppressed the glare by an order of magnitude and allowed improved imaging of a weak target. Finally, we demonstrated a method to image a moving target through scattering media noninvasively. Its principle roots are in the speckle-correlation-based imaging (SCI) invented by Ori Katz. We improved the technique and extended its application to bright field imaging of a moving target.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/Z9M32SRZ, author = {Ou, Xiaoze}, title = {Computational Microscopy: Breaking the Limit of Conventional Optics}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z9M32SRZ}, url = {https://resolver.caltech.edu/CaltechTHESIS:04282016-051723211}, abstract = {Computational imaging is flourishing thanks to the recent advancement in array photodetectors and image processing algorithms. This thesis presents Fourier ptychography, which is a computational imaging technique implemented in microscopy to break the limit of conventional optics. With the implementation of Fourier ptychography, the resolution of the imaging system can surpass the diffraction limit of the objective lens’s numerical aperture; the quantitative phase information of a sample can be reconstructed from intensity-only measurements; and the aberration of a microscope system can be characterized and computationally corrected. This computational microscopy technique enhances the performance of conventional optical systems and expands the scope of their applications.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/Z95Q4T1W, author = {Horstmeyer, Roarke William}, title = {Computational Microscopy: Turning Megapixels into Gigapixels}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z95Q4T1W}, url = {https://resolver.caltech.edu/CaltechTHESIS:10202015-173005082}, abstract = {The layout of a typical optical microscope has remained effectively unchanged over the past century. Besides the widespread adoption of digital focal plane arrays, relatively few innovations have helped improve standard imaging with bright-field microscopes. This thesis presents a new microscope imaging method, termed Fourier ptychography, which uses an LED to provide variable sample illumination and post-processing algorithms to recover useful sample information. Examples include increasing the resolution of megapixel-scale images to one gigapixel, measuring quantitative phase, achieving oil-immersion quality resolution without an immersion medium, and recovering complex three dimensional sample structure.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/Z99G5JSN, author = {Jang, Mooseok}, title = {Optical Phase Conjugation and Its Applications in Biology}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z99G5JSN}, url = {https://resolver.caltech.edu/CaltechTHESIS:05262016-142345346}, abstract = {Optical phase conjugation is a process where an incoming electromagnetic wave is reflected with a reversed phase. The propagation direction of an incoming beam (equivalently, local phase gradient) can thereby be precisely reversed by the phase conjugate beam. This intriguing effect, so called “time-reversal of electromagnetic waves,” allows cancellation of spatial distortion introduced into the incoming beam. Recently, this concept has provided a new avenue to overcome or utilize random scattering in the field of biophotonics.
This thesis discusses a number of interrelated topics regarding optical phase conjugation and its applications in biology. First, two examples of exploiting optical phase conjugation for light focusing are presented. The first example shows that the axial resolution can be improved based on the counter-propagating property of the phase-conjugate beam, and the second example demonstrates how the random scattering media can be used to enhance the flexibility in focusing range. We then discuss a new class of techniques that involves the use of guidestars in the phase conjugation process for deep tissue (> 1mm) light focusing and imaging. In the context of in vivo application, we model and estimate the penetration depth limit of one prominent example of this approach, time-reversed ultrasonically encoded (TRUE) optical focusing. Based on the analysis, we show that the iteration of phase conjugation operation can improve the contrast and resolution of the focal spot created inside deep tissue. We also present a new kind of guidestar-assisted method, time-reversed ultrasound microbubble encoded (TRUME) light focusing, which can focus light with sub-ultrasound wavelength resolution. At last, the effect of dynamic scatterers on time-reversal fidelity is studied to explore the possibility of applying the optical phase conjugation techniques in living tissue.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/Z9SF2T49, author = {Han, Chao}, title = {Wide Field-of-View Microscopes and Endoscopes for Time-Lapse Imaging and High-Throughput Screening}, school = {California Institute of Technology}, year = {2015}, doi = {10.7907/Z9SF2T49}, url = {https://resolver.caltech.edu/CaltechTHESIS:01302015-101318815}, abstract = {Wide field-of-view (FOV) microscopy is of high importance to biological research and clinical diagnosis where a high-throughput screening of samples is needed. This thesis presents the development of several novel wide FOV imaging technologies and demonstrates their capabilities in longitudinal imaging of living organisms, on the scale of viral plaques to live cells and tissues.
