All Technical Notes

These image files can be opened as Foreign Format files.

On the Macintosh:
Define a Foreign Format with the following settings:

Yes, IPLab for Windows is completely compatible with Windows XP and Windows 2000.

The IPLab installer automatically installs the Sentinel NT driver required to make the key/dongle work.

If you need to install them yourself, you will find the Sentinel NT installer on the IPLab distribution CD. Please read the simple installation directions that come with the installer.

IPLab calculates an object's area by measuring the number of piixels within the object.

If you tell IPLab the size of a pixel, then IPLab will automatically convert the area from pixels into the units you defined. For example, if you tell IPLab that a pixel is 2 µm tall and 2 µm wide, then IPLab will automatically report an object with a 10 pixel area as having an area of 40 µm2. You only need to define the pixel-to-units conversion factor once, but of course you can define multiple units for different objectives and imaging systems.

IPLab does measure the actual area of objects within the digital image. However, it is important to remember that CCD cameras and scanners always convert images into rectangular arrays of square pixels. Regardless, the measured area is the real area so long as the object is significantly larger than the size of a single pixel.

Multiply the image's width in pixels, its height in pixels, and the bit depth of the image.

Bit Depth:

• Byte, or 8 bit, images obviously have 1 byte for each pixel.
• Short Integer and Unsigned 16 images both have 2 bytes. Common digital cameras acquire 12 bits of data but store it in one of these 2 byte data types.
• Color 24 images have 3 bytes per pixel.
• Color 48 images have 6 bytes per pixel.

File size = (image width in pixels) x (image height in pixels) x (bytes per pixel)

Example #1:
Your monochrome digital camera acquires an image 1280 x 1024 pixels in size. The camera acquires 12 bit data, so the images are stored in Short Int or Unsigned 16 images, with 2 bytes per pixel.
Example File Size #1 = (1280 pixels) x (1024 pixels) x (2 bytes/pixel)
= 2,621,440 bytes
= ~2.6 MB

Example #2:
Your color video camera acquires an image 640 x 480 pixels in size. The camera and its frame grabber produce a color 24 image, with 3 bytes per pixel.
Example File Size #2 = (640 pixels) x (480 pixels) x (4 bytes/pixel)
= 921,600 bytes
= ~921 KB

1. First, you select the area you want to measure.
2. Next, you can choose which measurement to perform.
3. Finally, you measure!

All of the commands you need are in the Analyze menu, unless stated otherwise.

You're done! You may want to read the Analyze section of the IPLab User's Guide to learn how to perform more complex measurements.

The full error message is:

"The software needed to use the USB device 'USB Token' cannot be found. Please refer to the device documentation to install the necessary software."

The USB token is the key, or dongle, that makes IPLab work. To fix this, install IPLab version 3.2.4 or later. That will put the USBSentinel extension in the System: Extensions folder. After installing IPLab, you must restart the computer. You will then be able to use IPLab.

If you get a similar error message for a different USB device, you must install the extension that came with that piece of hardware. Restart the computer after installing the new extension. Please read the hardware's documentation.

By using a USB-to-serial-port adapter or a serial port PCI card.

Now that RS-232 serial ports are no longer included on the newest Macintosh computers, BD Biosciences Bioimaging has tested various third party devices that provide these necessary ports. The serial port PCI cards also work with older Macintoshes, in case you need more than the two built-in ports.

Remember to install the software supplied with the hardware.

PCI Solutions:

• Keyspan SX4 Pro Serial Card
• Creative Solutions Lightning PCI
• Megawolf Remus / 2

USB Solutions:

• Keyspan Twin Serial Adapter (USA28X) - this is an adapter that plugs into your USB port and provides two serial ports.

Within IPLab, use the Point Function command from the Math menu. "Point Function" means that the function you select will be applied to each pixel in the image.

1. In order to subtract a constant, use the first function, (ax + b)/c. The variable x represents the intensity value of the pixel.
2. In the bottom left corner of the Point Function dialog box, set a = 1 and c = 1.
3. Set b equal to the constant you want to subtract (i.e. 120).

IPLab can measure the mean intensity and the area using different units. If you set the units of distance with the Set XY Units command in IPLab, then the mena will not equal (measured sum / measured area) because the units will be different.

