PMA vs IL-8 Example: Part 1. Main Window Settings
What follows is a detailed explanation of how to generate results from CyteSeeer ViewRNA using the tutorial data for PMAvsIL-8. The images were obtained from 4 wells of a multiwell dish, representing a dose-response experiment to PMA (phorbol 12-myristate 13-acetate), in which each well was imaged both for nuclei and for RNA using the Panomics reagents:
Copy the tutorial image set to your hard drive
CyteSeer ViewRNA Tutorial Data set 1 is available either from the CyteSeer ViewRNA CD or can be downloaded from Vala’s web page. Please install it in an appropriate location for image data on your hard drive.
Start CyteSeer ViewRNA
Launch the program in a way that is appropriate for your operating system. The main menu will display. CyteSeer ViewRNA is designed in such a way, that most of the user’s interaction with the program to set up analysis will occur via the main window.
Select Image Naming Convention
Use the dropdown menu (right) that best matches your imaging platform. The Panomics naming convention is the default and a generic convention that can be used for most microscopes and high-content analysis instruments.
Select the Source Folder
Use the upper Browse button to navigate to the tutorial data set located in the folder titled PMAvsIL-8. When you have successfully directed CyteSeer ViewRNA to the images, the lower field will populate with the image set (e.g. wells C15, E15, G15, and I15).
Start CyteSeer ViewRNA
Select the Destination Folder. Use the lower Browse button to navigate to an appropriate location on your hard drive and create a destination folder. You can accomplish this within CyteSeer ViewRNA . For the purposes of this tutorial, name the folder TutorialDataanalysis. During the analysis, CyteSeer ViewRNA will create a series of subfolders within the destination folder, and files will be created that represent mask images as well as *.csv data files.
Select the Nulcear and RNA Channels. The Nuclear Channel and the RNA channel read “channel 0” and “channel 1”, respectively. The tutorial image set is named according to the Panomics naming convention. An additional channel can be analyzed if images are available.
Sensitivity. The system automatically determines which parts of the image should be used for the cell-by-cell analysis. The default sensitivity is 100% for all channels. Selecting a higher number will allow the program to recognize dimmer nuclei and RNA dots whereas entering smaller numbers will reduce the sensitivity of the system.
Well Definition. Match the number of Rows and Columns to the number of fields in each well. Since there is just one image per well, the Well Definition is 1 row by 1 column.
Nuclear Size. If CyteSeer is splitting the nuclear mask into two, enter a larger number for the Nuclear Size. For this data set, please enter a nuclear size of 16.
Enter values in the main window to match screenshot shown above.
Press the “Run” button to initiate the analysis. While CyteSeer ViewRNA is working a processing bar will be displayed in the lower right corner. On a typical XP computer the analysis will take a few minutes.
Proceed to the next lesson
