Comparative performance of airyscan and structured illumination superresolution microscopy in the study of the surface texture and 3D shape of pollen

The visualization of taxonomically diagnostic features of individual pollen grains can be a challenge for many ecologically and phylogenetically important pollen types. The resolution of traditional optical microscopy is limited by the diffraction of light (250 nm), while high resolution tools such as electron microscopy are limited by laborious preparation and imaging workflows. Airyscan confocal superresolution and structured illumination superresolution (SR-SIM) microscopy are powerful new tools for the study of nanoscale pollen morphology and three-dimensional structure that can overcome these basic limitations. This study demonstrates their utility in capturing morphological details below the diffraction limit of light. Using three distinct pollen morphotypes (Croton hirtus, Dactylis glomerata, and Helianthus sp.) and contrast-enhancing fluorescent staining, we were able to assess the effectiveness of the Airyscan and SR-SIM. We further demonstrate that these new superresolution methods can be easily applied to the study of fossil pollen material

Exploiting flow dynamics for super-resolution in contrast-enhanced ultrasound

Ultrasound localization microscopy offers new radiation-free diagnostic tools for vascular imaging deep within the tissue. Sequential localization of echoes returned from inert microbubbles with low-concentration within the bloodstream reveal the vasculature with capillary resolution. Despite its high spatial resolution, low microbubble concentrations dictate the acquisition of tens of thousands of images, over the course of several seconds to tens of seconds, to produce a single super-resolved image. %since each echo is required to be well separated from adjacent microbubbles. Such long acquisition times and stringent constraints on microbubble concentration are undesirable in many clinical scenarios. To address these restrictions, sparsity-based approaches have recently been developed. These methods reduce the total acquisition time dramatically, while maintaining good spatial resolution in settings with considerable microbubble overlap. %Yet, non of the reported methods exploit the fact that microbubbles actually flow within the bloodstream. % to improve recovery. Here, we further improve sparsity-based super-resolution ultrasound imaging by exploiting the inherent flow of microbubbles and utilize their motion kinematics. While doing so, we also provide quantitative measurements of microbubble velocities. Our method relies on simultaneous tracking and super-localization of individual microbubbles in a frame-by-frame manner, and as such, may be suitable for real-time implementation. We demonstrate the effectiveness of the proposed approach on both simulations and {\it in-vivo} contrast enhanced human prostate scans, acquired with a clinically approved scanner.Comment: 11 pages, 9 figure

Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells

Methods that enable the super-resolution imaging of intracellular proteins in live bacterial cells provide powerful tools for the study of prokaryotic cell biology. Photoswitchable organic dyes exhibit many of the photophysical properties needed for super-resolution imaging, including high brightness, photostability, and photon output, but most such dyes require organisms to be fixed and permeabilized if intracellular targets are to be labeled. We recently reported a general strategy for the chemoenzymatic labeling of bacterial proteins with azide-bearing fatty acids in live cells using the eukaryotic enzyme N-myristoyltransferase. Here we demonstrate the labeling of proteins in live Escherichia coli using cell-permeant bicyclononyne-functionalized photoswitchable rhodamine spirolactams. Single-molecule fluorescence measurements on model rhodamine spirolactam salts show that these dyes emit hundreds of photons per switching event. Super-resolution imaging was performed on bacterial chemotaxis proteins Tar and CheA and cell division proteins FtsZ and FtsA. High-resolution imaging of Tar revealed a helical pattern; imaging of FtsZ yielded banded patterns dispersed throughout the cell. The precision of radial and axial localization in reconstructed images approaches 15 and 30 nm, respectively. The simplicity of the method, which does not require redox imaging buffers, should make this approach broadly useful for imaging intracellular bacterial proteins in live cells with nanometer resolution