IPLab measures the mean as intensity units per pixel. It measures the area in the units of distance assigned by the Set XY Units command (for example, microns or millimeters).

You can define new distance units with the Define XY Units command, and you can apply the units to images by using the Set XY Units command. Both commands are in the Analysis menu.

IPLab will open only uncompressed TIFF files. If you wish to open a TIFF file with IPLab, please make sure that you turn off all compression options when the image within another application.

The image-sensing CCD can only capture a small area of the image that the microscope projects, so on the monitor you will see a smaller area at higher magnification.

If you need to widen your field on the computer, you will need a camera mount that has a reducing factor, typically 0.6x, 0.5x or 0.45x. BD Biosciences Bioimaging can help you select an appropriate mount. In addition, some microscope manufacturers produce variable magnification or zoom mounts. These let you customize the magnification and field size on your monitor.

Installing camera controls and extensions will add menus of commands to IPLab's menu bar. If IPLab cannot find these extensions, the menu will disappear. To find the extensions and replace the menu, answer these questions:

• Did you drag IPLab to the desktop?
   
  If so, you need to put it back in its folder and put an alias of it on the desktop instead.
• Is there more than one copy of IPLab on your computer?
   
  If so, one copy of IPLab might not have extensions installed. You might be running that copy, and that is why your menus are missing. It is best to have only one copy of IPLab on your disk. Otherwise you need to keep track of which copy of IPLab is running when you open an image.
• Have you just installed a new version of IPLab?
   
  Then you also need to install any other extensions that you own into the new IPLab folder.
• Have you read the manual?
   
  New versions of some extensions are put into different menus than in the past. For example, you will find the new Ratio Plus for Windows commands in their own Ratio menu. We no longer use the Ext. menu for anything.

You have to restart the computer, even though the installer didn't say that you needed to. This can happen when you install a camera that you previously used with the Evaluation version of IPLab.

The program is telling you to allocate more memory to it. Macintosh OS 9 and earlier allocates memory to the program when you launch the program. If you open too many images, the program will run out of memory and give you the "Insufficient Contiguous Memory" error.

Please do the following:

1. Quit the program if it is running.
2. From the Macintosh Finder, find the program. (For example, find IPLab in the "IPLab" folder).
3. Click once on the program's icon once to select it.
4. Choose the Get Info: Memory command from the File menu. If you only see Get Info, choose that.
5. Increase the Preferred Size value in the "Memory Requirements" section of the dialog.
   The amount to set depends on the amount of RAM in your machine, and the amount of memory used by other programs that you want to run at the same time.

It is very difficult to load exactly the same amount of material in every lane. In many experiments, you don't want to do this anyway. If, however, your intention is to compare the density of bands from lane to lane, you might want to examine data that are normalized. This is done by reporting either of the following fields:

• %IntOD. The percent integrated optical density value represents each peak (band) as a percentage of the sum of the integrated optical densities of all the detected bands in a given lane. That is to say, all of the bands' densities are summed, and each is then reported as a percent of the sum.
• %Lane IntOD. The percent lane integrated optical density values reports the density of each band as a percent of the summed total density of the entire lane, not just a percent of the detected bands.

The %IntOD and %Lane IntOD values will often be very close to one another, depending on whether or not all of the bands in a given lane have been detected.

The first question you must ask yourself is what you intend to do with the stored information, and exactly what pieces of information you wish to archive. In any given gel analysis, there are really only three data points which are scored:

• band MW,
• band density, and
• band migration (Rf).

You can convert some of these values into others, but they represent the basic information you obtain from a gel. Therefore, your first task is to decide exactly what information should be reported in your customized Report and saved in your Experiment file.

Gene Profiler will automatically store certain data fields in the database. These are: ExpID, SampleID, ConditionID, Lane, Band, MW, Bin, and Ubin. If you intend to send other data to the database, such as density, % IntOD, or Rf, you must modify the database prior to entering any data. To add a field to a database table, follow this procedure:

1. In the Gene Profiler Database Manager module, select File / New Database, and give it a name.
2. Highlight the Bands table and click on the View button.
3. Select Edit / Add New Field.
4. Press the Add button and supply the following information: name of the new field, type of data to be placed in the new field, number of decimal points, and source field in File / Reports where the data for the new field can be found.
5. Click OK and Close. You will now see that the new field has been added to the Bands table.