The ePetri Dish is a wide FOV on-chip bright-field microscope. Here we applied an ePetri platform for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm × 4 mm at 30 min intervals. A density-based clustering algorithm is used to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. We also demonstrate the capabilities of the ePetri in viral titer count and dynamically monitoring plaque formation, growth, and the influence of antiviral drugs.
We developed another wide FOV imaging technique, the Talbot microscope, for the fluorescence imaging of live cells. The Talbot microscope takes advantage of the Talbot effect and can generate a focal spot array to scan the fluorescence samples directly on-chip. It has a resolution of 1.2 μm and a FOV of ~13 mm2. We further upgraded the Talbot microscope for the long-term time-lapse fluorescence imaging of live cell cultures, and analyzed the cells’ dynamic response to an anticancer drug.
We present two wide FOV endoscopes for tissue imaging, named the AnCam and the PanCam. The AnCam is based on the contact image sensor (CIS) technology, and can scan the whole anal canal within 10 seconds with a resolution of 89 μm, a maximum FOV of 100 mm × 120 mm, and a depth-of-field (DOF) of 0.65 mm. We also demonstrate the performance of the AnCam in whole anal canal imaging in both animal models and real patients. In addition to this, the PanCam is based on a smartphone platform integrated with a panoramic annular lens (PAL), and can capture a FOV of 18 mm × 120 mm in a single shot with a resolution of 100─140 μm. In this work we demonstrate the PanCam’s performance in imaging a stained tissue sample.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/HNWJ-J182, author = {Lee, Seung Ah}, title = {Bright-Field and Fluorescence Chip-Scale Microscopy for Biological Imaging}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/HNWJ-J182}, url = {https://resolver.caltech.edu/CaltechTHESIS:02212014-174719213}, abstract = {Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.
Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.
Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.
Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.
Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/YNSN-8960, author = {Wang, Ying Min}, title = {Deep Tissue Fluorescence Imaging with Time-Reversed Light}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/YNSN-8960}, url = {https://resolver.caltech.edu/CaltechTHESIS:04282013-103938118}, abstract = {Advances in optical techniques have enabled many breakthroughs in biology and medicine. However, light scattering by biological tissues remains a great obstacle, restricting the use of optical methods to thin ex vivo sections or superficial layers in vivo. In this thesis, we present two related methods that overcome the optical depth limit—digital time reversal of ultrasound encoded light (digital TRUE) and time reversal of variance-encoded light (TROVE). These two techniques share the same principle of using acousto-optic beacons for time reversal optical focusing within highly scattering media, like biological tissues. Ultrasound, unlike light, is not significantly scattered in soft biological tissues, allowing for ultrasound focusing. In addition, a fraction of the scattered optical wavefront that passes through the ultrasound focus gets frequency-shifted via the acousto-optic effect, essentially creating a virtual source of frequency-shifted light within the tissue. The scattered ultrasound-tagged wavefront can be selectively measured outside the tissue and time-reversed to converge at the location of the ultrasound focus, enabling optical focusing within deep tissues. In digital TRUE, we time reverse ultrasound-tagged light with an optoelectronic time reversal device (the digital optical phase conjugate mirror, DOPC). The use of the DOPC enables high optical gain, allowing for high intensity optical focusing and focal fluorescence imaging in thick tissues at a lateral resolution of 36 µm by 52 µm. The resolution of the TRUE approach is fundamentally limited to that of the wavelength of ultrasound. The ultrasound focus (~ tens of microns wide) usually contains hundreds to thousands of optical modes, such that the scattered wavefront measured is a linear combination of the contributions of all these optical modes. In TROVE, we make use of our ability to digitally record, analyze and manipulate the scattered wavefront to demix the contributions of these spatial modes using variance encoding. In essence, we encode each spatial mode inside the scattering sample with a unique variance, allowing us to computationally derive the time reversal wavefront that corresponds to a single optical mode. In doing so, we uncouple the system resolution from the size of the ultrasound focus, demonstrating optical focusing and imaging between highly diffusing samples at an unprecedented, speckle-scale lateral resolution of ~ 5 µm. Our methods open up the possibility of fully exploiting the prowess and versatility of biomedical optics in deep tissues.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/Z9445JF5, author = {Ren, Jian}, title = {Endoscopic Optical Coherence Tomography: Design and Application}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/Z9445JF5}, url = {https://resolver.caltech.edu/CaltechTHESIS:06172013-155313179}, abstract = {
