Past Seminars

NCI Optical Microscopy Laboratory


Past Seminars (2014-2020)

Seminars in 2020

Tuesday, November 10, 2019
Dr. Eloise Grasset (Johns Hopkins U Med School). “3D imaging of the breast cancer metastasis; lessons from culture and tissues”

Metastasis is a complex process challenging to study in vivo since they occur deep inside the body over extended periods of time. To understand how cancer cells metastasize, we developed 3D imaging assays that recapitulate different steps of the metastatic cascade such as invasion and metastatic outgrowth1. Using these assays, we demonstrated that luminal breast cancer models typically retain an epithelial differentiation state, while triple negative breast cancer (TNBC) models display a hybrid E/M state that lead invasion ex vivo. We validated these results in vivo by analyzing whole tumor sections from mouse models, patient-derived xenografts and tumor microarray using an automated slide scanner microscope. We showed using single-cell RNA sequencing and immunofluorescence that basal breast cancer cells undergo EMT during invasion and MET during metastatic outgrowth ex vivo. We then demonstrated that the mesenchymal marker vimentin, which is acquired during EMT, suppressed invasion in multiple TNBC models, and metastasis formation in vivo, indicating that EMT is required for metastasis. Consistent with a MET in the distant organs, we showed that knocking down vimentin at the metastatic site, increased metastasis in vivo. However, a time-course analysis of the metastatic seeding and outgrowth using immunofluorescence revealed that cancer cells use EMT to disseminate at the metastatic site demonstrating that MET is advantageous but not required.

Tuesday, October 20, 2020
Dr. Ahmed Abdelfattah (Janelia/HHMI). “Genetically encoded voltage sensors for optical monitoring of the brain activity”

Voltage imaging provides unparalleled spatial and temporal resolution of the brain’s electrical signaling at the cellular and circuit levels. A longstanding challenge has been to develop genetically encoded voltage sensors to track membrane voltage from multiple neurons in behaving animals. However, brightness and signal to noise ratio have limited the utility of existing voltage sensors, especially in vivo. I will describe our work to engineer hybrid protein-small molecule sensors with improved brightness and photostability that allow imaging neural circuits in vivo. Using those sensors, we extend both productive imaging time and number of neurons imaged by more than 10 times in awake behaving animals.

Tuesday, January 21, 2020
Malgorzata Latallo (Johns Hopkins Sch. Med).

“Nuclear export and translation of circular repeat-containing intronic RNA in C9ORF72-ALS/FTD”
Amyotrophic lateral sclerosis (ALS) currently is an incurable disease caused by motor neuron degeneration. The most common cause of inheritable ALS is the expansion of hexanucleotide GGGGCC repeat in the first intron of C9ORF72 gene. RNA containing abnormally long GGGGCC repeats can form RNA foci, which may sequester essential RNA binding proteins. Alternatively, repetitive RNA can be exported from nucleus and translated via repeat associated non-ATG (RAN) translation to generate different poly dipeptide repeats (DPR). We now used single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat containing C9ORF72 RNAs. We demonstrated that the spliced intron was stabilized by the expanded repeats in a circular form due to defective debranching. The circular intronic RNA is exported to cytoplasm through the NXF1-NXT1 pathway and serves as the template for the unconventional RAN translation, which is elevated by stress. This study reveals an uncharacterized disease-causing RNA species and demonstrates the importance of gene context of repeat expansion and RNA spatial localization to understand disease etiology.

Seminars in 2019

Tuesday, December 10, 2019

Dr. Daniel Melters (NCI/NIH). “High-speed Atomic Force Microscopy reveals dynamic nature of chromatin fibers”

How do individual molecules behave? This question has is at the basis of the molecular biology. A wide range of techniques have been developed over the last several decades, but direct observation of the biophysical motions and actions of molecules remains elusive. Atomic force microscopy has allowed us to visualize individual molecules, but only as static images. With the recent development of high-speed atomic force microscopy, the dynamic behavior of individual molecules can now be visualized and quantified at near video-rate resolution. We can now see nucleosomes slide along DNA, for instance.

Tuesday, November 12, 2019
Dr. David Garcia Grisales (NCI/NIH; U. Maryland). “New Emergent Properties of Transcription Factor Dynamics and Their Interactions with Chromatin”

Single-molecule tracking allows the study of transcription factor dynamics in the nucleus, giving important information regarding the search and binding behavior of these proteins with chromatin in vivo. However, these experiments suffer from limitations due to photobleaching of the traced protein and pre-assumptions on exponential behavior required for data interpretation, potentially leading to serious artifacts. Here, we developed an improved method to account for photobleaching effects, theory-based models to accurately describe transcription factor dynamics, and an unbiased model selection approach to determine the best predicting model. A new biological interpretation of transcriptional regulation emerges from the proposed models wherein transcription factor searching and binding on the DNA and nuclear microenvironment heterogeneity result in a broad distribution of binding affinities and accounts for the power-law behavior of transcription factor residence times. Moreover, two types of confinement are discovered: one related with specific DNA interaction and a second one related to liquid-liquid phase separation. The latter shows the importance of phase separation in transcriptional regulation and gene expression.

