Browsing by Subject "Super-resolution"

The largest obstacle in nanoscale microscopy is the diffraction limit. Although several means of achieving sub-diffraction resolution exist, they all have shortcomings such as cost, complexity, and processing time, which make them impractical for widespread use. Additionally, these technologies struggle to find a balance between a high resolution and a large field of view. In this introduction of dissertation, we provide an overview of various microsphere based super resolution techniques that address the shortcomings of existing platforms and consistently achieve sub-diffraction resolutions. Initially, the theoretical basis of photonic nanojets, which make microsphere based super resolution imaging possible, are discussed. In the following sections, different type of acoustofluidic scanning techniques and intelligent nanoscope are explored. The introduction concludes with an emphasis on the limitless potential of this technology, and the wide range of possible biomedical applications.First, we have documented the development of an acoutofluidic scanning nanoscope that can achieve both high resolution and large field of view at the same time, which alleviates a long-existing shortcoming of conventional microscopes. The acoutofluidic scanning nanoscope developed here can serve as either an add-on component to expand the capability of a conventional microscope, or could be paired with low-cost imaging platforms to develop a stand-alone microscope for portable imaging. The acoutofluidic scanning nanoscope achieves high-resolution imaging without the need for conventional high-cost and bulky objectives with high numerical apertures. The field of view of the acoutofluidic scanning nanoscope is much larger than that from a conventional high numerical aperture objective lens, and it is able to achieve the same resolving power. The acoutofluidic scanning nanoscope automatically focuses and maintains a constant working distance during the scanning process thanks to the interaction of the microparticles with the liquid domain. The resolving power of the acoutofluidic scanning nanoscope can easily be adjusted by using microparticles of different sizes and refractive indices. Additionally, it may be possible to further improve the performance of this platform by exploring additional microparticle sizes and materials, in combination with various objectives. Altogether, we believe this acoutofluidic scanning nanoscope has potential to be integrated into a wide range of applications from portable nano-detection to biomedicine and microfluidics. Next, we developed a dual-camera acoustofluidic nanoscope with a seamless image merging algorithm (alpha blending process). This design allows us to precisely image both the sample and the microspheres simultaneously and accurately track the particle path and location. Therefore, the number of images required to capture the entire field of view (200 × 200 μm) by using our acoustofluidic scanning nanoscope is reduced by 55-fold compared with previous designs. Moreover, the image quality is also greatly improved by applying an alpha blending imaging technique, which is critical for accurately depicting and identifying nanoscale objects or processes. This dual-camera acoustofluidic nanoscope paves the way for enhanced nanoimaging with high resolution and a large field of view. Next, we developed an acoustofluidic scanning nanoscope via fluorescence amplification technique. Nanoscale fluorescence signal amplification is a significant feature for many biomedical and cell biology research area. Different types of fluorescence amplification techniques were studied; however, those technologies still need a complex process and rely on an elaborate optical system. To conquer these limitations, we developed an acoustofluidic scanning nanoscope via fluorescence amplification with hard PDMS membrane technique. The microsphere magnification by photonic nanojets effect with the hard PDMS could deliver certain focal distance to maximize the amplification. Moreover, a bidirectional acoustofluidic scanning device with an image processing also developed to perform 2D scanning of large field of view area. In the image processing procedure, we applied a correction of lens distortion to provide a restored distortion image. This fluorescence amplification via acoustofluidic nanoscope allow us to afford a nanoscale fluorescence imaging. Next, we developed an intelligent nanoscope that combines machine learning and microsphere array-based imaging to: (1) surpass the diffraction limit of the microscope objective with microsphere imaging to provide high-resolution images; (2) provide large field-of-view imaging without the sacrifice of resolution by utilizing a microsphere array; and (3) rapidly classify nanomaterials using a deep convolution neural network. The intelligent nanoscope delivers more than 46 magnified images from a single image frame so that we collected more than 1,000 images within 2 seconds. Moreover, the intelligent nanoscope achieves a 95% nanomaterial classification accuracy using 1,000 images of training sets, which is 45% more accurate than without the microsphere array. The intelligent nanoscope also achieves a 92% bacteria classification accuracy using 50,000 images of training sets, which is 35% more accurate than without the microsphere array. This platform accomplished rapid, accurate detection and classification of nanomaterials with miniscule size differences. The capabilities of this device wield the potential to further detect and classify smaller biological nanomaterial, such as viruses or extracellular vesicles. Lastly, this chapter serves a conclusion. Here, I discuss current issues regarding the acoustofluidic scanning nanoscope across review the current limitations of the technology and give suggestions for different direction of microsphere imaging. Moreover, I provide my perspective on the future development of acoustofluidic scanning nanoscope and potential new applications. I discuss how the technologies developed in this dissertation can be improved and applied to new applications in nanoimaging.

FtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that plays key roles in cytokinesis of almost all prokaryotes. FtsZ assembles into protofilaments (pfs), one subunit thick, and these pfs assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. FtsZ can be subdivided into 3 main functional regions: globular domain, C terminal (Ct) linker, and Ct peptide. The globular domain binds GTP to assembles the pfs. The extreme Ct peptide binds membrane proteins to allow cytoplasmic FtsZ to function at the inner membrane. The Ct linker connects the globular domain and Ct peptide. In the present studies, we used genetic and structural approaches to investigate the function of Escherichia coli (E. coli) FtsZ. We sought to examine three questions: (1) Are lateral bonds between pfs essential for the Z ring? (2) Can we improve direct visualization of FtsZ in vivo by engineering an FtsZ-FP fusion that can function as the sole source of FtsZ for cell division? (3) Is the divergent Ct linker of FtsZ an intrinsically disordered peptide (IDP)?

One model of the Z ring proposes that pfs associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of E. coli FtsZ by inserting either small peptides or whole FPs. Of the four lateral surfaces on FtsZ pfs, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and were identified as possible sites for essential lateral interactions. Another goal was to find a location within FtsZ that supported fusion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques.

The Ct linker is the most divergent region of FtsZ in both sequence and length. In E. coli FtsZ the Ct linker is 50 amino acids (aa), but for other FtsZ it can be as short as 37 aa or as long as 250 aa. The Ct linker has been hypothesized to be an IDP. In the present study, circular dichroism confirmed that isolated Ct linkers of E. coli (50 aa) and C. crescentus (175 aa) are IDPs. Limited trypsin proteolysis followed by mass spectrometry (LC-MS/MS) confirmed Ct linkers of E. coli (50 aa) and B. subtilis (47 aa) as IDPs even when still attached to the globular domain. In addition, we made chimeras, swapping the E. coli Ct linker for other peptides and proteins. Most chimeras allowed for normal cell division in E. coli, suggesting that IDPs with a length of 43 to 95 aa are tolerated, sequence has little importance, and electrostatic charge is unimportant. Several chimeras were purified to confirm the effect they had on pf assembly. We concluded that the Ct linker functions as a flexible tether allowing for force to be transferred from the FtsZ pf to the membrane to constrict the septum for division.

Optical microscopy plays a crucial role in the biological sciences for its ability to enable visualization of biological samples at sub-cellular levels. Many imaging subdivisions exist under this umbrella of general microscopy, and each are tailored towards specific design, contrast, and visualization constraints. Standard examples that have found widespread use include dark-field, phase-contrast, holographic, and fluorescent microscopies. However, a critical factor that physically limits the optical resolution of general microscopy is diffraction. Unfortunately, this “diffraction-limit” can prevent visualization of significant biologically relevant structures, which in turn can limit biological insights. In response to such a limit, several works have advanced the field of sub-diffraction resolution imaging, which consist of optical imaging techniques that seek to achieve imaging resolutions beyond that which is allowed by the diffraction-limit. This set of techniques can largely be divided into two classes. The first class of sub-diffraction techniques is targeted towards cases where the sample is coherently illuminated and diffracts into the imaging system’s aperture. For such cases, synthetic aperture (SA) is a popular choice and operates by using oblique illuminations to spatiotemporally synthesize a wider frequency support into the image than allowed by the diffraction limit. The second class of sub-diffraction techniques, often referred to as "super-resolution" techniques, typically utilize specialized fluorophores with either photoswitching or depletion capabilities. Photoactivated localization microscopy (PALM) is a super-resolution example that localizes photoswitchable fluorophores to sub-diffraction resolutions per acquisition, before combining into a final super-resolved image. Stimulated emission depletion (STED) is another super-resolution example that spatially modulates its excitation to narrow its optical point-spread-function. Unfortunately, SA and fluorescent super-resolution techniques are generally incompatible for sub-diffraction resolution fluorescent and coherent imaging, respectively – thus, a multimodal sub-diffraction imaging solution compatible with both coherent and fluorescent imaging has remained elusive.

In this dissertation, we demonstrate that structured illumination (SI) is a sub-diffraction technique compatible with both diffractive and fluorescent imaging. We first develop the theoretical framework that extends SI to coherent imaging and experimentally demonstrate SI’s capabilities for 2D sub-diffraction resolution imaging of coherently diffractive samples. Sub-diffraction resolution imaging based on scattering intensity and transmission-based quantitative-phase (QP) are shown. In addition, we show extend SI to 3D coherent imaging, and show applications of this towards 3D QP and refractive-index (RI) tomography. Finally, we show multimodal applications of SI that allow sub-diffraction resolution fluorescent and coherent imaging, which has great potential utility for the biological sciences.