This thesis presents an investigation on endoscopic optical coherence tomography (OCT). As a noninvasive imaging modality, OCT emerges as an increasingly important diagnostic tool for many clinical applications. Despite of many of its merits, such as high resolution and depth resolvability, a major limitation is the relatively shallow penetration depth in tissue (about 2∼3 mm). This is mainly due to tissue scattering and absorption. To overcome this limitation, people have been developing many different endoscopic OCT systems. By utilizing a minimally invasive endoscope, the OCT probing beam can be brought to the close vicinity of the tissue of interest and bypass the scattering of intervening tissues so that it can collect the reflected light signal from desired depth and provide a clear image representing the physiological structure of the region, which can not be disclosed by traditional OCT. In this thesis, three endoscope designs have been studied. While they rely on vastly different principles, they all converge to solve this long-standing problem.
A hand-held endoscope with manual scanning is first explored. When a user is holding a hand- held endoscope to examine samples, the movement of the device provides a natural scanning. We proposed and implemented an optical tracking system to estimate and record the trajectory of the device. By registering the OCT axial scan with the spatial information obtained from the tracking system, one can use this system to simply ‘paint’ a desired volume and get any arbitrary scanning pattern by manually waving the endoscope over the region of interest. The accuracy of the tracking system was measured to be about 10 microns, which is comparable to the lateral resolution of most OCT system. Targeted phantom sample and biological samples were manually scanned and the reconstructed images verified the method.
Next, we investigated a mechanical way to steer the beam in an OCT endoscope, which is termed as Paired-angle-rotation scanning (PARS). This concept was proposed by my colleague and we further developed this technology by enhancing the longevity of the device, reducing the diameter of the probe, and shrinking down the form factor of the hand-piece. Several families of probes have been designed and fabricated with various optical performances. They have been applied to different applications, including the collector channel examination for glaucoma stent implantation, and vitreous remnant detection during live animal vitrectomy.
Lastly a novel non-moving scanning method has been devised. This approach is based on the EO effect of a KTN crystal. With Ohmic contact of the electrodes, the KTN crystal can exhibit a special mode of EO effect, termed as space-charge-controlled electro-optic effect, where the carrier electron will be injected into the material via the Ohmic contact. By applying a high voltage across the material, a linear phase profile can be built under this mode, which in turn deflects the light beam passing through. We constructed a relay telescope to adapt the KTN deflector into a bench top OCT scanning system. One of major technical challenges for this system is the strong chromatic dispersion of KTN crystal within the wavelength band of OCT system. We investigated its impact on the acquired OCT images and proposed a new approach to estimate and compensate the actual dispersion. Comparing with traditional methods, the new method is more computational efficient and accurate. Some biological samples were scanned by this KTN based system. The acquired images justified the feasibility of the usage of this system into a endoscopy setting. My research above all aims to provide solutions to implement an OCT endoscope. As technology evolves from manual, to mechanical, and to electrical approaches, different solutions are presented. Since all have their own advantages and disadvantages, one has to determine the actual requirements and select the best fit for a specific application.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/WWFF-7S14, author = {Pang, Shuo}, title = {Fluorescence Optofluidic Microscopy and Fluorescence Microscopy Based on the Talbot Effect}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/WWFF-7S14}, url = {https://resolver.caltech.edu/CaltechTHESIS:04132013-185824588}, abstract = {Light microscopy has been one of the most common tools in biological research, because of its high resolution and non-invasive nature of the light. Due to its high sensitivity and specificity, fluorescence is one of the most important readout modes of light microscopy. This thesis presents two new fluorescence microscopic imaging techniques: fluorescence optofluidic microscopy and fluorescent Talbot microscopy. The designs of the two systems are fundamentally different from conventional microscopy, which makes compact and portable devices possible. The components of the devices are suitable for mass-production, making the microscopic imaging system more affordable for biological research and clinical diagnostics.
