April 28, 1998: Version 2.6 released.

Version 2.6 has more changes than any previous revision! Many of these changes were suggested by users. In addition, we are pleased to announce two new analysis platforms unique to FlowJo: Movies and Calibrated Parameters. A host of minor changes increase the power of FlowJo without increasing its complexity or interface. Finally, there were a few bugs that crept in to Version 2.5; these have been fixed as noted below.

Special note: Once again, the workspace file format has been modified. Any workspaces that you save with this version cannot be read by previous versions of FlowJo. Of course, version 2.6 can read old workspace files.

Major new features:

• QuickTime Movies. You can now generate a movie of any graph! The "third" dimension which controls the flow of the movie can either be time (if time is a parameter of the data); alternatively, it can be another parameter entirely. This latter tool is a powerful data exploration tool that allows you to view how one or two parameter plots vary as you scan across a different parameter, giving you essentially infinite gating resolution! This is a completely novel data exploration tool unique to FlowJo. For more information and examples, see the help pages on the Movie Platform.
• Calibrated Parameters. Using either a manually-defined calibration or by basing on a collected sample, you can calibrate any fluorescence parameter. This is useful for computing absolute numbers of molecules per cell. The automatic calibration works on sets of beads, where FlowJo will automatically find the peaks for each bead--you only have to enter the fluroescence value corresponding to each bead; it also works using a known calibration point (for instance, CD4-stained cells, where the assumption is that CD4-positive cells have 50,000 molecules). FlowJo creates a new parameter that is scaled to this absolute calibration: now your graphs are shown in terms of molecules (you can gate, for example, on cells with more than 10,000 molecules!); statistics of all types (median, mean, CV, etc) are given in units of molecules per cell. Of course, any calibration can be applied; for example, conversion to absolute number of photoelectrons for performing high-tech statistical calibration of flow cytometers. For more information, see the Calibration Platform.
• Magnetic Gates. Any gate can now be selected to be "magnetic." Magnetic gates have the property that they "move" towards the centroid of the events. You can set a lymphocyte gate to be magnetic, for example, and then you won't have to make the small sample-specific changes that can be necessary between samples. "Magnetic" gates are drawn with a special arrow in the graph which denotes exactly how far and in what direction they were moved. To make any gate magnetic (whether it is square, elliptical, or polygonal), select this option in the Graph Specification Dialog.

Minor new features (grouped by theme):

Graphs:

• The Y-axis label now appears rotated 90 degrees in all programs to which graphs are copied, even those that did not support this advanced PICT feature.
• Gates drawn in graphs when copied to other programs were sometimes not properly clipped to the graph region by those programs. FlowJo now manually clips the gates so that they don't overlap the boundaries of the graph.
• The default name for a new polygon is now generated to be unique; further, you are not allowed to enter a name that already exists.
• You can now use the "Zoom" box in the title bar of Resizeable graph windows to cycle between three standard (and square graph) sizes.
• Quadrant gates can now be moved together by selecting any quadrant gate and clicking on the shared vertex (the middle, quadrant point). When you click and drag this vertex, all four gates are moved together. Previously, you would have to shift-click all four gates to move them together.
• When you limit the number of dots drawn in a dot plot (via the Preferences window), FlowJo will now draw representative events from throughout the entire collection. Previously, FlowJo would only draw the first "n" gated events in the file. This would cut off dot plots of kinetic data.Note: We highly recommend the use of high resolution pseudo-color graphs in place of dot plots.

  Layouts & Tables:

• You can now print directly from the Layout View window. This provides a different functionality for printing layouts: the entire, "tiled" layout is printed together (you can select the print magnification via the "Page Setup..." button). When you choose to print from the layout editor directly, then each sample in the group is started on a new page (i.e., "report" generation). For more information, see the layout editor.
• The Layout View window has new controls to zoom in and out, in addition to selecting the magnification from the menu.
• When generating statistics in tables, the exact stain name does not have to match any more. This means that you can drag a statistic applied to "Fl-1", for example, and get values for all samples or populations which have the same gate irrespective of whether they have the same reagent in the FL-1 channel. Previously, the reagent had to match for the statistic to be entered into the table.

  Workspace:

• You may now save a default workspace view (which columns to display) for any new workspaces, via the Preferences.
• You can click & drag a marquee in the workspace window to select multiple rows (similar to the functionality in the Finder).
• A new preference allows you to display the system file name for the datafile in place of the "name" column in the workspace display. You may alternatively choose to display the file name in a separate column of the workspace by using the Column Manager.
• When exporting a group of samples, you may now choose to exclude uncompensated parameters. This is useful for when you want to create a folder of datafiles that are compensated and don't need to include the uncompensated data as well.

