The Presenters

Beth Cimini and Kyle Karhohs
Broad Institute of Harvard and MIT

Session Summary

Microscopy experiments have proven to be a powerful means of generating information-rich data for biological applications. From small-scale microscopy experiments to time-lapse movies and high-throughput screens, automatic image analysis is more objective and quantitative and less tedious than visual inspection. This course will introduce users to the free open-source image analysis program CellProfiler and its companion data exploration program CellProfiler Analyst. We will show how CellProfiler can be used to analyze a variety of types of imaging experiments. We will also briefly discuss the basic principles of supervised machine learning with CellProfiler Analyst in order to score complex and subtle phenotypes.

Session Outline

• Basics of image analysis.
• Methods of distinguishing objects of interest from the image background.
• Methods of separating clusters of touching objects.
• Obtaining measurements from the cell.
• Exporting measurements and images.

CMLE Credit: 1.0

The Presenters

Joanne Lannigan, MS
Director of the Flow Cytometry Core Facility
School of Medicine at the University of Virginia

Monica DeLay, MS
US Manager - Technical Application Support
Cytek Biosciences

Session Summary

In November 2016, the ISAC Shared Resource Laboratory (SRL) Task Force published a document outlining a set of “best practices” for SRLs, or "core facilities," to use as a general guide for achieving and maintaining standards of excellence in the services they provide. These best practices highlight several important areas that impact the efficiency, quality, and reproducibility of services provided by these facilities. The purpose of this tutorial is to provide guidance and generate discussion and feedback on how to best implement these “best practices” across a wide variety of these facilities.

Outline

1. Introduction of the "best practices" and what is hoped to be achieved through implementation.
2. Brief presentation and background information on each topic including SOPs, training and education, quality assurance, laboratory safety, data management, staffing, and operations.
3. Identification of potential roadblocks and hurdles and how to overcome them.
4. Benefits of implementation and adherence to these best practices.

CMLE Credit: 1.5

The Presenter

Caroyln Coyne, PhD
University of Pittsburgh

Session Summary

In eutherian organisms, the placenta acts as the key barrier between the maternal and fetal compartments and protects the developing fetus from the vertical transmission of viruses and other microorganisms. The human placenta is unlike any other human organ. Given its critical role in protecting the fetus, it must function not only as a barrier and conduit between the maternal and fetal microenvironments, but must also act as an active immunological tissue that responds to microbes present in the maternal circulation. 

Within the human hemochorial placenta, the frontlines of fetal protection are the syncytiotrophoblasts (SYNs), cells that cover the surface of the human placental villous trees. Very little is known about the cell intrinsic pathways that exist in SYNs to defend against viral infections, largely owing to the lack of appropriate cell-based systems to model the unique functional and morphological features of these specialized cells in vivo. We have shown previously that primary human SYNs isolated from full-term placentas constitutively release type III interferons (IFNs) to limit the replication of viruses, including Zika virus (ZIKV).

In addition, we have developed three-dimensional in vitro cell-line based models of human SYNs and second trimester human explant models that recapitulate the high resistance of SYNs to ZIKV infection and their constitutive release of type III IFNs. Collectively, these models provide new platforms to dissect the mechanisms involved in the protection of the human placenta from viral infections at many stages of human gestation.

CMLE Credit: 1.5

The Presenter

Jessica Mark Welch
Associate Scientist
University of Chicago

Session Summary

The spatial structure of complex natural microbiomes at the micron scale is largely unexplored, yet it is critical to understanding the interactions of individual taxa with one another and with the substrate or host tissue. Using ribosomal RNA sequence analysis and spectral imaging fluorescence in situ hybridization, we are investigating the micron-scale spatial organization of the human oral microbiome. We show that a limited set of bacterial taxa form the structural and spatial framework of dental plaque, consisting of a radially-organized, 10-taxon structure nucleated around the filamentous taxon Corynebacterium. Within this structure, individual taxa localize in ways consistent with their known metabolic requirements. Additional distinctive microbial consortia colonize other sites within the human mouth such as the tongue and buccal mucosa. These complex and highly-organized consortia illustrate how complex spatial organization can emerge from the micron-scale interactions of individual organisms. They provide a framework for understanding the spatial organization and community structure of the human microbiome and the micron-scale taxon-taxon associations of its component taxa.