Tuesday, October 15, 2019

Dr. Colenso Speer (U. Maryland). “Reconstructing neural circuits with volumetric super-resolution microscopy”

Light microscopy enables multi-color imaging, analysis, and live tracking of diverse cellular and molecular processes and is a vital tool for studying neural circuits. In the last decade, super-resolution microscopy techniques have extended the spatial resolution of optical imaging to the nanoscale and enabled new investigations of the molecular organization and structural properties of synapses in the nervous system. In this seminar, I will discuss approaches for applying STochastic Optical Reconstruction Microscopy (STORM) and Expansion Microscopy (Exm) to the analysis of synaptic structure and connectivity over volumetric areas of brain tissue. I will present a basic guide for researchers interested in applying super-resolution imaging in their own investigations and will cover both the strengths and limitations of the approaches. Considerations for optimizing sample preparation, super-resolution image quality, and automated data analysis will be discussed. Data demonstrating the imaging and analysis of synaptic inputs to identified neurons as well as approaches for synaptic protein labeling and cell type-specific labeling for super-resolution reconstruction will be presented. Volumetric super-resolution microscopy provides an important new class of structural and molecular imaging data that is complementary to connectomic reconstruction of neural circuits by electron microscopy

Tuesday, September 17, 2019

Dr. Brant Weinstein (NIH/NICHD) “Studying novel vascular-associated cell populations in the zebrafish using advanced imaging”.

Tuesday, June 18, 2019

Dr. Megan Rizzo (U. Maryland). “Polarization light sheet microscopy for homotransfer biosensor imaging”.

Homotransfer FLARE-type biosensors offer numerous advantages over classic heterotransfer FRET sensors, including polarized light readout and enhanced multiplexing capabilities. Even so, accurate FRET measurements require high signal-to-noise image capture conditions that limit data collection speeds and introduce phototoxicity concerns. These limitations can be overcome by constructing a polarization-capable inverted selective plane illumination microscope. The piSPIM technology enables video rate collection speeds of homotransfer reporters and long-term imaging over several hours without demonstrable phototoxicity.

Tuesday, May 21, 2019

Dr. Kem Sochacki (NIH/NHLBI). “Correlative fluorescence localization microscopy and platinum replica electron microscopy highlights the edge of clathrin mediated endocytosis”.

Clathrin mediated endocytosis (CME) is a major mode of internalization for eukaryotic cells. During this process, clathrin proteins form patches of honeycomb-shaped scaffolds that coat the internalizing membrane. Several dozens of membrane adapters and accessory proteins are intrinsic to the proper development of a clathrin-coated pit. Platinum replica electron microscopy allows high resolution (~2 nm) high contrast visualization of clathrin structures at the plasma membrane and has been a corner-stone imaging technique in the study of CME. Combining this classic technique with the more recently developed super-resolution fluorescence localization microscopy, we have mapped the positions of 19 key clathrin associated proteins with respect their ultrastructural topography at single coated pits. We find that obtaining the protein location and cellular ultrastructure with different imaging modes has many benefits. These include 1) an unhindered view of the ultrastructure surrounding proteins of interest and 2) straightforward computational analysis and statistics of protein distributions. Through this work we discovered that the edge of clathrin structures concentrates a hub of adaptors and accessory proteins which are integral to the fate of clathrin-mediated endocytosis.

Tuesday, April 23, 2019

Andrew Lauziere (U.Maryland/NIBIB) “Deep learning in imaging”

Deep learning describes a model paradigm that has quickly emerged as a leader across many fields. Microscopists in particular have found many cutting edge uses with the technology. A brief introduction to deep learning precedes basic models, and some applications. Specifically, the U-Net architecture produces state of the art segmentations in microscopy images all the while the same type of model can produce super resolution images. The Section on High Resolution Optical Imaging (HROI) is taking full advantage of this methodology to enhance and forward current projects.

