2015 Program Scientific Tutorials
Scientific Tutorials are open to all Full Congress
Registrants; no additional fees apply.

10:00 - 11:30

Fluorescent Proteins
Teresa Hawley, The George Washington University, USA
Bill Telford, National Institutes of Health (NIH), USA

John Nolan, Scintillon Institute, USA

In a scientific tutorial in 2011, one of us (W.T.) covered the fluorescent proteins (FPs) available at the time. As a follow-up, we will focus on recent developments in the field, including new FPs, new lasers, and the advances made in detecting FPs using spectral flow cytometry. We will discuss considerations for practical use of FPs and make recommendations for choice of FPs in flow cytometry. We will analyze FP data using fluorescence compensation for conventional flow cytometry and unmixing algorithms for spectral flow cytometry. Attendees are encouraged to bring along their own data.
Upon completion of this tutorial, the attendee will have an overview of the properties of commonly used FPs, the pertinent information to create optimal combinations of FPs, the knowledge to devise feasible detection strategies, and the skill to analyze FP data.

LABEL-FREE IMAGING: Ptychography-Label-Free Cytometry
Peter O'Toole, Univeresity of York, United Kingdom
Martin Humphry, Phasefocus, United Kingdom

This tutorial will cover the emerging area of label free microscopy, focusing on ptychography, but also introducing holography and other quantitative and semi-quantitative label free imaging approaches for cytometric analysis of cells. Examples of cell cycle, cell proliferation, cell death and live cell tracking will be given.

The student will leave with an insight in to what will possibly be the next big area of development in biological imaging. They will be able to understand the basic concepts, need and approaches available and leave enthused about the potential applications that this may open up for themselves and bioimaging in general.
Making Resource Technology a Career: Developing, Promoting and Retaining Core Leadership and Staff
Jonni Moore, University of Pennsylvania, USA

  1. The success of any shared resource lab is primarily dependent upon the quality of the resource scientists in the lab. It is recognized that this is a unique career where experience often plays a more important role than formal education and customer service is as important as technical knowledge. The “human capital” in a SRL is worth way more than the capital equipment, and deserves significant attention to maintain and develop. This tutorial will cover challenges and solutions to recruiting, developing and retaining high quality SRL staff and leadership.
  1. Participation in this session should provide the attendee with
  1. An understanding of how a resource scientist differs from a research scientist
  2. How to work with institutional human resources to create appropriate job classifications
  3. How to work with academic leadership to define faculty roles in SRLs
  4. How to work continuing education and staff development into your workflow and budgeting.
  5. How to identify and institute best practices to assure a rewarding work environment for SRL staff.
OMERO in Action
Will Moore, University of Dundee, United Kingdom
Petr Walcyzsko, University of Dundee, United Kingdom

In this tutorial, we will outline and demonstrate the OMERO platform, and show how you can use it to work with your microscopy and/or HCS data. In addition we will demonstrate some of the applications that have been released by OME and some of the integration with 3rd party tools, including:
  • OMERO.figure - fast figures from your OMERO images
  • OMERO.webtagging - automatically tag your data and search for tags
  • ImageJ - improved interaction with OMERO
We will also outline other applications and integration e.g. FLIM image data analysis, object tracking, Matlab Analysis tools, etc. We’ve designed OMERO to be as flexible as possible, and this has enabled its use in a range of imaging domains, including light and electron microscopy, high content screening. Come along to the workshop and bring your favourite data

12:45 - 14:15

RNA Flow Cytometry
Paul Wallace, Roswell Park Cancer Institute, USA
Steve McClellan, University of South Alabama, USA
Chris Groves, MedImmune, USA

