CYTOMETRY SEMINARS 2006-PRESENT

Ongoing series of Cytometry seminars for Immune Disease Institute and Harvard Medical School researchers (some abstracts seen below).

 PHOTOACTIVATABLE FLUORESCENT PROTEINS

Vladislav Verkhusha, Albert Einstein College of Medicine
Department of Anatomy and Structural Biology

Genetically encoded fusions of cellular proteins with a green fluorescent protein (GFP) from the jellyfish Aequorea victoria (class Hydrozoa) or with its multicolour homologues from class Anthozoa have become indispensable tools in cell biology. Such fusion proteins enable the non-invasive analysis of protein localization and dynamics in living cells owing to unique ability of GFP-like fluorescent proteins (FPs) and chromoproteins (CPs) to form chromophores autocatalytically without the involvement of external enzymes and cofactors except the molecular oxygen. Recently, a novel methodology has emerged with the development of so-called photoactivatable fluorescent proteins (PAFPs), which are capable of pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength, intensity and duration. Some PAFPs convert from very low-fluorescent (i.e., dark) to bright fluorescent state (photoactivation), others change fluorescence color (photoswitching or photoconversion). We use a general term "photoactivation" for all PAFPs since their applications are always based on drastic increase of the fluorescent signal. PAFPs seem to be an excellent tool for the precise optical labelling and spatiotemporal tracking of proteins, organelles and cells within living systems. PAFPs bring a new dimension to the kinetic microscopy of living cells, which was traditionally associated with fluorescence recovery after photobleaching (FRAP) approaches. We present the properties and in vivo applications for several PAFPs, including photoswitchable monomeric green-to-red Dendra and cyan-to-green PSCFP. We also discuss spectral and photochemical properties of several other new monomeric FPs.

After Cytometry Seminar
Howard Shapiro and Amit Tzur (Dept. System Biology)

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Cytometry in the Fast Lane: New Tools to Enable High Content Analysis  
Jonni Moore, University of Pennsylvania

The biotechnology revolution ushers in an era of high information content analytical methodologies that facilitate systems biology approaches to cell based- analysis. Flow cytometry is positioned to take full advantage of this era of high dimensional biology and can rapidly provide information that can impact research, discovery, translation and clinical practice. In this seminar, the presents will explore the concepts of high dimensional cytometry by discussing some new functional probes and computational approaches.

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NOVEL LASERS FOR FLOW CYTOMETRY:  COVERING THE VISIBLE SPECTRUM.
William Telford , NIH, NCI

Flow cytometry depends on laser light to excite our fluorescent probes.
Unfortunately, flow cytometers have traditionally been equipped with
very few lasers, usually just the usual blue-green 488 nm and
occasionally a red HeNe or diode. This has limited the range of
fluorescent probes we can analyze.  Recent advances in solid state laser
technology, however, now allow us to generate virtually any laser
wavelength we need.  This permits us to excite essentially any
fluorescent molecule, including the newest generation of expressible
fluorescent proteins.  Our laboratory has incorporated this technology
into working flow cytometers, giving investigators immediate access to this technology.  We discuss both lasers that emit both discrete
wavelengths, and white-light supercontinuum lasers that emit ALL
wavelengths simultaneously.   We also cover how they can be applied
to real-world applications in biomedical science.

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Novel Flow Cytometric Techniques for Analysis of Protein Phosphorylation and Signaling Networks
Robert Balderas, Vice-president of Research & Development,
BD BIOSCIENCES

Intracellular assays of signaling systems have been limited by the
inability to correlate functional subsets of cells in complex populations
on the basis of active protein states within the native context of the
cell. We demonstrate the ability to simultaneously monitor active protein states via phospho-epitope recognition in subpopulations of complex cell populations by multiparameter flow-cytometric analysis. Multi-dimensional assessment of active protein states, in combination with surface marker and other flow cytometric detectable parameters (ie, cytokines, apoptosis), can provide functional assessment on a single cell level that may have utility in clinical diagnostics and/or disease progression. Furthermore, the ability to profile both activating and inhibiting conditions of multiple protein states simultaneously within the cell in a rapid and parallel manner may be extended to pharmaceutical screening of compounds. Detailed examples for the utililty of assessing the signaling pathways using flow cytometry in  T/B cell and Cancer Models has been provided.

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HaloTag ®: Novel Protein Labeling Technology for Cell Imaging and Protein Analysis
Georgyi V. LOS , Imaging Group Leader, PROMEGA

Development of novel reporter systems for live cell imaging and protein analysis is essential for further advancement of life science research and drug discovery. Recently we have described a technology for site-specific covalent tethering of synthetic ligands to a reporting protein, HaloTag ®, in living cells and in vitro. The reporter protein is a genetically engineered catalytically inactive derivative of hydrolase, has globular structure and remains monomeric even at high concentrations. The ligands are small chemical tags capable of carrying a variety of functionalities, such as fluorescent labels, affinity handles, or attachments to a solid phase. The covalent bond forms rapidly under general physiological conditions, is highly specific, and essentially irreversible. Fusion of the HaloTag ® protein to cytosolic, membrane anchored, or transmembrane proteins does not interfere with native protein function. The open architecture of the technology ensures interchangeability of ligands, thereby facilitating a variety of functional studies (including imaging at different wavelengths and temporal or spatial separation of protein pools) without requiring changes to the underlying genetic construct. The stability of the bond between the HaloTag ® protein and HaloTag ® ligands allows imaging of live cells during long periods of time, imaging of fixed cells, stimulated emission depletion (STED) microscopy, and multiplexing with different cell/protein analysis techniques. The HaloTag ® protein retains activity upon fixation making possible covalent tethering of functional groups to fusion proteins in fixed cells. Attachment of the HaloTag ® ligands to different surfaces allows oriented, highly specific covalent immobilization of HaloTag ® fusion proteins. These surfaces can be used for detection of protein-protein and protein: DNA interactions.