In this window, the relative intensities of bands (on gels or blots) can be measured, and the number of colonies (on agar plates), cells or spots (in a microscopic field) can be determined. 
To open an image file, drag and drop the file into the image pane.  
Use the “print” button to print or to save into pdf, PostScript file the content of the image view or the table of folds.

  • Image adjustment
  • Labels
  • Grid for cells counting
  • Calculation of relative fold-change in selected bands/spots
  • Colonies/Cells/Spots counting

Image adjustment
To adjust the image use the “contrast” and “brightness” sliders. Use the “convert to grey”, “invert color” buttons to change the image correspondingly. To cancel all changes use the “cancel” button.

To label features or to write a title on the picture, enter text into the “label” field and press the button “paste”. The text can be moved to the required position by dragging it with the mouse (or trackpad). Use the color well to set the label color, and the “font size” popup menu to adjust the font size. To remove all the labels, use the “remove” button 

Grid for cells counting
To place a grid over the image, set the On/Off grid checkbox to On. The spacing of the grid lines can be adjusted with the scale slider. To set the grid color, use the color well.

Calculation of relative fold-change in selected bands/spots
Select the bands/spots to analyse by using the cursor to drag rectangle frames around them. Do this by clicking and dragging with the mouse down. To change the frame color, use the color well. 
To move a created frame, click inside the frame and drag.
To remove the frame, double-click inside the frame. To remove all frames use the “remove” button.
Before the calculation, select the aligned frames on interest, vertical or horizontal, e.g. whether in a gel lane or across different gel lanes. The frames will be numbered according to their vertical or horizontal alignment. To remove the alignment numbers, press the button “none”. If a frame is moved, the corresponding frame alignment numbers will be automatically redrawn according to the new frame positions.
Select the image background to be dark or light.
Use the “calculate” button to calculate the bands/spots intensities and fold-density changes. For each frame the local background will be subtracted. Press the button “remove” to clear the table and to remove the frames.

Colonies/Cells/Spots counting
The counting can be done for the whole image area or in the selected sectors. To draw the sectors switch on the “draw sectors” checkbox, then click around the target area. By this an open contour will be drawn. Double click to close the open contour and this will automatically generate a sector name in alphabetical order. 
The counting results for each sector and for the rest area as “Rest” will be displayed in the table. If no sectors are created the counting will be displayed in the table for the whole image area under the name “All”.
Use the sectors color well to set the color for the sectors.
To remove all sectors and to remove from the table the corresponding counts use the “remove all sectors” button. To remove a single sector set the “draw sectors” checkbox off and double click inside the sector. All the counts in the table will be automatically corrected. 
All counted colonies/cells/spots will be outlined. Use the contours color well to set the color for the contours. Falsely counted objects can be removed by outlining them (set the “draw sectors” checkbox off, click and drag a rectangle over them), and double clicking inside the outlined region (frame). After removal, the count will be immediately corrected. To remove all the contours and to clear the count use the “remove all contours” button.
      Colonies counting 
To count colonies on a culture plate, select the “colonies” section on segmented button (default). Then, convert the image into grey scale mode and press the “count” button. If the default settings of colony shade and background color are not optimal, then adjust the colony shade by mouse clicking inside a colony, and the background color by clicking outside the colonies. Adjust the colony color brightness using the HSB slider and press the “count” button again to see the new count. The counted colonies will be outlined. Merged colonies are not counted and can be added to the above single colony count by drawing a frame around the each colony within a close pair or cluster, in the same way as described above for the gel bands. To remove all the frames and to correct the counts use the “remove all frames” button. To remove a single frame, double-click inside the frame.

The picture of yeast colonies on an agar plate is courtesy of Mike Dyall-Smith. 

      Cells/Spots counting 
To count cells (or spots within cells), select the “cells” section on segmented button and set the cells/spots color as well as the background color. To set the colonies/cells/spots color activate the colonies/cells color well and click on a colony/cell. Use HSB slider to reduce the brightness for a grey scale image or both brightness and saturation for the color image. To set the background color activate the background color well and click on the image background. The next steps are the same as for the described above colony count. Play with brightness and saturation to get the best results.

The picture of immunofluorescently labeled cells is courtesy of Dr. Dmitri Lodygin, University of Goettingen. The cyan coloured spots are counted and outlined in blue.

Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s