Tuesday, March 19, 2019

Dr. Diana Stavreva (NCI/NIH) “Single cell transcription analysis in live cells”

Transcription factors (TFs) interact dynamically with genomic targets and genes are transcribed in a discontinuous pattern referred to as RNA bursting. However, it is unclear how TFs regulate bursting. To address this, we utilized several state-of-the-art approaches: 1) Custom imaging and analysis platform, which was developed by the High-throughput Imaging Facility at the NCI and used to characterize the real time synthesis of fluorescently labeled RNA from GR-regulated MMTV TSs in a high-throughput format. These experiments revealed that the RNA bursting pattern changes over time and is also ligand-specific. 2) Single-molecule tracking (SMT) method, which allowed us to characterize the changes in the intranuclear mobility of GR in living cells and to compare them to the MMTV-RNA bursting pattern under various treatment conditions. In these experiments we utilized a novel single-molecule tracking data analysis method developed in the lab. 3) 3D orbital tracking method (3DOT), which allows following the transcription sites (TSs) over time in 3D and simultaneous detection of the intensity of the GR binding and the RNA production at the transcribing locus. These experiments revealed a substantial delay between GR binding and RNA synthesis. We concluded that receptor dwell time at genome targets determines burst duration, while the fraction of transiently bound receptor molecules determines burst frequency. Using 3D tracking of TSs, we have directly correlated transcription factor binding and transcription initiation at the single promoter level in a mammalian system. Together, our data reveal a dynamic interplay between TF mobility and RNA bursting that is responsive to stimuli strength, type, modality, and duration.

Tuesday, January 15, 2019

Dr. Paul Tillberg (HHMI, Janelia) ” Expansion microscopy: scalable super-resolution imaging through uniform specimen expansion “

The Expansion Microscopy method allows effectively sub-diffraction limited optical imaging of biological specimens, without a super-resolution microscope. This is done by expanding the specimen uniformly in three dimensions. Expansion is achieved by embedding the tissue in an ultra-swellable gel, chemically linking biomolecules directly to the gel, digesting the tissue down to molecular-scale pieces, and using the swelling behavior of the gel material to pull the molecular fragments away from each other uniformly. This can be done on relatively thick tissue slices, up to at least 200 um thick. The method is easy to adopt and well-suited as a histology core facility offering, as it is compatible with existing antibody and fluorescent protein labeling protocols without modification. The gel material can be engineered to extend the original fourfold expansion (in each dimension) to seven-fold or more, while maintaining a simple, robust workflow.

Seminars in 2018

Tuesday, December 11, 2018

Dr. Quinggong Tang (U. Oklahoma) “Depth-resolved molecular characterization of tissues in 3D by Fluorescent Laminar Optical Tomography and its applications for cancer diagnostics”

Laminar optical tomography (LOT) is a mesoscopic three-dimensional (3D) optical imaging technique that can achieve both a resolution of 100-200 µm and a penetration depth of 2-3 mm based either on absorption or fluorescence contrast. Fluorescence laminar optical tomography (FLOT) can also provide large field-of-view (FOV) and high acquisition speed. All of these advantages make FLOT suitable for 3D depth-resolved imaging in tissue engineering, neuroscience, and oncology. In this study, by incorporating the high-dynamic-range (HDR) method widely used in digital cameras, we presented the HDR-FLOT. HDR-FLOT can moderate the limited dynamic range of the charge-coupled device-based system in FLOT and thus increase penetration depth and improve the ability to image fluorescent samples with a large concentration difference. For functional mapping of brain activities, we applied FLOT to record 3D neural activities evoked in the whisker system of mice by deflection of a single whisker in vivo. We utilized FLOT to investigate the cell viability, migration, and bone mineralization within bone tissue engineering scaffolds in situ, which allows depth-resolved molecular characterization of engineered tissues in 3D. Moreover, we investigated the feasibility of the multi-modal optical imaging approach including high-resolution optical coherence tomography (OCT) and high-sensitivity FLOT for structural and molecular imaging of colon tumors, which has demonstrated more accurate diagnosis with 88.23% (82.35%) for sensitivity (specificity) compared to either modality alone. We further applied the multi-modal imaging system to monitor the drug distribution and therapeutic effects during and after Photo-immunotherapy (PIT) in situ and in vivo, which is a novel low-side-effect targeted cancer therapy.

Tuesday, November 13, 2018

Dr. Amicia Elliott (NIH/NIMH) “Using light-sheet microscopy to study evoked motor sequence generation in Drosophila”

Motor sequences are important elements of everyday behavior, but how they are produced by central neural circuits is poorly understood. Fruit fly neural circuits drive complex motor sequences and are small enough to investigate at a brain-wide scale via emerging methods in microscopy. We use a critical behavioral sequence for the fruitfly, called ecdysis, to study the neural control of behavior. Ecdysis is required for molting at each developmental stage and consists of three serially executed, stereotyped behavioral programs at the pupal stage. The controlling neural circuit includes approximately 300 peptidergic neurons that express the Ecdysis Triggering Hormone receptor (ETHR) and are activated by peripheral release of Ecdysis Triggering Hormone (ETH) that leads to the behavioral sequence. Existing data indicate that specific subpopulations are required for each behavioral phase of ecdysis (1). However, the identities of the individual neurons that control each behavioral phase remain largely unknown, as do the mechanisms by which they regulate motor output. To achieve a detailed cellular-level understanding of the ecdysis circuit, we have built a light-sheet microscope that is capable of imaging the Drosophila pupal CNS rapidly at high resolution (2). We are currently using Ca2+ biosensors to monitor the neural activity in populations of motor neurons in excised brains in response to ETH.