Fluorescence optofluidic microscopy (FOFM) is capable of imaging fluorescent samples in fluid media. The FOFM employs an array of Fresnel zone plates (FZP) to generate an array of focused light spots within a microfluidic channel. As a sample flows through the channel and across the array of focused light spots, a filter-coated CMOS sensor collects the fluorescence emissions. The collected data can then be processed to render a fluorescence microscopic image. The resolution, which is determined by the focused light spot size, is experimentally measured to be 0.65 μm.
Fluorescence Talbot microscopy (FTM) is a fluorescence chip-scale microscopy technique that enables large field-of-view (FOV) and high-resolution imaging. The FTM method utilizes the Talbot effect to project a grid of focused excitation light spots onto the sample. The sample is placed on a filter-coated CMOS sensor chip. The fluorescence emissions associated with each focal spot are collected by the sensor chip and are composed into a sparsely sampled fluorescence image. By raster scanning the Talbot focal spot grid across the sample and collecting a sequence of sparse images, a filled-in high-resolution fluorescence image can be reconstructed. In contrast to a conventional microscope, a collection efficiency, resolution, and FOV are not tied to each other for this technique. The FOV of FTM is directly scalable. Our FTM prototype has demonstrated a resolution of 1.2 μm, and the collection efficiency equivalent to a conventional microscope objective with a 0.70 N.A. The FOV is 3.9 mm × 3.5 mm, which is 100 times larger than that of a 20X/0.40 N.A. conventional microscope objective. Due to its large FOV, high collection efficiency, compactness, and its potential for integration with other on-chip devices, FTM is suitable for diverse applications, such as point-of-care diagnostics, large-scale functional screens, and long-term automated imaging.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/SF6E-S775, author = {Zheng, Guoan}, title = {Innovations in Imaging System Design: Gigapixel, Chip-Scale and MultiFunctional Microscopy}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/SF6E-S775}, url = {https://resolver.caltech.edu/CaltechTHESIS:10102012-101657790}, abstract = {Microscopy imaging is of fundamental importance in diverse disciplines of science and technology. In a typical microscopy imaging platform, the light path can be generalized to the following steps: photons leave the light source, interact with the sample, and finally are detected by the image sensor. Based on such a light path, this thesis presents several new microscopy imaging techniques from three aspects: illumination design, sample manipulation, and imager modification.
