Accurate, precise modeling of cell proliferation kinetics from time-lapse imaging and automated image analysis of agar yeast culture arrays

Background Genome-wide mutant strain collections have increased demand for high throughput cellular phenotyping (HTCP). For example, investigators use HTCP to investigate interactions between gene deletion mutations and additional chemical or genetic perturbations by assessing differences in cell proliferation among the collection of 5000 S. cerevisiae gene deletion strains. Such studies have thus far been predominantly qualitative, using agar cell arrays to subjectively score growth differences. Quantitative systems level analysis of gene interactions would be enabled by more precise HTCP methods, such as kinetic analysis of cell proliferation in liquid culture by optical density. However, requirements for processing liquid cultures make them relatively cumbersome and low throughput compared to agar. To improve HTCP performance and advance capabilities for quantifying interactions, YeastXtract software was developed for automated analysis of cell array images. Results YeastXtract software was developed for kinetic growth curve analysis of spotted agar cultures. The accuracy and precision for image analysis of agar culture arrays was comparable to OD measurements of liquid cultures. Using YeastXtract, image intensity vs. biomass of spot cultures was linearly correlated over two orders of magnitude. Thus cell proliferation could be measured over about seven generations, including four to five generations of relatively constant exponential phase growth. Spot area normalization reduced the variation in measurements of total growth efficiency. A growth model, based on the logistic function, increased precision and accuracy of maximum specific rate measurements, compared to empirical methods. The logistic function model was also more robust against data sparseness, meaning that less data was required to obtain accurate, precise, quantitative growth phenotypes. Conclusion Microbial cultures spotted onto agar media are widely used for genotype-phenotype analysis, however quantitative HTCP methods capable of measuring kinetic growth rates have not been available previously. YeastXtract provides objective, automated, quantitative, image analysis of agar cell culture arrays. Fitting the resulting data to a logistic equation-based growth model yields robust, accurate growth rate information. These methods allow the incorporation of imaging and automated image analysis of cell arrays, grown on solid agar media, into HTCP-driven experimental approaches, such as global, quantitative analysis of gene interaction networks.

individual plates with culture spots circumscribed by ellipses and hence enables the user to quickly assess accuracy of spot detection. Sample images, screenshots of the application, and detailed instructions are available at the website. The program and the underlying source code can be downloaded free of cost and used under the Creative Commons Attribution-ShareAlike 2.5 license. The software has a modular design to facilitate modification and development of related imaging imaging applications.
YeastXtract takes as its input a time series of images of 8 x 12 cell arrays. Over time, the yeast cells grow and the culture spots become darker. YeastXtract analyzes the time-lapse images and outputs enumerated data for calculating cell proliferation phenotypes for each time series of data for a culture. To make the imaging process more efficient, plates are scanned together in groups of ten. The imaging rate is around 250 -300 plates per hour by this manual method.

System requirements
The basic system requirements to run YeastXtract are: ! Java 1.5 or better. Freely available at http://www.java.com. ! 512 MB or more physical memory recommended. ! For added performance, install the freely available Advanced Imaging (JAI) API (http://java.sun.com/products/java-media/jai/Java).

Installation Installation via Java WebStart
The easiest way to use YeastXtract is to not install it. For most system configurations, YeastXtract can be launched by clicking on the 'Launch YeastXtract' link at the top of this page. Users are advised to try this method of running YeastXtract first. Once you click on the link, a security dialog box will ask you whether or not you want to run the application. After you click the 'Run' button, Java Webstart will automatically download and install YeastXtract and all of its associated modules; this may take several minutes, depending on the speed of your network connection. If all goes well, a screen similar to the screenshot shown below should come up.
If this does not work or if you get an error message such as 'Cache viewer has unexpectedly quit,' you can try the stand-alone installer described in the next section.

Installation via stand-alone installer
Due to problems with Java Webstart, a minority of users might not be able to launch YeastXtract directly. Such users should still be able to run YeastXtract by downloading and installing a stand-alone version: ! Download and save the YeastXtract stand-alone installer by clicking on the link at the top of this page. ! Double-click on the file you just downloaded to start the installer application. ! The installer will ask for a target directory to install the application in. Make a note of this directory. ! After the installer exits, browse to the installation directory and double-click on the 'YeastExtractGUI' icon. ! If all goes well, a screen similar to the screenshot shown below should come up.