Tuesday, October 16, 2018

Dr. Kyung Lee (NIH/NCI) “Structure and Function of Mammalian Centrosomal Assemblies”

As the main microtubule-organizing center for animal cells, centrosomes are critically required for various cellular processes, including bipolar spindle formation and mitotic chromosome segregation. They are composed of two orthogonally arranged centrioles, which duplicate early in the cell cycle in a manner that takes place only once per each cycle. Accurate control of centriole numbers is essential for normal chromosome segregation and maintenance of genomic integrity. A growing body of evidence suggests that mammalian polo-like kinase 4 (Plk4) plays a key role in inducing centriole duplication. Plk4 localizes to distinct subcentrosomal sites by interacting with a centrosomal scaffold protein, Cep152. Interestingly, Cep152 tightly binds to another scaffold protein, Cep63, and this step appears to be critical to assemble a higher-order cylindrical architecture around a centriole. Notably, mutations in the Cep152 and Cep63 scaffolds are frequently linked to various human diseases, such as cancer, microcephaly, ciliopathy, and dwarfism. Therefore, investigating the molecular basis of how these scaffolds are assembled into a higher-order structure and how the assembled architecture promotes Plk4-dependent centriole duplication will be important not only to understand one of the most fundamental cellular processes of centrosomal organization but also to discover the causes of human genetic disorders associated with centrosome abnormalities. Our latest findings on the molecular nature and biological significance of the Cep152•Cep63 complex-generated cylindrical self-assembly will be discussed.

Tuesday, September 18, 2018

Dr. Mihaela Serpe (NIH/NICHD) “Molecular mechanisms of synapse assembly and homeostasis – lessons from genetics and imaging in flies”

The purpose of our research is to understand the mechanisms of synapse development and homeostasis. Using the Drosophila neuromuscular junction (NMJ) as a model for glutamatergic synapse, we focus on three key processes in synaptogenesis:  (1) trafficking of components to the proper site,  (2) organizing those components to build synaptic structures, and (3) maturation and homeostasis of the synapse to optimize its activity. We address the mechanisms underlying these processes using a comprehensive set of approaches including genetics, biochemistry, molecular biology, super resolution imaging and electrophysiology recordings in live animals and reconstituted systems. I will emphasize how microscopy techniques have enabled our recent accomplishments:

(1) identification of a key auxiliary protein of glutamatergic synapses, called Neto, that is essential for their development and function both in Drosophila and mammals, and the molecular dissection of its activities, and

(2) analysis of the action of the TGF-β pathway in synaptic plasticity, and in particular the discovery of a novel mechanism by which local, non-transcriptional BMP signaling directly modulates synapse structure and activity.

Tuesday, May 15, 2018

Dr. Philip Anfinrud (NIH/NIDDK) “Watching proteins function in real time via picosecond X-ray diffraction”

To understand how a protein functions, it is crucial to know the time-ordered sequence of structural changes associated with its function. To that end, we have developed numerous experimental techniques for characterizing structural changes in proteins over time scales ranging from femtoseconds to seconds. This talk will focus primarily on time-resolved X-ray studies performed on the BioCARS beamline at the Advanced Photon Source, which allowed us to characterize structural changes in proteins with 150-ps time resolution. We have used this capability to track the reversible photocycle of photoactive yellow protein following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore. Briefly, a picosecond laser pulse photoexcites pCA and triggers a structural change in the protein, which is probed with a suitably delayed picosecond X-ray pulse. When the protein is studied in a crystalline state, this “pump-probe” approach recovers time-resolved diffraction “snapshots” whose corresponding electron density maps can be stitched together into a real-time movie of the structural changes that ensue. However, the actual signaling state is not accessible in the crystalline state due to crystal packing constraints. This state is accessible in time-resolved small- and wide-angle X-ray scattering studies, which probe changes in the size, shape, and structure of the protein. These studies help provide a framework for understanding protein function, and for assessing and validating theoretical/computational approaches in protein biophysics. This research was supported in part by the Intramural Research Program of the NIH, NIDDK.

