The Defaults Window

This is a convenient tool for customising Image and its external processing modules. The default are handled on a "per project" basis :

When all your gels for a particular project are going to have the same characteristics, then you just set the parameters in this window once and all gels in this project will be assigned these parameters. This is handy, so you don't have to change the number of lanes everytime a new gel comes along, when you know that all the gels are going to have X number of lanes. Enter X in the textfield for "lane number :".

All parameters from the project default file are listed with textentry field attached to each parameters. A text field becomes active by clicking on it, which turns it yellow and allows you to type the new value.

After editing click on :-

[ OK ] - activate and save new parameters and close window
[Cancel] - quit without changing anything
[Apply] - just activate new values, but don't save

Note, that during editing of the parameters, all other windows of Image freeze until the editing is finished. This way it is made sure that Image doesn't get confused with new defaults while it is doing something that uses these defaults.

The upper half of the screen

All these values will be applied to a gel, when it is first opened up. This is meant to minimise typing, as most parameters are always the same throughout the project. However they can all be changed on a "per gel" basis in Step 1 in a similar fashion.

These are the parameters :

gel type :
ARG for autoradiographs
ABI for fluorescent fingerprinting on ABI machines
FLuorIM for agarose gels from the FluorImager
lane number : the total number of lanes (including marker lanes) on the gel.
marker repeat : number of lanes in one marker block = number of samples between two markers + 1
zone number : zones for lane tracking
using a big number will cope better with curvature of lanes, but will have less band in a zone to track on
a smaller number is advised on gels with less bands to track on.
dye number : only applies for ABI multiplexed gels (=4), 1 for all other gel types.
Component: only applies for ABI multiplexed gels. You may wish to view the dyes in a different order from the standard (B,G,Y,R). For example, if your standard is red, then this would be 4 1 2 3. This specifies the number of different dyes used per lane.
Max contig bands. Controls the number of bands that can be assigned to an undistinguished blob. Set this to 1 if you are worried about overcalling. Higher values like 3 will permit the calling of shoulders.
pixel ratio : scanning equipment is likely to gather better pixel resolution along the lanes than along the well axis. This number shows how much more. For Amersham scanned autoradiographs use 4.0, for FluorImager gels 1.0 and for ABI gels we found 15 to 18 to work well.
greyscale : the left and right values correspond to the bottom and top value in the greyramp tool. Enter the values that make the gel look best in Step 2, because the grey ramp is shifted in Step 3 and 4.
standard file : complete pathname of standard file. Click on the [file] button to locate it easier using the file chooser. Step 4 has a function to create a standard file.
geltraining file : The file written by BSTT to keep a database of training data. The initial default is where BSTT writes it by default as well.
name init scheme : In order to guess clone names you can specify the first clone name in Step 1, and the program then fills in all other names based on the first name and the naming scheme. If the clones are numbere by their tray position it is probably desiable to use the last two character of the first clone name, e.g. A1 to guess the others from that : A2, A3 ... A12, B1, B2 etc. The other option is simple linear increments. If the first clone name is "lane1", the others will be initialised with lane2, lane3, lane4 etc..
lock direction : The standard locking usually uses the loading well to lock upon (from left to right), but on an ABI gels it might be desirable to lock from the right, using the rightmost band as a starting point.
normalisation method : The default is linear. Non-linear normalisation would be used to map band positions from an ABI gel back to where they would have been on an ARG.
Band height model: If you have steadily decaying band heights, (e.g agarose gel, no glycerol), then set band height to decaying. If bands heights are random, then use random.

The lower half of the screen

These parameters here are applied to all the gels for that project, they can't be specified for a single gel. If gels should require different settings, they probably don't belong in the same project together. The image file type is really the only thing to worry about :

image file format:
Every gel file within one project will come from the same kind of data gathering tool, that writes a specific image format. The Amersham scanner writes a "dat" file, whereas PC or MAC based instruments, like the FluorImager are likely to produce a "tif" file. Click on the button with the required format. Image files other than the hidump format have to converted before they can be displayed. The image conversion program recognises the incoming file type by its file extension. The available choices are displayed as the row of buttons.
background subtraction:
In order to track the lanes on the image, the program finds band like features on the gel and fits a grid of lanes through it. For this feature finding the background noise of the image is removed by a process called background subtraction.
For each scan line the mean of pixel values over a sliding window is subtracted from each pixel. This is done n times. You can specify the size of that window in percentage of the gel length (in direction of the lanes). The iteration number specifies how many times to carry out the operation per scan line.
If the background varies a lot across the gel a smaller percentage than the default 20 is advised. A bigger iteration number might cause very strong blobs to be sperated out better for lanetracking, whereas a number smaller than 3 relies on very distinct features on a flat uniform background.