If you have other fields to add, repeat these steps. Then, when all of your tables have been modified to contain all of the fields you want filled, you can begin the process of importing the data into the new database.

Importing data into the database. Data are imported by Experiment files.

1. First, open the Gene Profiler software and display the images which contain the gel data you want to send to the database.
2. Then open each EXP experiment file for each image. These files must be activated before you use the Database Manager to import them into the database.
3. Select File / Import Experiments. You will be prompted to select:
   • the name of the experiment to import,
   • the ConditionID name which governs the experiment, and
   • select which lanes you want to import.

For multiplexed data sets (multiple primers, restriction digests, etc.) you must ensure that all samples are represented completely for each ConditionID, and that there are no gaps in the data sets. Otherwise, those samples will be scored differently than the others and they will not match properly.

IMPORTANT NOTE: SampleID names must be unique for each lane in the database. Gene Profiler requires a unique identifier to name each lane. This identifier is the SampleID name that you supply in the Samples Description dialog box. Be absolutely certain that each and every lane on each and every gel has a unique SampleID name. If you give the same SampleID to two different lanes, the software will assume they represent the same sample and will combine them. A convenient way to supply unique SampleID names is to use the date name, shown below.

13Mar96a, 14Apr95f, 23Aug94c, etc.

These names conform to the required 8-character (maximum) name. Multiple samples obtained on the same date can be differentiated using a, b, c, d, etc. The remainder of the sample name (extended name) can be added at the end of the date, as follows:

13Mar96a-P-aeruginosa, 14Apr95f-E-coli, 23Aug94c-H-influenzae

You may create unique SampleID names in any manner you desire.

The Gene Profiler Database Manager is based on the engine from Microsoft Access(TM). It is a typical relational database operating under the SQL syntax (Structured Query Language). All relational databases are constructed of various tables which contain columns (fields) of data. You may inspect any table by highlighting its name in the Tables list and clicking the View button. A window will pop up listing all the fields of data for that table. Each table contains different pieces of information, which can be cross-correlated using the proper SQL commands.

It is anticipated that you will want to retrieve specific pieces of information from your database without having to scroll through the entire list of data entries. This is made possible by creating SQL queries which ask for specific data to be retrieved from the database and displayed. In its simplest form, an SQL query is a statement written more or less in common English that specifies the nature of the information you wish to retrieve. SQL queries can be made very simple or very complex, depending on the specific nature of the information you need.

An example of a simple SQL query could be as follows. Let's say that you have a large database of bacterial isolates characterized by DNA fingerprinting using a PCR-RAPD technique. You notice that certain strains which appear to be isolated from a specific location always exhibit two bands of MW 10,300 and 5500 base pairs. You might become curious whether or not there are any other entries in your database which have banding patterns that contain these same two bands. Rather than scroll the entire Bands table, you could write the following SQL query:

SELECT SampleID, ExpID, MW

FROM Bands

WHERE MW = 10,300

UNION

SELECT SampleID, ExpID, MW

FROM Bands

WHERE MW = 5,500

This query would retrieve all entries having either of the two bands in question. Queries can be saved and modified for future use.

The Tables list in Gene Profiler Database Manager provides you with a means of examining all the data in your database. You should inspect the Bands table and the Match Absence / Presence table frequently so that the integrity of the data can be monitored. The Match Absence / Presence table contains the 0/1 strings which are important as export data to cluster analysis software. If you happen to make a mistake which results in missing data, this table will register a series of question marks (???????????????) where the missing data should be listed. This is your signal that an error has been made and that you need to investigate.

Gene Profiler offers two mechanisms for determining if bands match one another.

1. Binning is a method whereby the gel and all of its lanes are mathematically cut into slices where each slice represents a small range of molecular weights. The magnitude of this MW range is dependent upon the Tolerance value you specify, generally 0.5 - 2.0 %. Each slice, now representing a small range of MW, is termed a 'bin.' The number of bins can be determined empirically by the software, working on its own (Generated Bins), or, the number can be set by the user, if you want to define the number and range of each bin yourself (Universal Bins). When Match Bands by Binning is selected, the software performs a simultaneous match of all lanes. (Simultaneous matching is synonymous with binning).
    