Tuesday, April 17, 2018

Dr. Simona Patange (NIH/NCI)
“A living, single view of MYC’s effects on transcription”

How does a transcription factor transmit information to a gene in a single cell context? To answer this question we examine the oncogenic transcription factor c-MYC. MYC is upregulated in most human cancers, yielding a global increase in gene expression. However the mechanism by which MYC amplifies transcription, and why it promotes cancer, has remained notoriously elusive. To address this gap in understanding we seek a quantitative, single-cell view of how MYC modulates the kinetics of transcription. We use two recently-developed imaging techniques: 1) single-molecule FISH to quantify RNA in fixed cells, and 2) live cell imaging of transcription with the MS2-PP7 stem loop system to observe real-time RNA production at a given gene locus. We predict that if MYC amplifies gene expression, then we would observe either an increase in the frequency of transcription events, or the duration of transcription events, or a combination of the two. Our results looking at the effect of MYC perturbations on both an exogenous reporter and endogenous gene show that MYC increases the duration of transcription events— this results in greater gene expression, but also an increase in its heterogeneity. These findings provide living, single cell evidence of MYC as a global amplifier of gene expression and suggests that the mechanism is by stabilizing the active period of a gene. We speculate that the added consequence of MYC’s propensity to increase gene expression heterogeneity could drive cells to transiently populate pathological states and phenotypes that may ultimately make them susceptible to cancer.

Tuesday, March 20, 2018

Dr. Jie Xiao (Johns Hopkins U Sch Med)
“Quantitative superresolution imaging for bacterial cell biology”

Single-molecule localization based superresolution microscopy (SMLM) not only can reveal fine dimensional details of cellular structures beyond the diffraction limit, but also can provide lists of molecular coordinates to enable the critical ability of quantifying the number, clustering, complexity, colocalization and organization of biomolecules with 10-50 nm resolution. When coupled with genetic and biochemical investigations, these methods are powerful in accessing new information not possible before. In this talk I will discuss a few case studies in which SMLM allows us to understand the assembly, organization, function and dynamics of a variety of bacterial cellular structures.

Tuesday, January 16, 2018

Dr. Matthew Wooten (Johns Hopkins U Sch Med), “Super Resolution as a tool to study a potential role for DNA replication in establishing distinct epigenomes”

The primary function of DNA replication is to duplicate the genome. However, the process of DNA replication must also duplicate epigenetic information. Epigenetic mechanisms play a key role in altering chromatin structure and gene expression patterns, and in specifying and maintaining stem cell identity throughout cell division. Many types of stem cells have the ability to asymmetrically divide to give rise to one daughter cell capable of self-renewal and another daughter capable of differentiating.

Using a dual-color labeling system, we demonstrated that in the Drosophila germline H3 is inherited asymmetrically whereas the H3 variant H3.3 is inherited symmetrically. As H3 is incorporated during S-phase whereas H3.3 is incorporated in a replication-independent manner we hypothesize that old and new H3 are differentially incorporated into distinct sister chromatids during DNA replication.

Because the average diameter of replication forks in eukaryotic cells (~150 – 400 nm) is at or below the diffraction-limited resolution of conventional fluorescence light microscopy (250 – 300 nm), we used single molecule localization based super-resolution (SR) imaging to examine the spatial distribution of old and new H3 and H3.3 in interphase GSCs. Interestingly, we were able to observe that these two histone species show significantly different co-localization patterns throughout interphase.

We have developed a method using STED SR microscopy in tandem with the chromatin fiber technique to observe asymmetries in DNA replication as well as unique patterns in the distribution of H3 versus H3.3 in newly replicated sister chromatids. H3-labeled chromatin fibers show a significantly higher degree of asymmetry than do H3.3 chromatin fibers, which may be a basis for the distinct patterns of H3 and H3.3 inheritance at the genome-wide level. In addition, during replication-coupled nucleosome assembly, old histones preferentially associate with the leading strand whereas new histones preferentially associate with the lagging strand. Based on these data, we propose that the asymmetries inherent to the process of DNA replication serve to bias histone inheritance such that the leading strand preferentially inherits old histone and the lagging strand preferentially inherits new histones.

Seminars in 2017

Tuesday, December 12, 2017

Dr. Ulrike Boehm (NIH/NCI) “4Pi‐RESOLFT nanoscopy: Nanometer scale 3D fluorescence imaging in whole living cells”

Deep learning describes a model paradigm that has quickly emerged as a leader across many fields. Microscopists in particular have found many cutting edge uses with the technology. A brief introduction to deep learning precedes basic models, and some applications. Specifically, the U-Net architecture produces state of the art segmentations in microscopy images all the while the same type of model can produce super resolution images. The Section on High Resolution Optical Imaging (HROI) is taking full advantage of this methodology to enhance and forward current projects.

