Imaging Oral Presentations
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Session Type: Innovative Topics
Molecular Characterization of Leading Edge Protrusions in the Absence of Functional Arp2/3 Complex.Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. Classical models of leading edge protrusion rely on a treadmilling dendritic actin network that undergoes continuous assembly nucleated by the Arp2/3 complex, forming ruffling lamellipodia. Although the dendritic nucleation model has been rigorously evaluated in several computational studies, experimental evidence demonstrating a critical role for Arp2/3 in the generation of protrusive actin structures and cell motility has been far from clear. Most components of the pathway have been probed for their relevance by RNA interference or dominant-negative constructs. However, given that the Arp2/3 complex nucleates actin at nanomolar concentrations, even a dramatic knockdown could still leave behind a level sufficient to fully or partially support Arp2/3 complex-dependent functions. Our recent work renders the characterization of fibroblasts cells lacking functional the Arp2/3 complex. Characterization of the impact of the absence of functional Arp2/3 complex on these genetically matched cells included single cell spreading assays, wound healing assays, long-time single cell motility tracking, chemotaxis assays, fluorescence staining imaging with confocal or structured illumination microscopy [1.2]. ARPC3-/- fibroblasts maintained an ability to move but exhibited a strong defect in persistent directional migration in both wound healing and chemotaxis assays, while migrating at rates similar to wild-type cells. Here, we will highlight our advances on determining the molecular-level organization of the leading edge actin networks, through an integrated approach that employs electron cryo-tomography of whole mammalian cells in conjunction with correlative light microscopy. We show by correlative fluorescence and cryo-tomography that the nanometer-scale actin-network organization of smooth lamellipodia in wild-type cells is replaced by massive, bifurcating actin-based protrusions with fractal geometry linked to self-organized criticality. Agent-based modeling shows that the Arp2/3 complex suppresses the formation of these protrusions by locally fine-tuning actin network morphology, providing the switch for directional movement. References: 1. Suraneni P, Rubinstein B, Unruh JR, Durnin M, Hanein, D, and Li R (2012). The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. J Cell Biol. 197, 239-251.(2012) 2. Suraneni1 P, Fogelson B, Rubinstein B, Noguera P, Volkmann N, Hanein D, Mogilner A, Li R (2015). A mechanism of leading edge protrusion in the absence of Arp2/3 complex. Mol Biol Cell (2015). 3. Anderson KL, Page C, Swift MF, Suraneni P, Janssen ME, Pollard TD, Li R, Volkmann N, Hanein D. Nano-scale actin-network characterization of fibroblast cells lacking functional Arp2/3 complex. J Struct Biol. (2016). 4. This work was supported by NIH program project grant P01 GM098412 and R01CA179087 (DH, NV). NIH grants S10 OD012372 (DH) and P01 GM098412-S1(DH) funded the purchase of the Titan Krios TEM and Falcon II direct detection imaging device.
Session Type: Workshop
Optogenetic probes to reveal and control cell signallingBiomolecular engineering of improved fluorescent proteins (FPs) and innovative FP-based probes has been a major driving force behind advances in cell biology and neuroscience for the past two decades. Among these tools, FP-based reporters (i.e., FP-containing proteins that change their fluorescence intensity or color in response to a biochemical change) have uniquely revolutionized the ability of biologists to ‘see’ the otherwise invisible world of intracellular biology and neural signalling. In this seminar I will describe our most recent efforts to use protein engineering to make a new generation of versatile FP-based tools optimized for in vivo imaging of neural activity. Specifically, I will present our efforts to convert red and near-infrared FPs into reporters for calcium ion, membrane potential, and neurotransmitters. In addition, I will briefly describe our most recent efforts to exploit FPs for optogenetic control of protein activity and gene expression.
Lattice light sheet microscope: Technical considerations for core facilitiesLight sheet microscopy, one of the most significant technological advances in recent years in optical microscopy, has revolutionized the ability of biologists to visualize dynamic life processes in multicellular systems. By employing plane illumination, this technique greatly minimizes light exposure to the sample as it limits the excitation light to the focal plane. However, the field of view of a plane illumination system practically varies with the thickness of light sheet created by conventional, Gaussian beam laser. To achieve a reasonable field of view, Gaussian light sheets are too thick to facilitate the necessary resolution for subcellular imaging. By switching to Bessel beam laser, Chen et al. (Science 346: 1257998, 2014) creates a new kind of light sheets based on optical lattices that can be much thinner, achieving unprecedented axial resolution as well as signal-to-noise ratio. This breakthrough subsequently facilitated the engineering of a new class of plane illumination microscopes called the lattice light sheet microscopes (LLSMs). The Advanced Imaging Center (AIC) at Howard Hughes Medical Institute Janelia Research Campus became the world’s first imaging center to successfully offer the LLSM as a shared instrument. Recently, the LLSM has become more widely available through the sharing of the instrument blueprint by Janelia as well as via commercial sources. To help with the decision-making process for imaging center directors interested in acquiring the LLSM, the AIC will discuss its experience of offering the LLSM as a multi-user instrument, and examines the capabilities, limitations, operational pitfalls, as well as common misconceptions of this unique microscope.