   The binning method results in the creation of the ultimate digitization of a gel, the reduction of each lane's banding pattern into a binary code, a string of zeros and ones, called a '0/1' string or an Absence/Presence string. A zero represents an empty bin, and a one represents a bin with a band in it. Thus, a lane which has ten bands in it will be scored as a series of zeros and ones, depending on which bins contain the bands.
    
   Select Analyze / Match Bands. In the Match Bands dialog box, select Match all lanes simultaneously. Then click on the Match button. Go back to Analyze and select Match Statistics. Use the small arrow buttons to move the cursor from bin to bin. You will see matching bands circled in blue. The Match Statistics dialog box will tell you the size range of MW for each bin, plus the number of bins which were made during the matching phase.
    
   The binning method is a very commonly used technique for matching banding patterns, such as DNA fingerprinting. It is a simple way to reduce a complex image of a gel into a simple binary code which can be stored in a database. It is also the method of choice for generating data which will eventually be sent cluster analysis programs such as PHYLIP, PAUP, NTSYS, and TreeCon, for the purpose of measuring phylogenetic relatedness among individuals. The binning method is used for forensic purposes by law enforcement agencies interested in matching DNA types. It is also the method used by Gene Profiler for performing multiplexed RAPD and RFLP cluster analysis when multiple primers or restriction digests are used.
2. Pairwise matching is a method wherein a given lane is paired, one on one, with each and every other lane in the database, sequentially. The pairwise scoring is based upon how many bands are shared in common between the pair of lanes, giving rise to a Similarity Index or Band-Sharing Coefficient. The similarity index is simply the number of shared bands divided by the total number of detected bands found in both lanes. Once the software has matched the first lane against all others, it moves on the second lane in the database and matches it in a pairwise fashion against all others. This method proceeds until all lanes are exhausted. The Pairwise method is synonymous with matching two lanes at a time, and does not depend on creating bins.

PHYLIP, PAUP, NTSYS, and TreeCon are cluster analysis programs. They are established scientific programs used by many investigators for performing phylogenetic analysis and are not products of BD Biosciences Bioimaging. Gene Profiler has, however, been configured so that it is compatible with these programs. Data output from Gene Profiler can be fed directly into these programs so that you can generate dendrograms for measuring relatedness among individuals within study populations. Gene Profiler also has built-in control mechanisms for operating interfacing with these programs, making it very easy to pass data from one module to the other.

Most investigators who generate DNA fingerprints on organisms via RAPD, RFLP, and other typing methods are also interested in performing cluster analysis on the data. One of the most common methods available is the dendrogram. This diagrammatic representation allows you to visualize groups of individuals that have similar banding patterns. A phylogenetic inference can be established from the resulting dendrogram if it has been constructed with certain procedures available with PHYLIP, PAUP, NTSYS, and TreeCon. The bootstrap method allows you to reenter the data in an iterative fashion so as to randomize the order of entry of the data and to provide a measure of the confidence for each branch point.

A bug in the software corrupted RFLPDB.INI. This bug becomes evident if you change the physical dimensions and/or positions of the open windows in the Database Manager module. We suggest these two workarounds:

• Quit, Delete, Restart
  1. Quit the GeneProfiler/GeneImagIR program.
  2. Delete the file RFLPDB.INI.
  3. Start the application again.
• Change RFLPDB.INI to read-only.
  1. Go to Windows Explorer
  2. Locate RFLPDB.INI in the folder where you installed the software.
  3. Right-click on the RFLPDB.INI file.
  4. Select Properties.
  5. Select Read-Only.

This application can open only uncompressed TIFF files. Please make sure that you turn off all compression options when saving a TIFF file from another application if you wish to open that file with Gene Profiler / Gene ImagIR

(This solution is the same for both IPLab and ONE-Dscan/Mac.)

