2-D Version 6.4

January, 1999

Release Notes

Introduction

2-D version 6.4 has new features which will make database queries both simpler and more powerful, improve the efficiency (and therefore speed) of program operation, and improve spot detection. The improvements in efficiency will automatically re-format data created in earlier versions. This process is not reversible, so we recommend that you back up any project prior to using it in version 6.4.

New Database Features

1. Nomenclature

• All database questions are now called queries. Spot Question Templates are now Query Forms.
• The word operators in all Query Form queries have been replaced by symbols:
  • equal is now  =
  • not_equal is now  !=
  • less_than is now  <
  • greater_than is now  >
• Intensity Ratio has been changed to Expression Level.
• The Expression Level choices Increase, Decrease, and Change are now Up, Down, and Up or Down. These choices are now push-buttons, rather than a menu.
• Selection Range choices in all queries are now push-buttons, rather than a menu.

2. Selection Ranges

A new Selection Range: Exclusive Add, lets you find a commonly desired set of spots, without the need for complex queries or image copies. This set of spots is all spots that answered the previous query, plus all spots that answer the current query, but not all spots that answered both. In Boolean algebra, this set is the Union minus the Intersection. For example, if the first query is spots that are glycosylated, and the second query is spots that are phosphorylated, an Exclusive Add would be all spots that are either glycosylated or phosphorylated, but not both.

3. Query Implementation

Version 6.4 queries spots in original images (that is, in your samples), and if they pass the query, selects the matching spot in the composite image. Earlier versions do the reverse: spots in the composite are queried, and if they pass, selects the matching spots in the original images. The earlier implementation makes it impossible to select spots in subsets of images. For example, assume you have two control images and two treatment images matched to one composite. Use either Image Characteristics or Spot Characteristics to assign the values "Control", "Treatment", and "Composite" to all spots in the appropriate images. It is not possible to get a Matched Spots Report of just spots from "Treatment" images; such a query is possible in Version 6.4.

4. Simple Queries

A new choice: Data Value or Characteristic, lets you make simple queries about data values (integrated intensity, area, pI, or Mw), spot names, spot characteristics, or image characteristics without creating a Query Form.

5. Previous Query Results

A new choice: Previous Query Results, lets you restore (or re-select) the results of a previous query. This is especially useful when you make several queries, manually select a spot, and then want to see your query results again.

6. t-tests and ANOVA

t-test queries no longer compare the original images of two composites. They now use an image characteristic to define the two groups of images. Each group must have at least two images. If the image characteristic has three or more values an ANOVA (F-test) is done instead of a t-test.

7. AND and OR of Image and Spot Characteristics

If you have more than one Image or Spot Characteristic query in a Query Form, the queries are "ANDed" or "ORed" in the correct order. For example, assume we have an Image Characteristic "Treatment", with values "A", "B", "C", and "D". The Image Characteristic queries "Treatment = A" and "Treatment = B" will now be interpreted as "Treatment = A OR Treatment = B". In earlier versions these queries were interpreted as "Treatment = A AND Treatment = B". This interpretation was illogical, and no spots could be selected. Likewise, the queries "Treatment != A" and "Treatment != B" will now be interpreted as "Treatment != A AND Treatment != B" (that is, all Treatments except A or B). The queries "Treatment = A" and "Treatment != B" are illogical, and therefore not allowed.

8. Existence Queries

Existence queries, both directly from the Database menu and within Query Forms, now have another choice: Unique; this is equivalent to Expression Match = 1. The choices are now push-buttons, rather than a menu.

Efficiency Improvements

1. Separate Spot Boundary File

Version 6.4 stores spot boundaries in a separate file from the other spot information. This significantly reduces the amount of memory needed for each spot. Spot boundaries are represented by ellipses until you Read Bounds (from the menu within each image). After boundaries are in memory, you can Dismiss Bounds (also from the menu within each image) to free up memory. Any command which quantifies spots automatically reads the boundaries.

The separate boundary file is not created until you modify data for at least one spot, and subsequently save changes upon exiting Review.

2. Only Load Images in Use

Version 6.4 only loads data for images actually in use. This includes images you want to open, their composite (if it exists), and any other images which comprise that composite. All of these images are necessary to correctly define matching relationships and triangulation between images.

3. Spot Characteristic Re-format

Version 6.4 stores spot characteristics in significantly less space than earlier versions. This re-
format is not done until you modify data for at least one spot, and subsequently save changes upon exiting Review.

Other New Features

1. Web Maps

A new choice: Web Map is available in the Report menu. This uses a selected image and a spot characteristic (or the spot name) to create both an annotated JPEG image and associated URL statements. The URL statements are suitable for the "get" format.

2. Image Processing Filters

Two new choices: Gaussian and Least Squares are available in Image Processing Templates. These implement a 7x7 filter with Gaussian weights, and a 7x7 filter with Least Squares weights, respectively, for smoothing an image prior to spot detection. The filters are discussed in detail in "Biologist's perspective on analytical imaging systems as applied to protein gel electrophoresis", Patton, Journal of Chromatography Vol. A 698 (1995), pp. 55-87.

3. Spot Detection

The Minimum Spot Size parameter is now measured in pixels. There is a Multiple setting if you need values greater than 100 pixels. Earlier versions used a discontinuous step function measuring spot area in square millimeters. The new parameter should improve detection for most, if not all, of your samples.

4. Mark Axes

The Mark Axes command in the Edit Spots menu can now be used to define new spots, without the need to first select a spot. If you have a spot selected, Mark Axes works as it has in the past: the axes of the selected spot can be modified. However, if you have no spot selected, or more than one spot selected, Mark Axes will create a new spot whereever you define the axes. (This latter operation is only available if you have just one image icon selected).

5. Bug Fixes

We have fixed all bugs that we were aware of. This includes the inability of earlier versions to Add to Group.