Tuesday, November 14, 2017

Dr. Kandice Tanner (NIH/NCI) “Probing the physical properties of the microenvironment in vivo” 

Tissue is composed of heterogeneous biological components that modulate physical properties within the microenvironment. Transformation of the physico-chemical properties of the stromal microenvironment such as changes in the extracellular matrix (ECM) has been shown to be associated with cancer progression. Cells respond to both chemical and physical cues of the microenvironment. In tissue, chemical and mechanical cues are both modulated by changes in ligand density and localized tissue architecture. Hence, decoupling chemical cues from those due to the physical changes is non-trivial. What is needed is the ability to resolve and quantitate minute forces that cells sense in the local environment (on the order of microns) within thick tissue (in mm). To achieve this, we employ a method to quantitate absolute tissue mechanics using in vitro and in vivo models. We performed Active Microrheology by optical trapping in vivo, using in situ calibration to accurately apply and measure forces. With micrometer resolution at broadband frequencies and depths approaching 0.5 mm, we probed differential stresses and strains on force, time and length scales relevant to cellular processes in living zebrafish. We determined that proxy calibration methods overestimate complex moduli by as much as ~20 fold. While ECM hydrogels displayed rheological properties predicted for polymer networks, new models may be needed to describe the behavior of tissues observed. Finally, we validated our in vitro findings in an in vivo model using zebrafish as our model for metastasis. We believe that this platform can be used in elucidating the basic mechanisms that govern the role of material properties in mechanobiology.

Tuesday, October 17, 2017

Dr. Petr Kalab (Johns Hopkins U) “Fluorescence lifetime imaging microscopy (FLIM) for quantitative live-cell measurements with Forster resonance energy transfer (FRET) probes” 

Most of the commonly used fluorescence microscopy techniques today depend on the detection of the emission intensity of various fluorescent proteins, dyes or endogenous reports. In addition to the crucially useful separation of the emission and excitation wavelengths, the fluorescence process offers another exciting and so far underused opportunity to explore living cells through the fluorescence lifetimes of fluorophores. The fluorescence lifetime, which is independent of the emission intensity, is a measure of the time it takes before the excited fluorophore returns to its ground state. The interesting feature of fluorescence is that the energy dissipation from the excited state depends on the molecular condition of the fluorophores (protonation, oxygenation, etc.) and their environment (such as binding to fluorescent or non-fluorescent molecules). Because of that, the fluorescence lifetime imaging microscopy (FLIM) has the potential to provide real-time measurements of molecular interactions and biochemical reactions in live cells. The time-correlated single photon counting (TCSPC) method of FLIM provides better lifetime resolution and higher photon usage efficiency than its main alternative, the fluorescence polarization FLIM. We used the TCSPC FLIM with monomolecular FRET reporters to study the role of the small GTPase Ran in the regulation of mitosis, cell cycle or DNA repair. In those studies, the quantitative properties of the FLIM/FRET imaging gave us the particularly useful insights. FLIM provides a rigorous and quantitative method of FRET measurements and has several distinct advantages over the intensity-based methods, including relaxed concerns for spectral crosstalks (only donor emission is detected), insensitivity to sensor concentration (within reasonable limits) and the straightforward relationship between the measured lifetimes and FRET efficiency. The main limitations in FLIM/FRET, which should not be underestimated, is the need for a large number of photons (about 1000/bin) to resolve 2-3 exponential lifetimes that are typical for all fluorophores in the cellular environment. While the technology is constantly evolving, the application of FLIM/FRET requires careful consideration of the necessary temporal or spatial resolution. In this presentation, I will provide a brief overview of the main theoretical and namely practical aspects of FRET detection with FLIM. These topics will include the design of FRET sensors for FLIM, the important features of excitation sources and detectors, and the available FLIM computation methods, including the global analysis via the Phasor approach. Finally, since some of the data that I will discuss we acquired with the still fully functional equipment in the NCI imaging core, the talk could serve as an introduction to the users of the facility who are interested in applying FLIM in their research.

Tuesday, September 19, 2017

Dr. Steven S Vogel (NIH/NIAAA) “Ultra-Fast Long-Distance Energy Transfer Between Fluorescent Proteins”

An implicit and often unmentioned assumption of studies utilizing genetically encoded Green Fluorescent Protein, its derivatives, and structurally related fluorescent proteins (FPs), is that they behave like classical organic fluorophores. When conventional fluorophores are in close-proximity (< 1 nm) and/or are cooled to temperatures approaching absolute zero, coherent energy transfer (CET) may enable multiple fluorophores to behave as a single quantum entity. CET is thought to play a key role in photosynthesis, and vis-à-vis technology, may enable quantum computing. CET can manifest as ultra-fast long-distance energy transfer within fluorophore assemblies. Antibunching and Davydov splitting of circular dichroism (CD) spectra, uniquely quantum mechanical behaviors, are indicative of CET. Physiological temperatures extinguish CET by promoting rapid collisional dephasing of fluorophore vibrational modes (typically within 100 fs of photoexcitation). Moreover, because FP fluorophores are encased in a ß-barrel structure, proximities closer than 2 nm are not possible. Thus, CET between FPs at physiological temperatures is thought to be impossible. Nonetheless, time-resolved fluorescence anisotropy, fluorescence correlation spectroscopy, antibunching, and CD all indicate stronger than expected coupling between FPs. Paired-pulse correlation spectroscopy revealed that dephasing between coupled FPs occurs between 400-600 fs after photoexcitation, suggesting that the FP ß-barrel attenuates dephasing to allow CET.