Flow cytometry permits the simultaneous measurements of many biomarkers in individual cells from bulk populations. Until now analysis has been limited, however to primarily analysis of proteins and total DNA or highly abundant DNA sequences. Since most RNA gene transcripts are present at very low quantities our ability to detect these mRNA species by flow cytometry has been limited. In 1993, Patterson et. al. (Science, 1993. 260:976) used a PCR driven in situ hybridization technique to detect HIV mRNA in infected cells, however the stringent conditions required by this technique prevented its wide spread application to and development for flow cytometry. Two new techniques, PrimeFlowTM (Affymetrix/eBioscience) and SmartFlareTM (EMD Millipore) have recently been commercially introduced and are seeing increasing use among research scientists.
This tutorial will discuss:
  • The methodology leading up to the current techniques
  • The PrimeFlowTM branched DNA methodology and applications
  • The SmartFlareTM technique and procedures
  • Pitfalls, quality control and analysis strategies for both techniques
  • Methods to simultaneously combine mRNA measurements with labeling of surface and intracellular proteins
The PrimeFlowTM RNA assay system from Affymetrix/eBioscience allows for the determination of differential RNA expression within a mixed population of cells. The assay improves sensitivity and lowers background of fluorescent in situ hybridization (FISH) through the use of branched DNA signal amplification. Protocols and data will be presented showing the usefulness of this system to identify RNA expression on bone marrow B cells subsets. We will show a direct comparison of this method to a much more time consuming process of sorting cells for microarray analysis. This method provides a sophisticated molecular tool for the characterization of gene expression on rare cell subsets within tissue.
The SmartFlareTM system from EMD Millipore employs a gold nanoparticle that is actively endocytosed into most cell types.  In the cytoplasm, interaction with the specific mRNA target causes the generation of a fluorescent signal, which can be detected by flow cytometry or microscopy. Protocols and data will be presented showing the usefulness of this system to sort cells based on mRNA expression, for several applications such as isolation of cancer stem cells from tumors and optimizing shRNA or CRISPR knockdown clone selection.  This new technology now enables the non-destructive detection of intracellular epitopes in living cells.
By the end of this tutorial, attendees will have an understanding of which technique is most appropriate for their applications; how to apply them to their research and most importantly gain an understanding of the pitfalls and solutions from individuals who have learned these the hard way.

CellCognition: Image Analysis of Live Cell Imaging Data
Christoph Sommer, Institute of Molecular Biotecnology (IMBA), Austria

Automated time-lapse microscopy has become an enabling technology to monitor and quantify dynamics of cells and subcellular structures. A typical workflow to analyze time-lapse movies comprises the segmentation of cells, linking of cell objects over time by tracking, and classification of cellular phenotypes. In this tutorial session, the participants will be familiarized with common methods and workflows for the analysis of live cell imaging data. The tool for this tutorial session will be CellCognition (www.cellcognition.org), an open-source software platform for the analysis of live cell imaging data in the context of High-Content-Screening. The software contains a graphical user interface, which allows also non-experts to parameterize and run complex workflows on time-resolved image data.

Bridging Flow Cytometry with New Technologies
Monica DeLay, Cincinnati Children's Hospital Medical Center, USA
Sherry Thornton, Cincinnati Children's Hospital Medical Center, USA

Research projects frequently encompass technologies from more than one Shared Resource Lab (SRL).   Novel applications, such as single-cell RNASeq downstream of cell sorting, may require the involvement of several SRLs from sample collection to data analysis.  Often little to no communication between the client and the core staff occurs with regards to the experimental goals or the upstream and downstream processes related to a service request.  This tutorial will discuss applications for which multiple core technologies are needed to complete a research project and strategies for effective communication, cooperation and coordination between staff of different cores.   Upon conclusion of the tutorial, attendees will have specific tools to aid in the integration of new technologies from other cores into investigator-driven projects that utilize their SRL.

ASK THE EXPERTS: Quality Control in Image Cytometry
Michael Halter, National Institute of Standards and Technology (NIST), USA
Silas Leavesley, University of South Alabama, USA
Stephen Lockett, National Instituesof Health (NIH), USA

Quantitative fluorescence microscopy is an important tool for measuring cellular properties (i.e. image cytometry). The reproducibility of these measurements can be significantly improved by the use of standard instrument performance criteria, such as limit of detection sensitivity, linear dynamic range, spatial resolution (point spread function), and spectral response. Assuring that an instrument’s performance is comparable from day to day can provide evidence that an instrument is working reliably and facilitate the evaluation of quantitative image cytometry data. This tutorial will instruct students on practical and routine methods for evaluating the performance of a fluorescence microscope used for an image cytometry assay. When possible, reference materials for benchmarking the performance of a microscope will also be suggested. Students will also be provided with a checklist that can be used to assure that fluorescence microscopy image data is appropriate for quantitative analysis.

Cell Sorting: Fundamentals, Applications and Troubleshooting
Simon Monard, Walter+Eliza Hall Institute of Medical Research, Australia

The aim of this session is to provide participants with a solid background in the principles of cell sorting. Ideally the material is presented in a manner than encourages interactions with the participants and stimulates them to think about how cell sorting can be practically applied to help solve scientific problems.
The session will discuss how electrostatic cell sorters operate. Starting from basic principles, the hardware that is widely used will be covered followed by the practical constraints that hardware has on experimental design and the expected outcomes.
The types of sort strategies that can be made, the expected results and some of the reasons things go wrong, and what to do about them, will also be addressed.

