Live-Cell Imaging


 

Live-Cell Imaging

Day 1  |  Day 2 

MONDAY, SEPTEMBER 21

4:00-5:00 Conference Registration

5:00-6:00 Grand Opening Reception in the Exhibit Hall

 

TUESDAY, SEPTEMBER 22

7:00 am Conference Registration and Morning Coffee

7:30-8:15 Breakfast Presentation (Opportunity Available)

Contact Katelin Fitzgerald, Manager, Business Development,
at 781-972-5458 or kfitzgerald@healthtech.com.

 

Novel Probes and Biosensors

8:30-8:35 Chairperson’s Opening Remarks

8:35-9:00 Multidimensional Fluorescence Imaging for Cell Biology, High-Content Analysis and Label-Free Imaging

Paul French, Ph.D., Professor, Physics; Head, Photonics Group, Physics Department, Imperial College London

This talk will review our development and application of multidimensional fluorescence imaging (MDFI) technology, with an emphasis on fluorescence lifetime imaging (FLIM), implemented in microscopy, endoscopy and tomography. Applied to autofluorescence, MDFI can provide label-free molecular contrast in biological tissue for ex vivo and in vivo applications. For cell biology, high speed FLIM can be used to image the spatio-temporal organization of proteins and their interactions, including via FRET, for which we are working to improve the imaging speed and spatial resolution. For high-content analysis, we have developed an automated high-speed optically-sectioned FLIM multiwell plate reader applicable to fixed and live-cells. We have also developed a multiplexed FRET microscope capable of simultaneously imaging two different protein-protein interactions in a cell signaling network. For 3-D imaging of embryos and small organisms, we have developed a FLIM optical projection tomography (OPT) system and are working towards tomographic FLIM and FRET of cleared samples and of live animals for dynamic studies in vivo

9:00-9:25 Photoswitchable Probes for Cellular Imaging

Alexander D.Q. Li, Ph.D., Professor, Chemistry Department, Washington State University

This presentation highlights recent advances of our research dedicated to photoswitchable dyes, photoswitchable fluorescent nanoparticles and their applications in cellular imaging. Several strategies have been developed to enable nanoparticles with optically switchable emission properties, either fluorescence on/off or dual-alternating-color fluorescence photoswitching. Herein, we will discuss the underlying mechanisms of the fluorescence photoswitching that impart fluorescent probes including organic dyes, polymer nanoparticles, and quantum dots. The molecular mechanisms underpin the design of photoswitching nanoparticles. Among all possible applications, two approaches are emphasized. The first is to use photoswitchable dual-color nanoparticles to obtain unambiguous validation of the nano-probes and the other is to empower super-high resolution fluorescence imaging to resolve subcellular structures. Finally, we summarize the important areas regarding future research and development on photoswitchable fluorescent nano-probes.

9:25-9:50 Live Cell Imaging of Endogenous RNA Using Molecular Beacons

Andrew Tsourkas, Ph.D., Assistant Professor, Bioengineering and Radiology, Cellular & Molecular Imaging Group, Department of Bioengineering, University of Pennsylvania

With the recent development of novel techniques for imaging RNA in living cells, it is now possible to study the dynamics of RNA expression and regulation. In this presentation I will review recent developments in the use of 'molecular beacons' for live-cell RNA detection.  Common challenges faced by these fluorescent probes, such as probe design, delivery, and the ability to make quantitative measurements, will be discussed. It is expected that continued advancements in live cell imaging of RNA will open new and exciting opportunities in a wide range of biological and medical applications.

9:50-10:15 Dynamic Visualization of Signaling Activity in Living Cells

Jin Zhang, Ph.D., Associate Professor of Pharmacology, Neuroscience, Johns Hopkins University School of Medicine

The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation and dynamic modulation of the activities of protein kinases, phosphatases and second messengers. To achieve dynamic tracking of signaling activities in living cells, genetically encoded fluorescent reporters for protein kinases, second messengers such as cyclic AMP and phosphoinositides have been engineered. Their development and specific examples of their application will be discussed In this presentation.

