Use of YouScope to Implement Systematic Microscopy Protocols

Moritz Lang1, Fabian Rudolf1, Jörg Stelling1

1 Department of Biosystems Science and Engineering, ETH Zürich and Swiss Institute of Bioinformatics, Basel, Switzerland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.21
DOI:  10.1002/0471142727.mb1421s98
Online Posting Date:  April, 2012
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Abstract

Complex microscopy protocols, e.g., to dynamically track multiple signals in living cells under different conditions, are becoming more common. However, the implementation of complex protocols on modern, motorized microscopes often requires their reformulation into low‐level machine language. This recoding is a time‐consuming and error‐prone task that often requires advanced programming skills. This unit describes how to use the high level, open‐source microscope control platform YouScope to implement complex measurement protocols. Three protocols detail how to install and configure YouScope on a motorized microscope, how to use YouScope to quickly assess the quality of a sample, and how to set up imaging protocols for cells in a microplate. In addition to these protocols, descriptions are given for the use of various other tools YouScope provides to successfully accomplish various microscopy tasks. Curr. Protoc. Mol. Biol. 98:14.21.1‐14.21.23. © 2012 by John Wiley & Sons, Inc.

Keywords: microscopy; automation; screening; fluorescence; single cell

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Installation and Configuration of YouScope
  • Basic Protocol 2: Assessment of Quality of Sample Containing Fluorescent Cells Before Performing a Measurement
  • Basic Protocol 3: Imaging Cells in Multiple Wells of a Microplate
  • Alternate Protocol 1: Burst Imaging Measurement
  • Alternate Protocol 2: Concurrent Protocols Measurement
  • Alternate Protocol 3: Composed‐Imaging Measurement
  • Alternate Protocol 4: Using Scripts in JavaScript and Matlab to Direct YouScope
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Installation and Configuration of YouScope

  Materials
  • Personal computer with Windows XP, Vista or 7 (32‐ or 64‐bit), or a POSIX compatible operating system with sufficient virtual memory, and a large and fast hard drive to store measurement data (depending on the measurement performed, several GB of image data can be created per minute); attached screen with a resolution of at least 1280 × 1024
  • Supported motorized microscope connected to personal computer (see http://www.micro‐manager.org for a list of supported microscope devices)
  • Data sheets for devices attached to the microscope

Basic Protocol 2: Assessment of Quality of Sample Containing Fluorescent Cells Before Performing a Measurement

  Materials
  • Sample of cells to image in at least one well of a 96‐well microplate
  • Microscope with installed and configured YouScope (see protocol 1)

Basic Protocol 3: Imaging Cells in Multiple Wells of a Microplate

  Materials
  • Microplate with several wells filled with cells
  • Microscope with installed and configured YouScope (see protocol 1)

Alternate Protocol 1: Burst Imaging Measurement

  Materials
  • Microscope with installed and configured YouScope (see protocol 1)
  • Matlab (The MathWorks), release R2008a or later
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Figures

Videos

Literature Cited

   Carpenter, A.E., Jones, T.R., Lamprecht, M.R., Clarke, C., Kang, I.H., Friman, O., Guertin, D.A., Chang, J.H., Lindquist, R.A., Moffat, J., Golland, P., and Sabatini, D.M. 2006. CellProfiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7:R100.
   Chernomoretz, A., Bush, A., Yu, R., Gordan, A., and Colman‐Lerner, A. 2008. Using Cell‐ID 1.4 with R for microscope‐based cytometry. Curr. Protoc. Mol. Biol. 84:14.18.1‐14.18.27.
   Collins, T.J. 2007. ImageJ for microscopy. BioTechniques 43:S25‐S30.
   Edelstein, A., Amodaj, N., Hoover, K., Vale, R., and Stuurman, N. 2010. Computer control of microscopes using µManager. Curr. Protoc. Mol. Biol. 92:14.20.1‐14.20.17.
   Gordon, A., Colman‐Lerner, A., Chin, T.E., Benjamin, K.R., Yu, R.C., and Brent, R. 2007. Single‐cell quantification of molecules and rates using open‐source microscope‐based cytometry. Nat. Methods 4:175‐181.
   Kozak, K., Bauch, A., Csucs, G., Pylak, T., and Rinn, B. 2010. Towards a comprehensive open source platform for management and analysis of high content screening data. European Pharmaceutical Rev. 4:34‐38.
   Mitchison, T.J. 2005. Small‐molecule screening and profiling by using automated microscopy. ChemBioChem 6:33‐39.
   Stuurman, N., Amodaj, N., and Vale, R.D. 2007. Micro‐Manager: Open source software for light microscope imaging. Microsc. Today 15:42‐43.
   Taylor, D.L., Haskins, J.R., and Giuliano, K.A. 2007. High content screening, a powerful approach to systems cell biology and drug discovery. Human Press, Totowa, New Jersey.
Internet Resources
   http://www.youscope.org
  Homepage of YouScope. Possibility to download the newest version of the program as well as additional plug‐ins. Help and support section.
  http://valelab.ucsf.edu/∼MM/MMwiki/
  Possibility to download µManager. Since YouScope uses the device drivers developed as part of µManager, information on the installation procedures for specific devices can be found there. Furthermore, this homepage contains an active forum that discusses problems and their solutions of connecting various microscope devices.
  http://www.cisd.ethz.ch/software/openBIS
  OpenBIS is a free database solution to store microscopy images and their meta‐data.
  http://lbms.df.uba.ar
  CellID detects and tracks cells using out‐of‐focus images, and quantifies their fluorescence levels. Provides a graphical user interface and a specialized package for R.
  http://www.cellprofiler.org
  CellProfiler is an open‐source software to detect and track cells and quantify their fluorescence levels.
  http://research.microsoft.com/en‐us/um/redmond/groups/ivm/ICE/
  The Image Composite Editor from Microsoft Research can compose partly overlapping images.
  http://rsb.info.nih.gov/ij/
  Image analysis and processing tool especially suited for microscopy image analysis.
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