It is possible that the program does not have enough memory available to grab the image. When this happens, it tries to put up an alert to notify you of this situation. Sometimes there is not enough memory left to display the alert, so all you hear is a beep. Try closing some windows in the program, or assign more memory to the program.

Here's how to assign more memory to IPLab or ONE-Dscan/Mac:

1. Quit the program.
2. Open its folder.
3. Single-click on the program's icon to highlight it.
4. Select Get Info from the File menu.
5. Increase the Preferred Size of IPLab.

Error 65 is a timeout error. This means that the Snapper frame grabber board is waiting for data from the camera. Usually this means that the cables are not connected correctly.

If you are using version 1.2 (or higher) of the Orca acquire module, or any version of the Orca II acquire module, make sure that the BNC cable labeled 'O' at the end of the data cable is connected to the EXT. TRIG. port on the Orca controller.

The VirtualCam is a camera control which simulates a camera in software. You can use it to write scripts or demonstrate the software when you don't have a real camera available.

1. Open the File menu and select New Experiment at the top of the list.
2. For opening fluorescence images from a LI-COR imager, or other fluorescence imagers, set image File Type to LI-COR TIFF.
    
   Otherwise, select TIFF.
3. Browse to the desired folder on the hard drive.
4. Select your image file.
5. Click on Open.

Gene Profiler (a.k.a. GeneImagIR), ONE-Dscan/Win v.2x, and ZERO-Dscan could only open grayscale (monochrome), non-compressed TIFF files. They could not open color TIFF files or compressed TIFFs (such as LZW-compressed TIFFs).

1Dscan EX, which replaces ONE-Dscan/Win and ZERO-Dscan, does support color TIFF files. 1Dscan EX also supports:

• its own file format, the 1DScan EX (*.IPL) format, which stores extra information about the gel and the experiment;
• the IPLab Gel Experiment (*.IGE) format, which is useful for transferring experiment information between related images;
• foreign image files, including image files which may have been created under other operating systems. You can define foreign formats to suit your needs.

1Dscan EX still does not open compressed TIFFs, however.

If you need to, open a non-compliant image in another program that supports its format (e.g. Adobe Photoshop, LView Pro, etc.) and re-save the file as a non-compressed TIFF.

Using 1Dscan EX, you can open images over a network by using the Open commands (e.g. Open Chemi/Fluor, Open Autorad/Stained, or Open Chemi/Fluor Time Lapse) from the File menu or the top toolbar. Navigate to the network drive and select the image.

However, the older programs replaced by 1Dscan EX, ONE-Dscan/Win v.2x and ZERO-Dscan, cannot open images over a network. Gene Profiler/ImagIR also cannot open images over a network. For Gene Profiler/ImagIR and the old ONE-Dscan/Win v.2x and ZERO-Dscan, your TIFF files must be stored on a local drive and opened from there. Copy your image files to a folder on the local hard drive before you begin working.

You can also open images over a network using ONE-Dscan for Macintosh.

This is caused by the image's pixel values being inverted. Use a different Image File Type (TIFF, LI-COR TIFF, Fluorescence TIFF) when you first open the image.

Alternatively, you may use the Negate command in the Image menu. You must delete the lanes first (use the Edit menu, Delete Lanes).

If you have problems, open the TIFF file first using the New Experiment command. Then open the EXP file second.

A template stores experiment settings for later, repeated use.

A template is the same thing as an experiment file, ending with the extension .EXP. Gene Profiler / Gene ImageIR and ONE-Dscan for Windows create these files. When analyzing a new image, you can use the template to default to the band detection parameters saved in the template file:

1. Open the new image first, using the New Experiment command in the File menu.
2. Then open the desired template EXP file with the Template box checked.

To open and display a previous experiment, uncheck the Template box.

Gene Profiler / Gene ImageIR uses an auto-contrast function to improve the visibility of weak bands.

To turn off this function and use your own contrast settings, open the View menu, select Contrast, and then check the Full Dynamic Range box. Use the sliders to adjust contrast and brightness.

1. Right-click on the image and select Mark Lanes Location.
2. Drag a line down the mid-line of lane 1, and then repeat on the last lane. When you are done, you should see four red + signs: two at the top and two at the bottom.
3. If you make a mistake, right-click and select Delete Pair, and then start over.
4. When you have four + signs, right-click and select Define Lanes.
5. Enter the total number of lanes in the gel, including the empty lanes (spaces between loaded lanes).

