************************ * * * Operations Submenu * * * ************************ Analysis of an image typically requires the sequential stepping through this submenu. A message will appear in the Status Inofrmation Box if an attempt is made to perform a step which depends upon a prior step which has not been done. Descriptions of the steps appear below. **************** * * * Open Image * * * **************** An Image Flags Popup first appears. Its top button is set to "1" (marker) and should be left alone (the alternate {4 markers} is for SSRP images, but this code has not been completed). The next button is set to "No" and should be left as such unless Inheritance processing is to be acivated. Press "Load Image". A file browser appears on the screen and remains until an image is selected or the Cancel button is pressed. If an image is selected and is in a format which can be recognized by the program, the image will be displayed. If the format is not recognized, a popup menu appears for which one must enter the appropriate information (see the Help selection for Images). The time it takes to display the image depends upon the image size. Images with dimensions of 512 or less will appear faster than larger ones (see Help selection for Images). ****************** * * * Enter Params * * * ****************** A primary image belongs to class of images about which there is information to be used during routine analysis. Clicking the Enter Params button allows the fetching of this kind of information. The user species two kinds of files: "Gel Format" -- a file containing lane IDs. "Template Definition" -- a file containig pairs of values (mobility, base-pairs) corresponding to a standard against which an image's marker lanes are matched in order to warp an image into proper alignment. For either kind, one may use an existing file or create one on the spot. Separate help selections give details for creating these files. After clicking the Enter Params button, the user is led through a series of popup windows to specify the two files. (1) The first popup allows one to create a new gel-format file or use an existing one. If the choice is to use an existing one, (2) is popped up. If one prefers to create a new one, one specifies a header, the lane IDs, a directory path, and a file name. After clicking the "OK" button, the IDs are saved in the file specified and popup (3) appears. (2) The gel-format browser is popped up if one prefers to use an exisiting gel-format file. After selecting a file, one is given the opportunity to edit it for use with the current image via (3). (3) The lane-edit popup allows one to edit the chosen gel-format file. Changes are not permanent and apply only to the current image. There is a separate help file describing use of this popup. (4) Whether or not one chooses to edit the chosen gel-format file, the next popup lets one decide to create a new template-definition file or use an existing one. If the choice is to use an existing one, the template browser (5) appears for one to select an existing file. If one prefers to create a new one, mobilities and base-pairs are determined, the file is created, and control passes back to the operations menu. (5) From this template-definition file browser, one selects an exisiting template-definition file; control is then passed back to the operations menu. Also, at the upper right of the screen are a half dozen text fields for operator name, date of gel, etc., which the user can fill out now or at any time prior to saving results. These text fields are for information only and contribute nothing to the analysis. For SSRP images, a Sequence file is selected in lieu of the Template file. **************** * * * Crop Image * * * **************** Typically, only a portion of an image need be analyzed. Autoradiograms are frequently filled with notes around the perimeter of the area of interest. The crop box delineates that portion of the image to be analyzed. After clicking this button, move the pointer to the upper left portion of the region of interest. Then depress any pointer button and leave depressed while moving the pointer to the lower right of the region of interest. When the region has been bounded, release the depressed button. If the bounding box is not quite right, click the Crop Image button and repeat the process. *********** * * * Lanes * * * *********** Clicking this button will invoke the lane detection and demarcation algorithm. Once demarcated, the lanes be edited by removing or adding boundaries. The program checks to make sure the number of boundaries is even so that each lane has a left and right boundary. If the lane boundaries do not look quite right, repeat the process, beginning with the click of the Crop Box button. If a Comb-geometry file has been specified on the command line, lanes will be drawn with according to the specifications of the Comb-geometry file rather than automatic lane detection. If the lanes appear to be in error, first check to make sure the crop box begins at the left edge of the leftmost lane and extends to the right edge of the rightmost lane. Then check the Comb-geometry file. *************** * * * Std Bands * * * *************** Clicking this button will attempt to detect bands in the marker lanes. Before doing so, however, the program will pop up the template of marker bands belonging to this class of images. The user is free to accept the template as is or to delete template bands which may prove troublesome to the normalize process. There are no guidelines for editing the template; a working knowledge of the class of images dictates the editing procedure. Once the template has been edited or accepted in full, the program detects bands in the marker lanes. Before closing out, the user is prompted to edit these bands; bands may be deleted (MB2) or added (MB1). Once the marker bands have been accepted (MB3), this Std Bands button may be re-activated if further editing is needed. In the case of SSRP images, the template edit procedure is ignored; and the template bands are represented by the bands in the first group of ACGT lanes. After standard bands have been accepted, consider "Save Results" which will save everything done to the pre-normalized image. Once "Normalize" is clicked, results for the pre-normalized image are lost, though they are saved in the normalized image. *************** * * * Normalize * * * *************** If marker bands have been detected, clicking this button forces the program to apply a spatial normalization algorithm to the primary image. The result is a new image which has been "normalized" or warped to a standard. Depending upon the size of the image, this routine may take 5-20 seconds; occasional messages will appear in the Status Information Box. *************** * * * Exp Bands * * * *************** If the primary image displayed has been normalized, clicking this button will force the program to detect bands in the experimental lanes. They will be colored differently from the bands in the marker lanes. A Bands Process Menu pops up, giving you several choices for detecting the bands in the experimental lanes. "Automatic" lets the program do fully automatic detection; you may edit its results. "Semi-automatic" lets you move the cursor in the vicinity of a band and click; the program looks for peak intensity in a small window near your click and postions the band there. "Manual" lets you click where you want each band to be. "Edit" is the same as "Automatic". "Clear" lets you remove all bands and start over. "Cancel" exits from experimental band detection. If the "Automatic" option is chosen, after the program has done its detection, you may edit its results: bands may be deleted (MB2) or inserted (MB1). By which ever method is chosen, clicking MB3 accepts the results and returns program control to the Operations menu If experimental bands have already been detected, clicking the Exp Bands button still allows editing of displayed bands. This time, however, a Semi-automatic popup allows the enabling or disabling of the semi-automatic method. All experimental bands may be deleted with the Reset Bands button in the Options submenu. Follow directions in the Status Information Box for edit actions. ****************** * * * Save Results * * * ****************** Depending upon the kind of image and states of special flags, accumulated results are saved in appropriate files. The primary image remains displayed for further investigation. ***************** * * * Close Image * * * ***************** The primary image and any associated windows disappear. The primary image window, lane ID window, fragment-size window, tracking box, and the Image Information Region go blank; any profile graphs associated with the primary image disappear. Unless the Save Results button was depressed prior to closing the primary image, any accumulated results are lost. When the primary image is closed, another may be opened. ********** * * * Done * * * ********** If selected, the Operations submenu disappears, and control is returned to the main commands menu.