The first design strategy involves the active control of the illumination sources. Based on this strategy, we demonstrate a simple and cost-effective imaging method, termed Non-interferometric Aperture-synthesizing Microscopy (NAM), for breaking the spatial-bandwidth product barrier of a conventional microscope. We show that the NAM method is capable of providing two orders of magnitude higher throughput for most existing bright-field microscopes without involving any mechanical scanning. Based on NAM, we report the implementation of a 1.6 gigapixel microscope with a maximum numerical aperture of 0.5, a field-of-view of 120 mm2, and a resolution-invariant imaging depth of 0.3 mm. This platform is fast (acquisition time of ~ 3 minutes), free from chromatic aberration, capable for phase imaging, and, most importantly, compatible with most existing microscopes. High quality color images of histology slides were acquired by using such a platform for demonstration. The proposed NAM method provides a robust way to transform the problem of high-throughput microscopy from one that is tied to physical limitations of the optics to one that is computationally solvable. The active control of illumination sources can also be adapted for chip-scale microscopy imaging. To this end, we present a lensless microscopy solution termed ePetri-dish. This ePetri-dish platform can automatically perform high resolution (~ 0.66 micron) microscopy imaging over a large field-of-view (6 mm × 4 mm). This new approach is fully capable of working with cell cultures or any samples in which cells/bacteria may be contiguously connected, and thus, it can significantly improve Petri-dish-based cell/bacteria culture experiments. With this approach providing a low-cost and disposable microscopy solution, we can start to transit Petri-dish-based experiments from the traditionally labor-intensive process to an automated and streamlined process.
The second strategy in design considerations is to manipulate the sample. We present a fully on-chip, lensless, sub-pixel resolving optofluidic microscope (SROFM). This device utilizes microfluidic flow to deliver specimens directly across an image sensor to generate a sequence of low-resolution projection images, where resolution is limited by the sensor’s pixel size. This image sequence is then processed to reconstruct a single high-resolution image, where features beyond the Nyquist rate of the LR images are resolved. We demonstrate the device’s capabilities by imaging microspheres, protist Euglena gracilis, and Entamoeba invadens cysts with sub-cellular resolution.
The third accessing point in design considerations is the image sensor. Imager modification is an emerging technique that performs pre-detection light field manipulation. We present two novel optical structure designs: surface-wave-enabled darkfield aperture (SWEDA) and light field sensor. These structures can be directly incorporated onto optical sensors to accomplish pre-detection background suppression and wavefront sensing. We further demonstrate SWEDA’s ability to boost the detection sensitivity, with a contrast enhancement of 27 dB.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/GKW9-QR51, author = {Lee, Lap Man}, title = {The Implementation of Optofluidic Microscopy on a Chip Scale and Its Potential Applications in Biology}, school = {California Institute of Technology}, year = {2012}, doi = {10.7907/GKW9-QR51}, url = {https://resolver.caltech.edu/CaltechTHESIS:10192011-190918785}, abstract = {This thesis presents an effort to miniaturize conventional optical microscopy to a chip level using microfluidic technology. Modern compound microscopes use a set of bulk glass lenses to form magnified images from biological objects. This limits the possibility of shrinking the size of a microscope system. The invention of micro/nanofabrication technology gives hope to engineers who want to rethink the way we build optical microscopes. This advancement can fundamentally reform the way clinicians and biologists conduct microscopy. Optofluidic microscopy (OFM) is a miniaturized optical imaging method which utilizes a microfluidic flow to deliver biological samples across a 1-D or 2-D array of sampling points defined in a microfluidic channel for optical scanning. The optical information of these sampling points is collected by a CMOS imaging sensor on the bottom of the microfluidic channel. Although the size of the OFM device is as small as a US dime, it can render high resolution images of less than 1 μm with quality comparable to that of a bulky, standard optical microscope. OFM has a good potential in various biological applications. For example, the integration of an OFM system with high-speed hydrodynamic focusing technology will allow very large scale imaging-based analysis of cells or microorganisms; the compactness and low cost nature of OFM systems can enable portable or even disposable biomedical diagnostic tools for future telemedicine and personalized health care.