1. Mark the lane locations by creating (dragging) two lines along the mid-lines of the first and last lanes.
2. Enter the total number of lanes in the gel; count loaded lanes as well as empty lanes.
3. If necessary, edit the positions of the lane lines by right-clicking on the gel and selecting Edit Lane Lines. The easiest way to edit the lines is to click once on the center of any band where you want the lane line to pass through.
4. Set the band detection threshold by double-clicking on the lane, and then use the lane profile window that pops open to set the peak detection threshold. To do that, drag the straight blue line left or right to intercept the peaks but not the background.

Note: the software menus are context sensitive. When in doubt as to what to do next, point at the object of interest in the gel image and right-click. The pop-up menu will guide you to the best selections.

Use 2-3 marker lanes to correct for gel smile (distortion).

1. Before beginning the calibration process, make sure the software detected the same number of standard marker bands in each standard lane. This is easy to check by double-clicking on the lanes.
2. Use the Edit MW Standards dialog box to create a list of standard MW values. You should have the same number of values listed as the number of bands detected in each standard lane.
3. Then press the Apply Stds button.

Try Piecewise Linear first. It will provide "match tolerance" values in user-defined units of bp or kDa. If you select Log Piecewise Linear, the tolerance is given in units of percent.

You must have at least two standard lanes to perform gel desmiling automatically. You may also use "known" bands as internal standards in the sample lanes.

1. Locate a sample band of known MW that is near a standard lane.
2. Right-click and select Edit MW Stds.
3. Select Add a De-smile Line.
4. Left-click on the "known" sample band.
5. Right-click and select Add/Update Stds.
6. Left-click on another sample band of the same MW (a co-migrating band).
7. Click on OK to confirm the MW value of the known band.
8. Repeat this process with 2-3 additional "known" bands, as needed.

The red-colored iso-MW lines will correct the curvature or slant in the gel.

Complete the editing of lane lines, detection thresholds, and band marking before you start to measure band intensity. It is also a good idea to save the experiment as you go, and update it as you make changes.

1. Use the Edit Detected Bands command to add/remove band markers, as needed.
2. Right-click and select Calc. Band Intensity.
3. Use Gaussian fit if the bands are poorly resolved, or Drop-Line if you have well-separated peaks, or if you have just one band to measure in each lane.
4. To adjust the baseline (background subtraction), right-click on the lane profile and select Edit Baseline.
5. Move/add/delete the deflection points along the baseline, as needed.
6. If the baseline routinely appears to be set too high, use the Edit Band Detection Parameters dialog box to lower the sensitivity of the auto-baseline function by sliding the control slider to the left.

1. Open the File menu and select Reports.
2. In most instances, the Band Report will be the most useful.
3. Click the Fields button and select the fields you want to report.
4. When your list of fields is complete, you may edit the field names and set the number of decimals to be reported.
5. For most measurements of band intensity, select the fields named "IntOD" or "%IntOD". IntOD refers to the integrated mean density; %IntOD refers to the integrated OD expressed as a percentage of all the marked bands in a lane.

To perform MW determination, your gel needs at least one standard (marker) lane; ideally, it should have two or three standard lanes. Three standard lanes (left, middle, and right) will give you the best, most accurate MW data.

After you mark the lanes on your gel, please follow these instructions to calibrate the MW. (Click the 1D Auto Analysis button if you need to mark the lanes.)

1. In 1Dscan EX, click the Calibrate MW button. We will do all of our work within the Calibrate MW dialog box.
   You can also choose the Calibrate MW command from the Lane Profile menu.
2. First, you should identify the standard lanes.
   1. Click the Edit Std Lanes button within the Calibrate MW dialog box.
   2. Click on each lane that should be a standard lane, highlighting it blue.
   3. Click OK.
3. Create a list of the standards present in your marker lanes.
   1. Click on the Edit Stds button, which is right above the Edit Std Lanes button.
   2. Type the MW value for each standard in the Standard box, and click Add.
      You must add the same number of standards as are in each standard lane.
   3. Specify whether the MWs should be Ascending or Descending in this gel.
   4. Click OK.
   5. Now click the Apply Stds button to assign each standard MW to a band in the standards lanes.
   6. Select the Curve Fitting method you want (Piecewise Linear is best).
   7. Finally, click the Calibrate button. 1Dscan EX will calculate the MW of each band.