10:15-11:15 Networking Coffee Break with Exhibit and Poster Viewing

 

Technology Showcase: Live-Cell Imaging

        Sponsored by
Cyntellect
11:15-11:45 Analysis of Every Cell in Every Well:  Rapid, Whole-Well Brightfield and Fluorescence Imaging of Live Cells without Edge Effects

Fred Koller, Ph.D., President & CEO, Cyntellect, Inc.
Cyntellect provides novel technology platforms for cell analysis, purification, and processing that utilize large-field F-theta scanning lens and galvanometer scanning mirror technologies to rapidly process living cells in situ within cell culture flasks and microplates. The company's new Celigo™ adherent cell cytometer is a benchtop, easy-to-use system that integrates fast label-free brightfield and fluorescence cellular imaging, providing unique whole-well uniform illumination and imaging to analyze ‘every cell in every well.’ These unique capabilities enable systematic analysis of all cells within wells faster than many systems can analyze a small region in the center of a well. Case studies will be shown to demonstrate rapid, label-free cell counting and cell proliferation/growth tracking for culture management and compound screening; colony counting and characterization for stem cells and embryoid bodies; automated quantification of cell secretion from individual live cells; fluorescent analysis of cell health and marker expression assays; as well as the ability to image and quantify large multicellular structures. These combined attributes of Celigo create a powerful and flexible platform for high-throughput, robust cellular analysis.

 
Sponsored byGE


11:45-12:00 New Development in High Content Analysis, Introducing IN Cell Analyzer 2000
Dan Collins, Applications Specialist, GE Healthcare
The IN Cell Analyzer 2000 is the latest addition to the High Content imaging instrumentation arena. It is an impressively enabling platform designed around ease of use, reliability and superior image quality. The new platform expands on the capabilities of the previous generation hardware by delivering a suite of enhanced image restoration techniques, new functionality, with improved hardware and software features. We will discuss the product benefits and provide example of how the new features will enable a wider range of applications.




12:00 pm -12:30 Sponsored Presentations (Opportunities Available)
Contact Katelin Fitzgerald, Manager, Business Development,
at 781-972-5458 or kfitzgerald@healthtech.com.

12:30-2:00 Lunch on your own

 

FLIM-FRET

2:00-2:05 Chairperson’s Opening Remarks

2:05-2:30 FLIM-FRET Microscopy: Red Fluorescent Proteins as an Acceptor

Ammasi Periasamy, Ph.D., Director, Keck Center for Cellular Imaging, Department of Biology, University of Virginia

Currently, most commonly used visible fluorescent proteins for live-cell FRET (Förster resonance energy transfer) studies are the Cerulean and Venus variants of the cyan and yellow fluorescent proteins. Recently, we characterized an ideal donor, the monomeric teal fluorescent protein (mTFP), which is excitable using the commonly available 457(8) nm argon laser line. We used Teal as a donor for various red fluorescent proteins as acceptors including tdTomato, mKO2, mOrange2, mTagRFP, mKate. We have employed a “FRET standard” genetic construct to minimize variability in separation distances and positioning of the fused donor and acceptor FPs. Using fluorescence lifetime (FLIM-FRET) measurements in living cells expressing the fused proteins, we have characterized both sensitized acceptor emission and the change in the donor lifetime distribution as a result of quenching for each of the fused FP pairings. These FRET sensors may be useful in screening the protein molecules in living cells. 

2:30-2:55 Monitoring Dynamic Protein Interactions in the Living Cell Nucleus

Richard N. Day, Ph.D., Professor, Department of Cellular & Integrative Physiology, Indiana University School of Medicine

The mobility of transcription factors and coregulatory proteins within the nuclear compartment reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. We are using Förster resonance energy transfer (FRET)-based microscopy approaches to define these network interactions in living cells. These methods are being used to measure the dynamic interactions between the homeodomain transcription factor Pit-1 and the CCAAT/enhancer binding protein alpha (C/EBPα) in the nucleus of living mouse pituitary cells. In addition, we have monitored dynamic interactions between C/EBPα and the heterochromatin protein-1 alpha (HP1α) in regions of the centromeric heterochromatin in the pituitary cells.