Tuesday, June 20, 2017

Dr. Ronald N. Germain (NIH/NIAID) “Imaging Immunity – Developing a Spatiotemporal Understanding of Host Defense Using Intravital Dynamic and Multiplex Static Microscopy”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar of Dr. Ronald Germain (NIH/NIAID) about two-photon dynamic intravital and static 3D tissue imaging. For the latter Dr. Germain perfected clarification techniques and developed software that allows quantitative multichannel analysis of cellular distribution in 3D in whole organs. In combination, these two techniques lead to deep understanding of how the tissues are organized, and how this organization is dynamically maintained and adapted to microenvironment. Stunning work, stunning resource!

Tuesday, May 16, 2017

Dr. Vinay Swaminathan (NIH/NHLBI) “Co-alignment and orientation of activated integrins in focal adhesions of migrating cells studied by fluorescence polarization”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar of Dr. Vinay Swaminathan (NIH/NHLBI) about dynamic integrin response to physical and chemical information. Techniques of fluorescence polarization microscopy led to amazing discovery: integrins come to attention (co-align) obeying the trumpet of directional mechanical force. Come to this seminar to learn whether measurements of molecular anisotropy are applicable to your favorite biological system.

Tuesday, April 18, 2017

Dr. Ville Paakinaho (NIH/NCI) “Single-Molecule Imaging and the future of simultaneous tracking of Multiple Transcription Factors”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar of Dr. Ville Paakinaho (NIH/NCI) about real-time dynamics of Transcription Factor action studied by the state-of-the-art Single Molecule Tracking. In these pioneer studies Glucocorticoid Receptor (GR) and other TF were tagged with HaloTag and SNAP-tag and observed on custom-built microscope with a special illumination technique (HILO). Don’t miss this presentation about the exciting new technique! Isn’t it surprising how dynamic the life of the cellular molecules is?

Tuesday, March 21, 2017

Dr. Lakshmi Balagopalan (NIH/NCI) “Oh The Places LAT Goes!”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar of Dr. Lakshmi Balagopalan (NIH/NCI) about the mechanism of microcluster formation in activated T-cells. This is an excellent example of the application of cutting-edge high-resolution microscopy for kinetics of T-cell vesicle formation (Lattice LSM and TIRF-SIM). Where Biochemistry makes educated guesses, Microscopy observes!

Tuesday, January 17, 2017

Dr. Elisabeth Finn (NIH/NCI) “Examining genome-wide patterns of DNA:DNA interaction via high throughput imaging”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar of Dr. Elisabeth FInn (NIH/NCI) about genome organization and its cell-to-cell variation revealed by high-throughput FISH.

Seminars in 2016

Tuesday, November 22, 2016

Dr. Martin Schnermann (NIH/NCI) “Near IR uncaging chemistry: discovery and applications”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about a new antibody-based drug-delivery method, based on “uncaging” of biologically active molecules by near-IR light. The use of tissue penetrant near-IR wavelengths enables in vivo applications. Dr. Schnermann (NCI) applied chemical remodeling to cyanines and developed novel cyanine fluorophores with improved properties for drug uncaging, in vivo optical imaging, and super resolution microscopy. Amazing resource!

Tuesday, October 18, 2016

Dr. Luke Lavis (HHMI, Janelia Farms) “Building brighter dyes for single-molecule imaging”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about fantastic advancements in the field of labeling live proteins in situ. Reseach of Dr. Lavis opened a new era in live imaging by permitting bright, stable and reliable labeling of protein fusions to Halo Tag, SNAP tag and CLIP tag. Single-molecule imaging is made possible by this technological achievement. Ladies and Gentlemen, PhD and MD, please greet our future Nobel laureate!

Tuesday, September 20, 2016

Dr. Jason Yi (NIH/NCI) “madSTORM: a super resolution technique for large-scale multiplexing at single molecule accuracy”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about madly successful superresolution strategy developed in CCR. madSTORM allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM was applied to an activated T cell to localize 25 epitopes, 14 of which are on components of the same multi-molecular T cell receptor complex. Please, come! Especially if you fancy a big multisubunit complex of your own!