Problems
YeastXtract has been tested on Windows XP, Mac OS X, and several flavors of Linux and has been found to work flawlessly on systems that meet the above-stated requirements. If for some reason you are having problems installing or running YeastXtract, please do not hesitate to contact us. We will work with you to get the program running on your machine and also fix any compatibility issues that you may uncover.

Image Analysis
Quick start To analyze a set of images with YeastXtract, follow these steps: 1. Start the YeastXtract application. If you have Windows XP, a window similar to the following screenshot should be displayed. If you are using another operating system, the format of the interface should be very similar.
2. You will need to add some images to analyze first. Click on the 'Add File' button to add an image to your analysis set. A preview shot of the image you added will be shown in the 'Selected Image' pane below. The full path and name of the image file will be added to the 'Image Files (TIFF)' list. 3. Repeat this process until you've added all of the files that you want analyzed. Make sure that the files are listed in increasing order with respect to time; i.e. the image for the latest timepoint of your experiment should be the last file in the list. You can re-arrange this order by using the 'Move Up' and 'Move Down' buttons. 4. Next, you have to specify a temporary directory. YeastXtract generates a number of intermediary images and hence requires a location to store them. We recommend that you create a new folder on your computer and delete it after you complete your analysis. To specify a temporary folder, click on 'Browse' and use the interface to choose your preferred folder. 5. Next, you will specify the number of plates you want analyzed. For the sample dataset, this number will be 3. 6. Simply click the 'Start Analysis' button to begin image analysis. If you want to be entertained while you're waiting for analysis to complete, click on the 'Last generated image' tab and you'll see a delayed slideshow of images created by YeastXtract.

Output format
When image analysis is complete, YeastXtract will display some text in the 'Output' box. You will notice that YeastXtract conveniently displays information in the same 8 X 12 format used in the actual plates. ! The first section will contain the spot areas for the culture spots. Each number in this array format denotes the area in pixels of the corresponding culture spot. Note that the area is the area of the culture at the final time point.
! After the spot-area measurements will be the actual intensities of the culture spots. These intensities are again in array format and are ordered by time point.
To export this data, click and drag inside the 'Output' box to highlight all of its contents. Press 'Ctrl-C' and then open your favorite text editor or Microsoft Excel and press 'Ctrl-V' to paste.

Spot detection
Before looking at the data more closely, you will probably want to make some quick assessments about the accuracy of YeastXtract. To do so, click on the 'Spot Detection' tab in the main window. Next, choose a plate number from the drop-down list. Note that you cannot choose a plate that you did not analyze. The 'Preview' pane should something like the following: As shown in the above figure, YeastXtract displays an image of the specified plate and overlays ellipses calculated during its spot detection phase. If your images were in the correct format, you should see ellipses tightly circumscribing the culture spots. To get more information about a culture spot, simply click on it!

Culture spots
If you are considering using YeastXtract, you are probably most interested in getting the numerical information from the 'Output' box. However, it is often useful to look at the actual images to perform quick checks, etc. YeastXtract enables you to do just that via its 'Spot Level Information' pane.
Time-lapse images of culture spots ! First click on the 'Spot Level Information' tab. ! Next, use the three drop-down lists to specify the particular culture from the particular plate you want to look at. ! Click on 'Show Image.' YeastXtract will display the image of the specified culture in time-lapse format.
Note that there is no gray background on these images. This is because that background has been subtracted and the resultant image is shown. This feature enables you to quickly assess whether the background calculation ran into some issues.

Growth curves
! To look at the growth plot for a particular culture follow the first two bullets of the preceding section. Then click on the 'Plot Growth Curve' button. You may wonder how growth curves are plotted versus time if you did not specify it. YeastXtract uses the time-stamp on the files themselves to ascertain this information. The growth curve plotting feature is for display purposes only; in other words, as long as you specify the images in order of increasing time in the beginning, the actual time-stamps do not affect the analysis values shown in the output box.