14:30 - 16:00

Forensic Flow Cytometry: Multicolor
Pratip Chattopadhyay, National Institutes of Health (NIH), USA
Jennifer Wilshire, Memorial Sloan-Kettering Cancern Center, USA

Troubleshooting is a critical skill in flow cytometry.  In this tutorial, we will demonstrate how to troubleshoot flow cytometry data through the use of case studies and real-life examples.  We will cover common issues in staining, compensation, and data analysis and discuss practical methods to avoid these issues.

"SUPER" RESOLUTION: Fluorescence Microscopy
Rainer Heintzmann, Leibniz Institute of Photonic Technology, Germany

This tutorial covers superresolution microscopy from the basic concepts to some recent schemes. It will include methods such as stimulated emission depletion (STED) or photoactivated localization microscopy (PALM), which received the Nobel price for chemistry in 2014. However, other techniques such as linear and non-linear structured illumination microscopy (SIM) or optical reassignment (OPRA) will also be covered. Finally, an implementation of light-sheet microscopy, which is not really superresolution microscopy, but allows the high-resolution imaging of large 3-dimensional sample volumes is discussed.

The student will gain a good understanding of Abbe’s theory of resolution and a feeling of several ways, how to circumvent this limit in fluorescence microscopy.

Flow Cytometry Data Management - drinking from the firehose (without getting wet)
Wade Rogers, University of Pennsylvania, USA

Flow Cytometry is a high-content technology, and unique in the complexity, not to mention the sheer volume of data of data that it generates.  Typical experiments in the “pre-digital” era averaged in the 1-10 Mbyte range, whereas experiments done on modern digital instruments average over 100 times as much, and some extend well above 1000-fold larger.  Techniques that were once adequate to manage data were not scalable, now leaving researchers and core facilities in a quandary: how do they store, organize, transfer, back up, archive and analyze these very large volumes of data?
In this workshop we will get into the way-back machine and travel back to the time when storing and distributing data on CDs seemed so much better than floppy disks or ZIP drives.  We will then pose the problem of today’s data in the context of yesterday’s technology and finally illustrate with examples some solutions that current computing technology, together with innovative software, have to offer.
After the tutorial, the participant will understand the full dimension of the challenge of data management, and will be conversant in the technologies and strategies that can be utilized to effectively “drink from the firehose”.

Processing Images Using the Free and Open-Source Software Icy
Fabrice de Chaumont, Institut Pasteur, France

This tutorial presents how to investigate an image, by extracting quantitative information. This tutorial is presented as an interactive study which is performed live, using Icy. The audience will participate and will propose interpretation of the problem, and of course, there will be a lot of traps! It covers a large number of topics: understanding the nature of noise in the image, understanding the interest of different representations of the images: the richness of 2D and 3D rendering in different modalities, the use of color maps and the practical use of histograms. In a second step, more advanced algorithms such as wavelets for spot detection and MHT for tracking of particles are also covered.
During this presentation, a number of Icy’s functionalities are covered: visualization, use of ROI, histogram, look up table, running and installing plugins, scripting in JavaScript and Python, graphical scripting using protocols, management of data, method reusability and how to reach unknown functionalities.
Students coming to this session will learn in a didactic and ludic way why the noise is so important in the images and what the good practices of the image analysis are. In this interactive session, they will discover that one needs to deeply understand his/her data before performing an analysis. Each step performed during this tutorial is reproducible since the software and the data are free and available for download. At the end of the session, the attendants can perform and extend their analysis directly on their laptops.

Flow Cytometry Biosafety
Kevin Holmes, NIAID, National Institutes of Health (NIH), USA
Hank Pletcher, University of Pennsylvania, USA
Phil Hogarth, Animal Health and Veterinary Laboratories Agency, United Kingdom
Robert Wadley, Mater Medical Research Institute, Australia
Simon Monard, WEHI, Australia
Steve P. Perfetto, VRC, NIAID, National Institutes of Health (NIH), USA
Ingrid Schmid, UCLA, USA

This tutorial will provide a summary of biosafety principles as they apply to flow cytometry and cell sorting, with emphasis on the new ISAC Cell Sorter Biosafety Standards.

This tutorial will also provide a forum in which to discuss with experts in the field, specific scenarios that operators or core facility managers encounter.

After participation in this tutorial the attendee should have a clearer understanding of the principles and practices of biosafety as it pertains to flow cytometry, in particular cell sorting.  Additionally, the attendee will have a list of resources to aid in risk assessment and the development of Standard Operating procedures in their own lab.
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