Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

Grain growth in protoplanetary disks

The majority of young, low-mass stars are surrounded by optically thick accretion disks. These circumstellar disks provide large reservoirs of gas and dust that will eventually be transformed into planetary systems. Theory and observations suggest that the earliest stage toward planet formation in a protoplanetary disk is the growth of particles, from sub-micron-sized grains to centimeter- sized pebbles. Theory indicates that small interstellar grains are well coupled into the gas and are incorporated to the disk during the proto-stellar collapse. These dust particles settle toward the disk mid-plane and simultaneously grow through collisional coagulation in a very short timescale. Observationally, grain growth can be inferred by measuring the spectral energy distribution at long wavelengths, which traces the continuum dust emission spectrum and hence the dust opacity. Several observational studies have indicated that the dust component in protoplanetary disks has evolved as compared to interstellar medium dust particles, suggesting at least 4 orders of magnitude in particle- size growth. However, the limited angular resolution and poor sensitivity of previous observations has not allowed for further exploration of this astrophysical process.

As part of my thesis, I embarked in an observational program to search for evidence of radial variations in the dust properties across a protoplanetary disk, which may be indicative of grain growth. By making use of high angular resolution observations obtained with CARMA, VLA, and SMA, I searched for radial variations in the dust opacity inside protoplanetary disks. These observations span more than an order of magnitude in wavelength (from sub-millimeter to centimeter wavelengths) and attain spatial resolutions down to 20 AU. I characterized the radial distribution of the circumstellar material and constrained radial variations of the dust opacity spectral index, which may originate from particle growth in these circumstellar disks. Furthermore, I compared these observational constraints with simple physical models of grain evolution that include collisional coagulation, fragmentation, and the interaction of these grains with the gaseous disk (the radial drift problem). For the parameters explored, these observational constraints are in agreement with a population of grains limited in size by radial drift. Finally, I also discuss future endeavors with forthcoming ALMA observations.

Understanding planet formation through high precision photometry and spectroscopy

From studies of protoplanetary disks to extrasolar planets and planetary debris, we aim to understand the full evolution of a planetary system. Observational constraints from ground- and space-based instrumentation allows us to measure the properties of objects near and far and are central to developing this understanding. We present here three observational campaigns that, when combined with theoretical models, reveal characteristics of different stages and remnants of planet formation. The Kuiper Belt provides evidence of chemical and dynamical activity that reveals clues to its primordial environment and subsequent evolution. Large samples of this population can only be assembled at optical wavelengths, with thermal measurements at infrared and sub-mm wavelengths currently available for only the largest and closest bodies. We measure the size and shape of one particular object precisely here, in hopes of better understanding its unique dynamical history and layered composition.

Molecular organic chemistry is one of the most fundamental and widespread facets of the universe, and plays a key role in planet formation. A host of carbon-containing molecules vibrationally emit in the near-infrared when excited by warm gas, T~1000 K. The NIRSPEC instrument at the W.M. Keck Observatory is uniquely configured to study large ranges of this wavelength region at high spectral resolution. Using this facility we present studies of warm CO gas in protoplanetary disks, with a new code for precise excitation modeling. A parameterized suite of models demonstrates the abilities of the code and matches observational constraints such as line strength and shape. We use the models to probe various disk parameters as well, which are easily extensible to others with known disk emission spectra such as water, carbon dioxide, acetylene, and hydrogen cyanide.

Lastly, the existence of molecules in extrasolar planets can also be studied with NIRSPEC and reveals a great deal about the evolution of the protoplanetary gas. The species we observe in protoplanetary disks are also often present in exoplanet atmospheres, and are abundant in Earth's atmosphere as well. Thus, a sophisticated telluric removal code is necessary to analyze these high dynamic range, high-resolution spectra. We present observations of a hot Jupiter, revealing water in its atmosphere and demonstrating a new technique for exoplanet mass determination and atmospheric characterization. We will also be applying this atmospheric removal code to the aforementioned disk observations, to improve our data analysis and probe less abundant species. Guiding models using observations is the only way to develop an accurate understanding of the timescales and processes involved. The futures of the modeling and of the observations are bright, and the end goal of realizing a unified model of planet formation will require both theory and data, from a diverse collection of sources.

From molecules to organs : Microscopy and multi-scale nature of development

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.