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/7Q7B-1E27, author = {McDowell, Emily Jayne}, title = {Low Optical Signal Detection in Biological Materials: SNR Considerations and Novel Techniques}, school = {California Institute of Technology}, year = {2010}, doi = {10.7907/7Q7B-1E27}, url = {https://resolver.caltech.edu/CaltechTHESIS:11012009-234712901}, abstract = {Light scattering poses significant challenges for biomedical optical imaging techniques. Diffuse scattering scrambles wavefront information, confounding easy analysis of signals reflected from or transmitted through biological tissues. For optical imaging techniques that employ only unscattered light components, the penetration depth is severely limited. In this thesis, we develop and discuss two general methods for dealing with large levels of light scattering in tissue. The first involves optimization of the signal-to-noise ratio (SNR) of coherence domain optical tomography techniques. The majority of the signal measured in these techniques is singly scattered. Thus, an improvement in SNR will improve the penetration depth of the system by picking out the weak signal contribution from increasing depths that would otherwise be buried in noise. We show that the SNR can be optimized in terms of image reconstruction algorithms, and in terms of detection parameters. An important detection parameter, the integration time, determines the dominant noise source of the measurement, and can be varied to obtain the maximal SNR. A second general method that will be discussed involves the time-reversal of scattered light components in tissues through the process of optical phase conjugation (OPC). OPC has long been used to remove optical aberrations and distortions, but has never before been applied to light scattering in tissues. We show that we are capable of time reversing light scattering in both chicken tissue sections and tissue phantoms, and characterize both the amplitude and resolution trends of the process. Finally, we provide the first successful results of OPC in living tissues.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/BJY0-NJ69, author = {Cui, Xiquan}, title = {Optofluidic Microscopy and Wavefront Microscopy: Innovations in Biological Imaging}, school = {California Institute of Technology}, year = {2010}, doi = {10.7907/BJY0-NJ69}, url = {https://resolver.caltech.edu/CaltechTHESIS:12032009-160213018}, abstract = {This thesis presents two new microscopic imaging techniques: the optofluidic microscopy (OFM) and the wavefront microscopy (WM). By integrating optical functionalities onto a single semiconductor chip, these inventions could reduce the cost and improve the efficiency and quality of microscopic imaging in biological research and clinical diagnostics. First, OFM utilizes a microfluidic flow to deliver cellular samples across array(s) of micron-sized apertures defined on a metal-coated CMOS image sensor to acquire direct projection images of the samples. Although the OFM prototype is as small as a dime, it can render high resolution images (~1 µm) with comparable quality to those of a bulky standard optical microscope. OFM has great potential in revolutionizing the way we use microscopes. For example, the availability of tens or even hundreds of microscopes on a single chip will allow massively paralleled imaging of large populations of cells or microorganisms; the compactness and low cost of the OFM can enable portable and even disposable biomedical diagnostic tools for future telemedicine and personalized health care. Second, we present a new microscopy concept - WM. Wavefront image sensor (WIS) is the enabling component of WM. By monitoring the tightly confined transmitted light spots through a 2D aperture grid (spacing = 11 µm, diameter = 6 µm) fabricated on a CMOS image sensor in a high Fresnel number regime, we can accurately measure both intensity and phase front variations (a measured normalized phase gradient sensitivity of 0.1 mrad under the typical working condition - 1.0 second total signal accumulation time and 9.2 µW/cm^2 light intensity on the sensor) of a wavefront separately and quantitatively. Therefore, researchers and clinicians can incorporate pure phase imaging into their current microscope systems by simply adding the WIS in place of the conventional camera. When combined with adaptive optics strategies, this technology will facilitate deep tissue imaging using multiphoton microscopy.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/6H07-PA44, author = {Wu, Jigang}, title = {Coherence Domain Optical Imaging Techniques}, school = {California Institute of Technology}, year = {2009}, doi = {10.7907/6H07-PA44}, url = {https://resolver.caltech.edu/CaltechETD:etd-12112008-102138}, abstract = {
Coherence domain optical imaging techniques have been developing quickly in the past few decades after the invention of laser. In this thesis, I will report the imaging methods that constitute my research projects during these years of graduate studies, including paired-angle-rotation scanning (PARS) forward-imaging probe for optical coherence tomography (OCT), full-field phase imaging technique based on harmonically matched diffraction grating (G1G2 grating), and Fresnel zone plate (FZP) based optifluidic microscopy (OFM). Compared with conventional optical microscopy, the coherence domain optical imaging has many advantages and greatly extends the application of imaging techniques.