Studies of Single-Molecule Dynamics in Microorganisms

Fluorescence microscopy is one of the most extensively used techniques in the life sciences. Considering the non-invasive sample preparation, enabling live-cell compliant imaging, and the specific fluorescence labeling, allowing for a specific visualization of virtually any cellular compound, it is possible to localize even a single molecule in living cells. This makes modern fluorescence microscopy a powerful toolbox. In the recent decades, the development of new, "super-resolution" fluorescence microscopy techniques, which surpass the diffraction limit, revolutionized the field. Single-Molecule Localization Microscopy (SMLM) is a class of super-resolution microscopy methods and it enables resolution of down to tens of nanometers. SMLM methods like Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), Ground-State Depletion followed by Individual Molecule Return (GSDIM) and Point Accumulation for Imaging in Nanoscale Topography (PAINT) have allowed to investigate both, the intracellular spatial organization of proteins and to observe their real-time dynamics at the single-molecule level in live cells. The focus of this thesis was the development of novel tools and strategies for live-cell SingleParticle Tracking PALM (sptPALM) imaging and implementing them for biological research. In the first part of this thesis, I describe the development of new Photoconvertible Fluorescent Proteins (pcFPs) which are optimized for sptPALM lowering the phototoxic damage caused by the imaging procedure. Furthermore, we show that we can utilize them together with Photoactivatable Fluorescent Proteins (paFPs) to enable multi-target labeling and read-out in a single color channel, which significantly simplifies the sample preparation and imaging routines as well as data analysis of multi-color PALM imaging of live cells. In parallel to developing new fluorescent proteins, I developed a high throughput data analysis pipeline. I have implemented this pipeline in my second project, described in the second part of this thesis, where I have investigated the protein organization and dynamics of the CRISPR-Cas antiviral defense mechanism of bacteria in vivo at a high spatiotemporal level with the sptPALM approach. I was successful to show the differences in the target search dynamics of the CRISPR effector complexes as well as of single Cas proteins for different target complementarities. I have also first data describing longer-lasting bound-times between effector complex and their potential targets in vivo, for which only in vitro data has been available till today. In summary, this thesis is a significant contribution for both, the advances of current sptPALM imaging methods, as well as for the understanding of the native behavior of CRISPR-Cas systems in vivo

Subaru and Gemini High Spatial Resolution Infrared 18 Micron Imaging Observations of Nearby Luminous Infrared Galaxies

We present the results of a ground-based, high spatial resolution infrared 18 micron imaging study of nearby luminous infrared galaxies (LIRGs), using the Subaru 8.2-m and Gemini South 8.1-m telescopes. The diffraction-limited images routinely achieved with these telescopes in the Q-band (17-23 micron) allow us to investigate the detailed spatial distribution of infrared emission in these LIRGs. We then investigate whether the emission surface brightnesses are modest, as observed in starbursts, or are so high that luminous active galactic nuclei (AGNs; high emission surface brightness energy sources) are indicated. The sample consists of 18 luminous buried AGN candidates and starburst-classified LIRGs identified in earlier infrared spectroscopy. We find that the infrared 18 micron emission from the buried AGN candidates is generally compact, and the estimated emission surface brightnesses are high, sometimes exceeding the maximum value observed in and theoretically predicted for a starburst phenomenon. The starburst-classified LIRGs usually display spatially extended 18 micron emission and the estimated emission surface brightnesses are modest, within the range sustained by a starburst phenomenon. The general agreement between infrared spectroscopic and imaging energy diagnostic methods suggests that both are useful tools for understanding the hidden energy sources of the dusty LIRG population.Comment: 17 pages, 3 figures, accepted for publication in AJ (No. 141, 2011 May issue). Higher resolution version is available at http://optik2.mtk.nao.ac.jp/~imanishi/Paper/20um/20um.pd

Cryoimaging-Microscopy Implementation for 3D Optical Imaging

The structures and biochemistry properties of biological tissues are mostly affected by diseases. The visualization of organ structure and biochemistry helps in early detection and progression monitoring of diseases. Although, 2D imaging has traditionally been used to gain information from the tissue, it does not accurately represent many of the structures and functions. There currently exists a need for sensitive and specific methods to show detailed information about the structure of the tissue with high resolution and in 3D. The potential advantage of the high resolution 3D images is the ability to accurately probe structural and biochemical properties of the tissue. Not only the changes in structure, but also the changes in temporal physiological responses affected by oxidative stress (OS) at cellular levels. Thus, it would be valuable to detect the cellular metabolic states, which play a key role in understanding the pathogenesis of the disease, and to develop instruments to detect high resolution 3D images of the tissue. The objective of this research is to develop a second generation fluorescence optical imaging instrument to image the cellular redox state in 3D, in control and diseases conditions. I have improved upon one of optical instrument, called cryoimager software and hardware wise to enable higher resolution images. This higher resolution imaging resembles the microscopy capability in cryo temperatures for high resolution 3D imaging. In conclusion, high resolution optical instrumentation combined with signal and image processing tools provide quantitative physiological and structural information of diseased tissue