2:55-3:20 Quantitative Molecular Imaging in Living Cells via FLIM

Mary-Ann Mycek, Ph.D., Associate Professor & Associate Chair, Biomedical Engineering; Faculty Member, Applied Physics Program; Core Member, Comprehensive Cancer Center, University of Michigan

Fluorescence lifetime imaging microscopy (FLIM) employs fluorophore lifetime, rather than fluorescence intensity, for image contrast. Compared to intensity-based methods, lifetime imaging requires less calibration and/or correction for fluorophore concentration, photo-bleaching, and other artifacts that affect intensity measurements. FLIM has been employed to probe the microenvironments of endogenous and exogenous fluorophores, including measurements of cellular metabolic co-factors, pH, dissolved gas concentration, and molecular interactions via FRET. Several applications of FLIM for quantitative, live-cell imaging will be described, including studies of cellular metabolic pathways, improved FRET detection of oncogene association, microfluidic bioreactor characterization for continuous cell culture, and improved precision for low-light FLIM imaging. 

3:20-4:15 Networking Refreshment Break with Exhibit and Poster Viewing

4:15-4:40 Monitoring Dynamic GPCR Signaling Using Fluorescence Microscopy, FRET Imaging and Single Molecule Analyses

Tian Jin, Ph.D., Chief, Chemotaxis Signal Section; Investigator, NIAID, National Institutes of Health

How a eukaryotic cell translates the small concentration difference of a chemoattractant on its surface into highly polarized intracellular responses is a fundamental question in chemotaxis. Chemoattractants are detected by G-protein coupled receptors (GPCRs). Binding of chemoattractants to GPCRs induces the dissociation of heterotrimeric G-protein into Gα and Gβγ subunits. To fully understand chemotaxis, it is essential to quantitatively measure dynamic changes of chemoattractant concentrations around cells, activation of heterotrimeric G-proteins, and mobility of GPCR and G-protein subunits in the cell membrane. Here, I will introduce fluorescence imaging methods, including fluorescence resonance energy transfer (FRET) imaging and a single molecule analysis, to determine the dynamic properties of GPCR signaling in single live-cells. 

4:40-5:05 A High-Contrast Method for Detecting Intermolecular FRET between Fluorescent Proteins

Mark Rizzo, Ph.D., Assistant Professor, Department of Physiology, University of Maryland School of Medicine

Protein-protein interactions can be detected in living cells by incorporating Förster resonance energy transfer (FRET) measurements into live-cell microscopy. However, conventional ratio-imaging approaches are poorly suited towards detecting fluorescent protein-labeled constructs because of the problems introduced by fluorescence crosstalk. Here we discuss a new method for detecting FRET based on the observation that FRET fluorescence is depolarized. This method for FRET detection is simple to implement, high contrast, and free of artifacts that give a false-positive indication of FRET.

5:05-5:30 FRET-Based Determination of Structure and Distribution of Protein Complexes in Living Cells

Valerica Raicu, Ph.D., Assistant Professor, Departments of Physics and Biological Sciences, University of Wisconsin

This talk will present recent progress in the development of Förster (or Fluorescence) Resonance Energy Transfer (FRET) into a fully quantitative method for determination of structure and localization in living cells of protein complexes. I will begin by identifying the main requirements that any quantitative FRET technology for in vivo investigations should meet. These will be discussed in the broader context of information extraction from fluorescence images of molecular distributions undergoing continuous changes. I will then describe a method for imaging protein complex distributions and for determination of the size and geometry of such complexes in living cells. The talk will conclude with an overview of our recent results obtained using this method to study oligomeric complexes of some G protein-coupled receptors in vivo.

5:30 Close of Day


For more information, please contact:
Julia Boguslavsky, Executive Director, Conferences
Cambridge Healthtech Institute
E-mail: juliab@healthtech.com

For sponsorship information, please contact:
Katelin Fitzgerald, Manager, Business Development
Cambridge Healthtech Institute
Phone: 781-972-5458; E-mail: kfitzgerald@healthtech.com



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