OCT, based on low-coherence interferometry, is a high-resolution imaging technique that has been successfully applied to many biomedical applications. The development of various probes for OCT further made this technique applicable to endoscopic imaging. In the project of PARS-OCT probe, I have developed a forward-imaging probe based on two rotating angle-cut GRIN lenses. The diameter of the first prototype PARS-OCT probe that I made is 1.65 mm. My colleagues further built a probe with diameter of 0.82 mm. To our knowledge, this is the smallest forward-imaging probe that has been reported. The first prototype probe was characterized and successfully used to acquire OCT images of a Xenopus laevis tadpole.
Full-field phase imaging techniques are important for metrology and can also obtain high-resolution images for biological samples, especially transparent samples such as living cells. We have developed a novel full-field phase imaging technique based on the G1G2 grating. The G1G2 interferometry uses the G1G2 grating as a beam splitter/combiner and can confer nontrivial phase shift between output interference signals. Thus the phase and intensity information of the sample can be obtained by processing the two direct CCD images acquired at the output ports of the G1G2 grating. The details of this technique are explained in this thesis, and the phase imaging results for standard phase objects and biological samples are also shown.
OFM is a novel high-resolution and low-cost chip-level microscope developed by our group several years ago. Combining the unique imaging concept and microfluidic techniques, OFM system can be potentially useful to many biomedical applications, such as cytometry, blood parasite diagnosis, and water quality inspection. In the project of FZP-OFM, I applied the FZP to project the OFM aperture array onto an imaging sensor for OFM imaging. In this way, the sensor and the aperture array can be separated and will be useful for some situations. To demonstrate its capability, the FZP-OFM system was used to acquire OFM images of the protist Euglena gracilis.
The studies in my research show the possibility of the application of various coherence domain optical imaging techniques in biomedical area, which is the primary objective of this thesis.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, } @phdthesis{10.7907/JAYF-RX26, author = {Heng, Xin}, title = {Optofluidic Microscopy: Technology Development and Its Applications in Biology}, school = {California Institute of Technology}, year = {2008}, doi = {10.7907/JAYF-RX26}, url = {https://resolver.caltech.edu/CaltechETD:etd-12182007-163333}, abstract = {The Optofluidic Microscope (OFM) is a new imaging platform based upon nanoapertures that are fabricated on planar metallic film, whilst microfluidic delivery technology is used to transport the objects-of-interest. The planar nature of OFM makes it ideal to integrate with other micro total analysis systems, such as cell sorters or cell culturing chambers. Furthermore, a variety of imaging functionalities, such as differential phase contrast, fluorescence, and Raman spectroscopy can potentially fit into a single OFM device.
This thesis reports on the early technology development of Optofluidic Microscopy. I have built a variety of off-chip prototypes of OFM that all possess different functionalities. These OFM prototypes include 1D array OFM, hydraulically pumped OFM, 2D nanoaperture grid OFM, super high-resolution OFM, OFM coupled with optical tweezer actuation, fluorescent OFM, electrokinetic enabled OFM, etc.
I applied the first OFM prototype in imaging Caenorhabditis elegans (C. elegans) larvae and characterizing different genotypes. Later on, the microscopy properties of OFM, such as the optical resolution and the depth of field, were thoroughly investigated both experimentally and theoretically. More recently, I successfully combined optical tweezers with a grid-based OFM prototype, which was then used in high-resolution imaging of microspheres and a few biological samples. In addition, preliminary results on fluorescence OFM imaging were also demonstrated.
I trust that these functionalities, after being demonstrated off-chip, can be readily fabricated and then assembled as a complete on-chip OFM. It will eventually enable a real “microscale microscope on a chip”.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Yang, Changhuei}, }