Advancing fluorescent contrast agent recovery methods for surgical guidance applications

Fluorescence-guided surgery (FGS) utilizes fluorescent contrast agents and specialized optical instruments to assist surgeons in intraoperatively identifying tissue-specific characteristics, such as perfusion, malignancy, and molecular function. In doing so, FGS represents a powerful surgical navigation tool for solving clinical challenges not easily addressed by other conventional imaging methods. With growing translational efforts, major hurdles within the FGS field include: insufficient tools for understanding contrast agent uptake behaviors, the inability to image tissue beyond a couple millimeters, and lastly, performance limitations of currently-approved contrast agents in accurately and rapidly labeling disease. The developments presented within this thesis aim to address such shortcomings. Current preclinical fluorescence imaging tools often sacrifice either 3D scale or spatial resolution. To address this gap in high-resolution, whole-body preclinical imaging tools available, the crux of this work lays on the development of a hyperspectral cryo-imaging system and image-processing techniques to accurately recapitulate high-resolution, 3D biodistributions in whole-animal experiments. Specifically, the goal is to correct each cryo-imaging dataset such that it becomes a useful reporter for whole-body biodistributions in relevant disease models. To investigate potential benefits of seeing deeper during FGS, we investigated short-wave infrared imaging (SWIR) for recovering fluorescence beyond the conventional top few millimeters. Through phantom, preclinical, and clinical SWIR imaging, we were able to 1) validate the capability of SWIR imaging with conventional NIR-I fluorophores, 2) demonstrate the translational benefits of SWIR-ICG angiography in a large animal model, and 3) detect micro-dose levels of an EGFR-targeted NIR-I probe during a Phase 0 clinical trial. Lastly, we evaluated contrast agent performances for FGS glioma resection and breast cancer margin assessment. To evaluate glioma-labeling performance of untargeted contrast agents, 3D agent biodistributions were compared voxel-by-voxel to gold-standard Gd-MRI and pathology slides. Finally, building on expertise in dual-probe ratiometric imaging at Dartmouth, a 10-pt clinical pilot study was carried out to assess the technique’s efficacy for rapid margin assessment. In summary, this thesis serves to advance FGS by introducing novel fluorescence imaging devices, techniques, and agents which overcome challenges in understanding whole-body agent biodistributions, recovering agent distributions at greater depths, and verifying agents’ performance for specific FGS applications

A framework for the statistical analysis of mass spectrometry imaging experiments

Mass spectrometry (MS) imaging is a powerful investigation technique for a wide range of biological applications such as molecular histology of tissue, whole body sections, and bacterial films , and biomedical applications such as cancer diagnosis. MS imaging visualizes the spatial distribution of molecular ions in a sample by repeatedly collecting mass spectra across its surface, resulting in complex, high-dimensional imaging datasets. Two of the primary goals of statistical analysis of MS imaging experiments are classification (for supervised experiments), i.e. assigning pixels to pre-defined classes based on their spectral profiles, and segmentation (for unsupervised experiments), i.e. assigning pixels to newly discovered segments with relatively homogenous and distinct spectral profiles. To accomplish these goals, this research provides both statistical methods and statistical computing tools. First, we propose a novel spatial shrunken centroids framework for performing classification and segmentation of MS imaging experiments with feature selection. Spatial shrunken centroids combines spatial smoothing with statistical regularization in a model-based framework appropriate for both supervised and unsupervised settings. Second, we provide Cardinal, a free and open-source R package for processing, visualization, and statistical analysis of MS imaging experiments. Cardinal is the first R package designed specifically for MS imaging, and the first software for MS imaging that focuses specifically on experiments and statistical analysis. In addition to providing tools for statistical analysis, it also provides infrastructure to enable other statisticians to more easily develop new methods for MS imaging experiments. Lastly, to enable scalability of Cardinal to larger-than-memory datasets, we provide matter, a free and open-source R package for statistical computing with structured datasets-on-disk, such as MS imaging data files. Together, spatial shrunken centroids, Cardinal, and matter aim to allow scalable statistical analysis for high-resolution, high-throughput MS imaging experiments