Writing successful shared instrument grants for imaging instrumentsMicroscopy instruments are costly to purchase and the imaging technologies are evolving so rapidly that most institutions cannot afford to stay technologically abreast with internal funds. Instrumentation grants at the federal level thus become a critical source of funding to meet this challenge. There are two main grant funding sources for the acquisition of shared microscopy instruments: The Major Research Instrument (MRI) grant from the National Science Foundation and the Shared Instrumentation Grant (SIG) from the National Institutes of Health. Effective planning for a shared instrumentation grant typically starts almost a year prior to submission – surveying the user base for the needed technology, arrange for instrument demo, and request internal cost-share funds or institutional commitment with the leadership of the home institution. A critical step is to get the identified “Major Users” to write a description of their research projects for the grant. These elements must be in hand before the actual writing of the grant can ensue. These instrumentation grants are competitive and the nuances of how these applications are evaluated and scored may not be obvious to many core directors on their first application. Missteps made in any of these grant elements will jeopardize the chances of success. In this presentation, the evaluation criteria, strategic planning, best practices, commonly seen mistakes, will be discussed. We will also walk through a case study.
Admin Issues Facing Light Microscopy CoresThis workshop will focus on administrative issues specific to light microscopy core facilities. Kurt Anderson recently moved from the Beatson Institute in Glasgow to the Crick Institute in London, and he will describe his experiences starting a large new light microscopy core facility. Kurt will explain his vision for a light microscopy facility fully integrated into a larger infrastructure network including electron microscopy and computational data analysis. From the initial blueprints to the selection of instrumentation and hiring of staff, Kurt will detail how he put together what promises to be one of the most highly dynamic light microscopy core facilities, within one of the most elite research institutions, in the world. Phil Hockberger from Northwestern University will speak about light microscopy core facilities from both his perceptive as one of the first light microscopists to develop a world class NIH funded two-photon microscopy facility, as well as his more recent work overseeing all core facilities at a large research intensive university. Phil will focus on issues such as best practices for core staff in professional development, and marketing to and engaging with facility users. Phil will speak about the past, present and future development of professional core facility scientist career paths, and various routes to promotion within and beyond individual core facilities, as well productive relationships with faculty and administrative oversight and support mechanisms. Finally, Phil will describe routes for facility sustainability and growth, including strategies for success in research collaborations and grant support from various mechanisms including for upgrading and purchasing of both replacement and novel light microscopy instrumentation.
Session Type: Research Group
LMRG Study 3: 3D QC Samples for Evaluation of Confocal MicroscopesHere we present the third study of the Association of Biomolecular Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). In LMRG, our goal is to promote scientific exchange between researchers, specifically those in core facilities in order to increase our general knowledge and experience. We seek to provide a forum for multi-site experiments exploring “standards” for the field of light microscopy. The study is aimed at creating a 3D biologically relevant test slide and imaging protocol to test for 1) System resolution and distortions in 2D and 3D, 2) the dependence of intensity quantification and image signal to noise of the microscope on imaging depth and 3) the dependence of the microscope sensitivity on imaging depth.
Case Studies in Modern Bioimage Analysis: 3D, Machine Learning, and Super ResolutionRecent advances in 3D imaging and the increasing popularity of 3D model systems have resulted in a proliferation of higher-dimensional microscopy data. Developments in 2D and 3D super-resolution have produced data at unprecedented length scales. Simultaneously, machine learning has become increasingly dominant within the computer vision field. The confluence of these recent trends has made it an exciting time to be a bioimage analyst. We will present several short vignettes highlighting these trends: 3D analysis ranging from millimeter-scale tissue samples to nanoscale subcellular structures. Supervised deep learning for cellular classification from phase-contrast images. And finally, unsupervised machine learning for STORM super-resolution data analysis and phenotypic profiling.
Session Type: Scientific Session
Recent advances in super-resolutionSuper-resolution microscopy is a new and dynamic field that is currently finding its useful application in Biological research. Advanced instruments and techniques are increasingly accessible to Biological researchers at imaging facilities that are open to external users. At the same time, the super-resolution field itself is rapidly evolving. New methods - and groundbreaking new twists to existing methods - are constantly being developed. Super-resolution methodologies that improve resolution in all three spatial dimensions (3D) - such as 3D Structured Illumination Microscopy (3D SIM), 3D Stimulated Emission Depletion (3D STED) and interferometric Photoactivated and Localization Microscopy (iPALM) - are particularly interesting since structures and features in a biological specimen are most often distributed not simply next to each other but also above and below. Other crucial parameters in applied biomicroscopy are contrast and optical sectioning capability. These can often be more difficult to tackle than insufficient resolution. Finally, in the new emerging field of live-cell super-resolution imaging, major limiting factors are light dose and acquisition rate. Acquisition rate becomes especially challenging in 3D imaging where this information is obtained by scanning. In my work I am currently addressing this issue in an imaging system that combines 3D SIM with multifocus microscopy (MFM) technology to provide 3D super-resolution live-cell imaging capacity of dynamic biological processes.
