Using Cell‐ID 1.4 with R for Microscope‐Based Cytometry

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposely defocused transmission image (sometimes referred to as bright?field or BF) is acquired to segment the image and locate each cell. Fluorescence images (one for each of the color channels to be analyzed) are then acquired by conventional wide?field epifluorescence or confocal microscopy. This method uses the image?processing capabilities of Cell?ID and data analysis by the statistical programming framework R, which is supplemented with a package of routines for analyzing Cell?ID output. Both Cell?ID and the analysis package are open?source. Curr. Protoc. Mol. Biol. 100:14.18.1?14.18.26. © 2012 by John Wiley & Sons, Inc.

Keywords: image processing; fluorescence microscopy; Cell?ID; R

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Introduction
Basic Protocol 1: Extracting Quantitative Information from Single Cells
Alternate Protocol 1: Measuring FRET in Single Cells Using a Beam Splitter
Support Protocol 1: Obtaining and Installing Cell‐ID and R
Support Protocol 2: Preparing Yeast and Mammalian Cells for Imaging
Support Protocol 3: Calculating Nuclear and Plasma Membrane CFP‐YFP FRET Using Split Images
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Extracting Quantitative Information from Single Cells

Materials

Cells of interest affixed to the bottom of multiwell glass‐bottom plates or on slides ( protocol 4 )
Optically appropriate support for cells (multiwell plates are preferred over slides and cover slips for easy access)

Fluorescence microscope with the following features:
- Inverted (to allow access to live cells during imaging over time), preferable
- Stage with motorized z control, preferable to manual focus for convenience and reproducibility
- Stage with motorized xy control (for live imaging of multiple fields of view, e.g., in neighboring wells with cells treated differently), optional
- Motorized shutter (for precisely controlling exposure time of fluorescence illumination), optional for bright‐field illumination
- High‐numerical‐aperture (NA; e.g., >1.2 for 60× objective), chromatically corrected (at least PlanApo) objective to capture as much light as possible, especially important for work that requires subcellular colocalization
- Black‐and‐white (not color for quantitative imaging), cooled CCD camera (when using non‐confocal microscope)
- Appropriate filter cubes or wheels (motorized filter cube turret for complete automation of imaging)
- PC to run the microscope
- Acquisition software, e.g., Metamorph (Molecular Devices), ImagePro (Media Cybernetics), or the open‐source software µManager (unit 14.20 ; http://www.micro‐manager.org) or YouScope (http://www.youscope.org)
Images in TIFF format (gray‐level TIFF files of 8 and 16 bits); time‐related information can be extracted from the Metamorph‐generated image files (see below)
VCell‐ID and Cell‐ID (Gordon et al., ) (included in Microsoft Windows distribution of VCell‐ID)
R (R‐Development‐Team, ) and the package Rcell (see protocol 3 )
PC workstation with UNIX, LINUX, or Windows XP or higher operating systems; or Apple workstation with Mac OS X operating systems, Xcode and X11 installed
For oil immersion with high‐NA objectives: glass‐bottom, coverslip‐thin 96‐ or 384‐well plates (available from Arctic White)
For air or water immersion objectives: plastic or glass‐bottom multiwell plates

Alternate Protocol 1: Measuring FRET in Single Cells Using a Beam Splitter

Image splitter with an appropriate dichroic mirror and top and bottom emission filters, e.g., for CFP/YFP FRET, a Dual View image splitter (Optical Insights/Photometrics, http://www.photometrics.de) fitted with the following filters and dichroic mirrors (Chroma): CFP emission, 480/30M; YFP emission, 535/20M; dichroic mirror, DX505.
Fluorescence microscope: same as in protocol 1 , except remove the emission filter from the cube used to excite the donor fluorophore (e.g., for CFP/YFP FRET, remove the emission filter from the CFP cube); referred to as the FRET cube

Support Protocol 1: Obtaining and Installing Cell‐ID and R

Materials

PC workstation with UNIX, LINUX, or Windows XP or higher operating systems; or Apple workstation with Mac OS X operating systems and X11 installed

Support Protocol 2: Preparing Yeast and Mammalian Cells for Imaging

Materials

Yeast cultures
Appropriate growth medium for required yeast auxotrophies
Synthetic medium (also known as complete minimal medium; see unit 13.1 ) supplemented with all amino acids (except for those omissions required to select for plasmids, if any) and 20 µg/ml of adenine
1 mg/ml concanavalin A (Sigma) in phosphate‐buffered saline (PBS; appendix 22 )
CaCl₂
MgCl₂
Yeast cell culture medium containing an appropriate stimulus
2% or 4% (w/v) paraformaldehyde in PBS ( appendix 22 ) pH 7, 4°C
LB medium (unit 1.1 ) or YPD medium (unit 13.1 )
PBS ( appendix 22 )
1 mg/ml poly‐L‐lysine solution (Sigma)
Mammalian cell cultures (e.g., HEK293, 3T3, HeLa)
Mammalian cell culture medium containing an appropriate stimulus

50‐ml glass tubes, sterile
30°C incubator
Rotating wheel or shaker for test tubes
Imaging plate appropriate for microscope
96‐ or 384‐well glass‐bottom plates
96‐well plastic cell culture plates
Temperature and gas‐controlled incubator mounted on the microscope (see protocol 1 ) for imaging live mammalian cells

Additional reagents and equipment for trypsinizing adherent cells (e.g., see appendix 3F )

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Figures

Figure 14.18.1 Examples of yeast and mammalian cells processed by Cell‐ID. (A ) From left to right: yeast in focus, slightly defocused (note the dark ring on the border of the cells), and the same cells after Cell‐ID has identified each one and traced their borders correctly. Bar = 5 µm. Magnification: 60×. (B ) HEK293 cells fixed, trypsinized, and imaged (see and ), before (left) and after (right) Cell‐ID has located them in the image. Bar = 15 µm. Magnification: 20×.

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Figure 14.18.2 The main VCell‐ID window with the Load Images window open, before selecting the folder containing the images.

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Figure 14.18.3 The main VCell‐ID window with the Image Setup window open, after applying the settings shown in Figure . On the main window at the left, is the directory tree with BF_Position04.tif selected. That image is shown in the central window.

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Figure 14.18.4 The main VCell‐ID window with Segmentation Setup open, after running Cell‐ID with the parameters shown. Note that on the tree directory there are new images, the out.tif files created by Cell‐ID. On the central window the cells have been found by Cell‐ID. Note the presence of several structures in the background wrongly identified by Cell‐ID as cells.

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Figure 14.18.5 The effect of changing the value of “background reject factor.” The same image was processed by Cell‐ID using two different values for this variable, 0.2 and 0.8 (left and right, respectively). Note that with 0.8 fewer spurious cells were found.

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Figure 14.18.6 Plotting with R. The upper left window contains an output of the show.img function. The upper right window shows a plot created with cplot. The bottom window shows the R console, with executed commands.

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Videos

Literature Cited

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