Tuesday, April 19, 2016

Dr. Diego Presman (NCI/NIH) “Analysis of Glucocorticoid Receptor Dynamics by Number and Brightness and Single-Molecule Tracking methods”

Can you imagine such a treat! A hunt for single molecules! Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about dynamic binding behavior and oligomerization of glucocorticoid receptors. Single molecule tracking will tell you important things about biophysical characteristics of transcriptional factors. And we want NUMBERS! Prepare yourself for a wild ride on the waves of state-of the art techniques!

Tuesday, March 22, 2016

Dr. Kenneth Jacobson (NIH/NIDDK) “Fluorescent Ligands of GPCRs: Adenosine and P2Y Receptors”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about fluorescent probes for adenosine receptors – important proteins involved in inflammation.

Tuesday, January 19, 2016

Dr. Jadranka Loncarek (NIH/NCI) “Centriole engagement: a matter of maturity?”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about super resolution, light and electron microscopy in studies of centrosome anomalies and centrosome cycle.

Seminars in 2015

Tuesday, December 15, 2015

Dr. Jan Wisniewski (Janelia Farms) “Instant 3D imaging with Multi-Focus Microscope”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about tracking of molecules in 4D made possible by innovative technique allowing simultaneous acquisition of multiple z-sections (multifocus microscopy). Until recently we had to sacrifice either speed or resolution for the low-light time-lapse fluorescent imaging, but now we may have it all and really observe individual molecules in their natural habitat…. And do some 3D PALM on the side.

Tuesday, November 18th, 2015

Dr. Dan Larson (NCI/NIH, LRBGE) “A single-molecule view of the central dogma.”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about single-molecule view on transcription. Portraits of individual mRNA will be provided, as well as info about the best hunting places for the observation of gene expression in live single cells with highest temporal and spatial resolution.

Tuesday, October 20, 2015

Dr. Christopher Westlake (NIH/NCI) “Cell antenna assembly studied by advanced fluorescence and electron microscopy imaging”

WHAT CAN Immuno Fluorescence AND Electron Microscopy TELL US ABOUT Rab SMALL GTPases? Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar on the role of Rab small GTPases and associated Rab effectors and GTPase regulators, SNAREs and membrane shaping proteins in ciliogenesis. Results are important for research in Polycystic kidney disease and other ciliopathies. This is a perfect example of how Electron and Light microscopy may be used for a structural and functional dissection of intracellular pathways.

Tuesday, September 15, 2015

Dr. Rolf Swenson (NIH/NHLBI) “The chemistry of optical imaging agents, experiences from the Imaging Probe Development Center”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar about NIH Imaging Probe Development Center (IPDC) that provides support for advanced molecular technologies. IPDC scientists can synthesize requested probes that are published in literature, but commercially unavailable, or are completely novel. They can do it for us – isn’t it wonderful? And do you, personally, know that such resource is at hands distance? If not, come to this talk!

Tuesday, May 19, 2015

Dr. Keir Neuman (NIH/NHLBI) “Background-free imaging in vivo with fluorescent nanodiamonds applied to lymph node imaging”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar on the topic of tissue imaging studied by a state-of the art fluorescent biomarkers – nanodiamonds. If you think you don’t need them nanodiamonds, think again! The most important potential biological applications of fluorescent nanodiamonds include (1) biomolecular labeling, (2) cellular imaging, (3) tumor targeting, (4) single particle tracking, (5) long-term in vivo monitoring. And… nanodiamonds are within a reach for the NIH community.

Tuesday, April 21, 2015

Dr. Thomas Ried (NIH/NCI) “The NIH 4D Nucleome Initiative”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar on the topic of 4D nucleome. The NUCLEUS, naked, transparent – all the guts visible and classified, in its past, present, and future! Can we do it? We can do it!

Tuesday, March 17, 2015

Dr. Daniela Malide (NIH/NHLBI) “In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy”

Light Microscopy Interest Group (LMIG) organizers invite you to attend a seminar on the topic of clonal tracking of hematopoietic stem and progenitor cells studied by a genetic combinatorial marking in five shiny colors! Yes, you can track five colors!

Tuesday, January 20, 2015

Dr. Prabuddha Sengupta (NICHD/NIH, CBMB) “Mammalian plasma membrane remodeling studied by quantitative point localization microscopy”

LMIG organizers invite you to attend a seminar on the topic of HIV biogenesis studied by a state-of the art microscopy technique. Single molecule superrersolution (point localization) microscopy based data acquisition and image analysis strategies to highlight the details of the molecular mechanism of individual steps of the viral assembly process.

Seminars in 2014

Tuesday, December 16, 2014

Dr. Valentin Magidson (NCI/NIH, CCR/OD) “Insights into chromosome segregation enabled by multimodal microscopy”

Tuesday, October 14th, 2014

Dr. Jiji Chen (NCI/NIH, CCR/OD) “Single Molecule Imaging for Cellular Dynamics and Function”