Low-noise THz Niobium SIS Mixers

This thesis describes the development of low-noise heterodyne receivers at THz frequencies for submillimeter astronomy using Nb-based superconductor-insulator-superconductor (SIS) tunneling junctions. The mixers utilize a quasi-optical configuration which consists of a planar twin-slot antenna and antisymmetrically-fed two-junctions on an antireflection-coated silicon hyperhemispherical lens. On-chip integrated tuning circuits, in the form of microstrip lines, are used to obtain maximum coupling efficiency in the designed frequency band. To reduce the rf losses in the integrated tuning circuits above the superconducting Nb gap frequency (~ 700 GHz), normal-metal Al is used to replace Nb as the tuning circuits.

To account the rf losses in the micros trip lines, we calculated the surface impedance of the AI films using the nonlocal anomalous skin effect for finite thickness films. Nb films were calculated using the Mattis-Bardeen theory in the extreme anomalous limit. Our calculations show that the losses of the Al and Nb microstrip lines are about equal at 830 GHz. For Al-wiring and Nb-wiring mixers both optimized at 1050 GHz, the RF coupling efficiency of Al-wiring mixer is higher than that of Nb-wiring one by almost 50%. We have designed both Nb-wiring and Al-wiring mixers below and above the gap frequency.

A Fourier transform spectrometer (FTS) has been constructed especially for the study of the frequency response of SIS receivers. This FTS features large aperture size (10 inch) and high frequency resolution (114 MHz). The FTS spectra, obtained using the SIS receivers as direct detectors on the FTS, agree quite well with our theoretical simulations. We have also, for the first time, measured the FTS heterodyne response of an SIS mixer at sufficiently high resolution to resolve the LO and the sidebands. Heterodyne measurements of our SIS receivers with Nb-wiring or Al-wiring have yielded results which arc among the best reported to date for broadband heterodyne receivers. The Nb-wiring mixers, covering 400 - 850 GHz band with four separate fixed-tuned mixers, have uncorrected DSB receiver noise temperature around 5hv/k_b to 700 GHz, and better than 540 K at 808 GHz. An Al-wiring mixer designed for 1050 GHz band has an uncorrected DSB receiver noise temperature 840 K at 1042 GHz and 2.5 K bath temperature. Mixer performance analysis shows that Nb junctions can work well up to twice the gap frequency and the major cause of loss above the gap frequency is the rf losses in the microstrip tuning structures. Further advances in THz SIS mixers may be possible using circuits fabricated with higher-gap superconductors such as NbN. However, this will require high-quality films with low RF surface resistance at THz frequencies.

Volatiles in Protoplanetary Disks

Planets are assembled from the gas, dust, and ice in the accretion disks that encircle young stars. Ices of chemical compounds with low condensation temperatures (<200 K), the so-called volatiles, dominate the solid mass reservoir from which planetesimals are formed and are thus available to build the protoplanetary cores of gas/ice giant planets. It has long been thought that the regions near the condensation fronts of volatiles are preferential birth sites of planets. Moreover, the main volatiles in disks are also the main C-and O-containing species in (exo)planetary atmospheres. Understanding the distribution of volatiles in disks and their role in planet-formation processes is therefore of great interest.

This thesis addresses two fundamental questions concerning the nature of volatiles in planet-forming disks: (1) how are volatiles distributed throughout a disk, and (2) how can we use volatiles to probe planet-forming processes in disks? We tackle the first question in two complementary ways. We have developed a novel super-resolution method to constrain the radial distribution of volatiles throughout a disk by combining multi-wavelength spectra. Thanks to the ordered velocity and temperature profiles in disks, we find that detailed constraints can be derived even with spatially and spectrally unresolved data -- provided a wide range of energy levels are sampled. We also employ high-spatial resolution interferometric images at (sub)mm frequencies using the Atacama Large Millimeter Array (ALMA) to directly measure the radial distribution of volatiles.

For the second question, we combine volatile gas emission measurements with those of the dust continuum emission or extinction to understand dust growth mechanisms in disks and disk instabilities at planet-forming distances from the central star. Our observations and models support the idea that the water vapor can be concentrated in regions near its condensation front at certain evolutionary stages in the lifetime of protoplanetary disks, and that fast pebble growth is likely to occur near the condensation fronts of various volatile species.

Wide Field-of-view Microscopes and Endoscopes for Time-lapse Imaging and High-throughput Screening

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.