Correct gel distortion by selecting a group of co-migrating bands to be used as "internal standards."

1. Click on the Correct Gel Distortion button (from the right toolbar).
2. Click on the New Group button within the Correct Gel Distortion dialog box. The "group" will be the set of bands that migrate the same distance in the gel.
3. In the Select Operation section, choose the Add Point to Group and Snap to Nearest Band options.
4. In the Display section, choose Lines to connect the co-migrating bands in this group. This step only affects how the group is marked.
5. Click on several bands that make up a single co-migration group. You must be certain that the chosen bands all represent the same MW value.
6. Make new groups as needed in order to correct for the gel's distortion.
7. When you are done, click Close.

This will correct the calculated molecular weights for the bands in between the standard lanes.

In 1Dscan EX, you can generate a report by using one of three buttons: the 1D Reports, Area-Density, or Colony Counting button from the right toolbar. It will, of course, depend upon the type of report you need.

For a full answer, please see the Tutorials chapter of the 1Dscan EX User's Guide. Briefly, however:

• To generate a 1D Lane Profile report:
  1. First, mark the lanes on the gel. Click the 1D Auto Analysis button (right toolbar) to automatically detect and mark the lanes and bands.
  2. Next, you may wish to calibrate the MW and concentrations. To do so, use the Calibrate MW and 1D Concentration buttons.
  3. Click the 1D Reports button. A pop-up menu will appear; choose Report Options to set up your report.
  4. Click the 1D Reports button again. This time, choose your report format from the pop-up menu. 1Dscan EX will create the report and display it to you in a spreadsheet-like window.
• To export a 1D report:
  1. While you are looking at the 1D report window, choose the Export Report to Text File command from the File menu.
  2. You can import this text file from within Microsoft Excel® or you can open it in any other spreadsheet or graphing program.
• To generate an Area-Density or Colony Counting report:
  1. Click on the appropriate button on the right toolbar.
  2. Use the Area-Density or Colony Counting dialog box's features to set up the analysis.
  3. Click the Measure Objects button at the bottom of the dialog box. (In the case of Colony Counting, you can also click the Count Objects button.) This will generate a spreadsheet-like window of data.
• To export an Area-Density or Colony Counting report:
  1. Click the Export to Excel button, which is at the top of the report window. This will work with Microsoft Excel® version 2002 or later.
  2. You can also use the Export Report to Text File command from the File menu.
  3. If you do not have Microsoft Excel® version 2002 or later, export the report to text and then import the text file from within Excel.

1Dscan EX has two Open functions depending on the type of data: chemi/fluorescence or autorad/stained. Opening an image as one or the other affects the measurements that 1Dscan EX performs.

• If the image represents a chemiluminescent or fluorescent blot or gel, such as ECL or ethidium bromide, either:
  • Click on the Open Chemi/Fluorescence button (top toolbar) , or
  • Open the File menu and select the Open Chemi/Fluor command.
• If the image represents a scanned X-ray film, or a gel/blot stained with a visible stain such as Coomassie blue, then either:
  • Click on the Open Autorad/Stained button (top toolbar) , or
  • Select the Open Autorad/Stained command from the File menu.

If you open the image with the wrong Open command, simply correct the matter by selecting the correct type of image from the top of the window. The window's frame contains a drop box that lets you pick Chemi/Fluorescence or Autorad/Stained.

The valleys will be marked as peaks (bands) when the image is incorrectly identified as Chemi/Fluorescence instead of Autorad/Stained, or vice versa. This can happen when you use the wrong Open command to open the image. However, it is easy to fix. Click on the Image Tag drop box at the top of the image window. Choose the correct type of image: Chemi/Fluorescence or Autorad/Stained, depending on the type of gel. This tag tells 1Dscan EX whether the peaks should have high or low pixel intensities.