CMLE Credit: 0.5

The Presenters

Holger Hennig, PhD and Minh Doan, PhD
Broad Institute of Harvard an MIT
Department of System Biology and Bioinformatics
University of Rostock, Germany

Session Summary

Imaging flow cytometry (IFC) enables the high-throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high-content, information-rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. 

In this tutorial, we demonstrate a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated image files (.cif) from an imaging flow cytometer are generated with the software IDEAS from Millipore. The .cif files are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analyzed with cutting-edge machine learning and clustering approaches using ‘‘user-friendly” platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data sets.

Session Outline

• Generating image tiles from .cif files and importing the images tiles into CellProfiler.
• CellProfiler pipeline for object segmentation and extraction of hundreds of morphological features per cell.
• Machine learning using CellProfiler Analyst.
• Machine learning using custom scripts (e.g., R and Python)

CMLE Credit: 1.5

The Presenter

Bruce Bagwell, MD, PhD
Verity Software House

Session Summary

In this tutorial, a step-by-step explanation of how t-SNE works will be presented. The tutorial will present some of the problems with the original implementation which dramatically limited its potential resolution of cytometry-defined cellular populations. A new variant of the method called Verity Cen-se will be presented that is considerably faster than the original method and presents populations in unprecedented high resolution. Several examples and experiments demonstrating the value of this tool will also be presented.

CMLE Credit: 1.5

The Presenter

Gillian Isabelle, PhD

Session Summary

This tutorial is aimed at scientists in academia who are considering starting a company to commercialize their inventions but have limited experience in doing so. The goal of the tutorial is to describe what a value proposition is and how a startup can define, defend, and demonstrate its value proposition. The format will be interactive, incorporating real-world examples, as well as discussion among attendees.

CMLE Credit: 1.0

The Presenter

P. Rogers, J. Bochicchio, and C. Nusbaum
Broad Institute of MIT and Harvard

Session Objectives

• To learn the best practices for sorting single cells on different cell sorters for the range of genomics applications.
• To understand how to set up your facility to facilitate single cell sorting experiments.
• To understand how post-sorting handling and storage can affect cells in downstream analyses.
• To recognize warning signs of degraded cells in library construction.
• To get a view of the most commonly used techniques in single cell analyses, including RNAseq, DropSeq, and 10x.
• To learn how to troubleshoot when processes fail.

CMLE Credit: 1.5

The Presenters

Gelo Dela Cruz
Danish Stem Cell Center
Denmark

Peter Lopez
NYU School of Medicine
New York

Michael Thomson
Monash Health Translation Precinct
Australia

Session Summary

In establishing a new SRL Facility, or comprehensively upgrading an existing one, many factors have to be considered beyond the scope of purchasing an instrument and hiring someone to operate it. There is a wealth of expertise and published literature that can act as a guide, but major challenges arise in order to successfully implement the best practices needed to have a functional and productive SRL. It is the task of the new facility manager to gather detailed information about the need for the core facility and refine this knowledge to get a realistic image and plan to set up and equip the shared resource lab.

Session Overview

• Selecting the right manager and support staff.
• Ensuring the dedicated space fulfils the needs for now and the future, depending on the space allocated, or the space that can be made.
• Understanding your user base, the direction of the institute, the access to other SRLs, and basing instrument purchases and infrastructure construction around these.
• Funding considerations: allocating and ensuring money streams to fulfil the needs and wants of the SRL.
• Ancillary equipment and infrastructure considerations beyond flow cytometers.

CMLE Credit: 1.5

The Presenter

Brian Capaldo, PhD
Staff Scientist
National Cancer Institute

Session Summary

This course seeks to provide an introduction to R programming and the cytometry-focused tools in the Bioconductor libraries.

I. Overview of R and Bioconductor

1. Installing R.
2. Installing libraries in R.
3. Installing Bioconductor libraries in R.
4. Introduction to cytometry packages available in Bioconductor.
5. Introduction to cytometry packages not available in Bioconductor.

II. Basics of Flow Cytometry Standard file manipulation in R

III. The power of Bioconductor vignettes

1. Using a vignette to analyze your own data.
2. Stringing vignettes together to create your own analysis pipelines.

IV. Further resources to explore

1. CyTOF Forum.
2. Bioconductor mailing list.
3. GitHub, SourceForge, and Reddit discussion boards.

V. Conclusions/Discussion

CMLE Credit: 1.5