Linking single-molecule dynamics to local cell activityAdvances in single molecule microscopy have made it possible to obtain high-resolution maps of the inside of cells. However, technical difficulties still limit the ability to obtain dense fields of single molecules in live cells. Even more challenging is that the disparity in the spatial and temporal scales pose make it difficult to explicitly connect molecular behaviors to cellular behaviors. Here we present an integrated, multi-scale live-cell imaging and data analysis framework that explicitly links transient spatiotemporal modulation of receptor density and mobility to cellular activity. Using integrin adhesion receptors we demonstrate variations in molecular behavior can predict localized cell protrusion. Integrins are the key receptors mediating cell-matrix connections and play a critical role in mediating linkages to the cytoskeleton essential for cell migration. Our framework uncovered an integrin spatial gradient across the entire morphologically active region of the cell, with the highest density and slowest diffusion simultaneously occurring at the cell edge. Moreover, we discovered transient increases in density and reductions in speed that indicated the onset of local cell protrusion. Despite inherent heterogeneity of stochastically sampled molecules, our approach is also capable of linking the behavior to cell behavior using >90% of the molecules imaged. Through the use of receptor mutants we demonstrate that the distribution and mobility of receptors rely on unique binding domains. Moreover, our imaging and analysis framework demonstrates the ability to define unique molecular signatures dependent upon receptor conformational state. Thus, our studies demonstrate an explicit coupling between individual molecular dynamics and local cellular events, providing a paradigm for dissecting the molecular behaviors underlie the cellular functions observed with conventional microscopes.
IsoView: High-speed, Live Imaging of Large Biological Specimens with Isotropic Spatial ResolutionTo image fast cellular dynamics at an isotropic, sub-cellular resolution across a large specimen, with high imaging speeds and minimal photo-damage, we recently developed isotropic multiview (IsoView) light-sheet microscopy. IsoView microscopy images large specimens at a high spatio-temporal resolution via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. The four-views in combination yield a system resolution of 450 nm in all three dimensions after a high-throughput multiview-deconvolution. IsoView enables longitudinal in vivo imaging of fast dynamic processes, such as cell movements in an entire developing embryo and neuronal activity throughout an entire brain or nervous system. Using IsoView microscopy, we performed whole-animal functional imaging of Drosophila embryos and larvae at a spatial resolution of 1.1-2.5 microns and at a temporal resolution of 2 Hz for up to 9 hours. We also performed whole-brain functional imaging in larval zebrafish and multicolor imaging of fast cellular dynamics across entire, gastrulating Drosophila embryos with isotropic, sub-cellular resolution. Compared with conventional light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Additionally, IsoView microscopy effectively doubles the penetration depth and provides sub-second temporal resolution for specimens 400-fold larger than could previously be imaged with other high-resolution light-sheet techniques.
The hidden life of protein zombies and their role in agingAge is the major risk factor for the development of neurodegenerative diseases such as Alzheimer’s disease (AD). Currently, AD alone impacts the lives of approximately 5 million Americans and their families. The disorder imposes an immense emotional burden on family members and caretakers. Compounding the problem is the reality that the number of patients will increase more than two-fold in the next 30 years and impose a financial cost of more than $1 trillion per year. One can hardly imagine the negative consequences for the wellbeing of our economies, our families and the future of mankind. The only proper response to this formidable challenge is to combat it through efforts that extend from the care of individual patients to the discovery of effective therapeutics to treat, and ideally, prevent it. We discovered a class of extremely long-lived proteins (LLPs) in the adult brain that functionally decline during aging. We speculate that biochemical changes and subsequent deterioration of LLPs may be responsible for the age-related impairment of cognitive performance and the onset/progression of neurodegenerative disorders such as AD. Proposed experiments will allow us to decipher the mechanisms underlying the functional integrity of LLPs and determine how they relate to pathologies in the brain.
Imaging Drosophila Brain ActivityUnderstanding how the action of neurons, synapses, circuits and the interaction between different brain regions underlie brain function and dysfunction is a core challenge for neuroscience. Remarkable advances in brain imaging technologies, such as two-photon microscopy and genetically encoded activity sensors, have opened new avenues for linking neural activity to behavior. However, animals from insects to mammals exhibit ever-changing patterns of brain activity in response to the same stimulation, depending on the state of the brain and the body. The action of neuromodulators – biogenic amines, neuropeptides, and hormones – mediates rapid and slow state shifts over a timescale of seconds to minutes or even hours. Thus, it is imperative to develop a non-invasive imaging system to maintain an intact neuromodulatory system. The fruit fly Drosophila melanogaster is an attractive model organism for studying the neuronal basis of behavior. However, most imaging studies in Drosophila require surgical removal of the cuticle to create a window for light penetration, which disrupts the intricate neuromodulatory system. Unfortunately, the infrared laser at the wavelengths used for two-photon excitation of currently available activity probes is absorbed by the pigmented cuticle. Here we demonstrate the ability to monitor neural activity in flies with intact cuticle at cellular and subcellular resolution. The use of three-photon excitation overcomes the heating problem associated with two-photon excitation.