  Kinetics:

• Copying the table from the Kinetics window now includes a one-line descriptive title with the statistics.
• The Kinetics table has the following additional statistics: Elapsed time (for any given time slice interval), Average value, and Area under the curve.
• The Kinetics window now displays the function name and options in the window.

  Derived Parameters:

• You may now select multiple samples or a group when choosing the "Derived Parameters..." platform; any derived parameters you create will be added to all selected samples (or all samples in a group).
• You may add a calibrated parameter using a previously-defined calibration standard. See the information on the new Calibration Platform.

  General:

• Holding down the option key while clicking on the menu bar will change the names (and functions) of some of the menu items. Previously, the function was altered via the option key, but you did not see this in the menu. Also, the option-modifier works with the menu key command (for example, option-command-W closes all open graph windows if a graph window is front-most).
• Many number-entry fields have now been modified to accept only characters that are part of numbers ("0" to "9", ".", "-", and "e" for exponent).
• There is now a "Help" menu, consistent with MacOS 8 implementation.

Bug fixes:

• Dragging a statistic into a table where the lengths of the names of all of the ancestor nodes was more than 63 characters caused FlowJo to crash. This has been fixed.
• Occasionally, the vertical scale of a histogram did not correspond to the data. This has been fixed.
• "L"-shaped gates, in which every corner was exactly 90 degrees, would be computed as if they were square. This has been fixed; these gates now compute properly.
• Printing from the layout editor would sometimes corrupt the system fonts in the window titles & menu bar. This was been fixed.
• In version 2.5.1 (but not 2.5.3), copying from the layout view window would copy the graphs scaled to the view display rather than always at 100%. Graphs are now always copied at full scale.
• Opening a workspace that is currently open by another station gives a more descriptive error message and does not disable the menu bar.
• Extremely large sample views (more than 2,000 rows of samples, gates, & statistics) are now handled properly.

Known bugs & workarounds

• Rarely, FlowJo gets into a state where some or all of the menus in the menu bar are inappropriately dimmed. If this happens, switch to another application (like the Finder) and back.

February 22, 1998: Version 2.5.1 released.

Special note: With version 2.5, the workspace file format has been modified. Therefore, any workspaces that you save with FlowJo v2.5 can not be read by previous versions of FlowJo. Of course, version 2.5 can read old workspace files. Note: Version 2.5.1 is slightly modified from 2.5, which was distributed on CD-ROMs. The differences between these versions is noted by the parenthetical addition "(v2.5.1)".

Major new features:

• Kinetics Platform. If you have time-dependent data (or just want to assess the stability of your sample during a run), you can launch a sophisticated platform that calculates a variety of statistics and graphs to display data as a function of time. You can calculate means, medians, percentiles, and percent of responding cells as a function of time. Both the data and the graphs can then be exported for further analysis or display in other programs. See the pages on Kinetics for more information.
• Derived Parameters. FlowJo can now calculate a limited set of functions on collected parameters. You can create new parameters which are the ratio of other parameters, or are log/linear conversions. You can also add a parameter representing time, with the assumption that the event rate during sample collection was constant. For more information, see the pages about derived parameters.
• Demonstration Mode. You can now run FlowJo without a serial number or dongle in order to evaluate it. FlowJo now has a "demonstration mode." In this mode, you have access to all of the features and capabilities of FlowJo; however, you can only read the data files provided by Tree Star, Inc. We provide a variety of different types of Demonstration data files to explore many of the new features of FlowJo. Of course, you can still obtain a free 60-day trial serial number that will let you use the fully functional FlowJo on any data collected by any flow cytometer (as long as it is in FCS format).
• Background processing. FlowJo will now work well when it is run in the background. You can switch to other applications while FlowJo generates layouts, performs calculations, etc., and continue smooth operation for both applications (v2.5.1).

Minor new features:

• Workspace grid. The sample and group lists in the workspace can be drawn with a very light blue grid. This can make viewing the lists much easier. You can turn the grid on and off via the preferences dialog.
• New Preferences. In addition to the workspace grid display option, you can now choose two additional preferences: (1) automatic hiding of the floating tools window (for graph windows), whenever the graph window is not in the front (the floating window is automatically shown again when the graph window becomes front-most); and (2) auto-tint gates as they are created (when off, the gates are not tinted by default).
• Formatting of statistics. In the workspace and table editor, numbers are now shown with approximately 4 significant figures. If they are less than 0.001 in absolute magnitude, they are shown in engineering notation (for example, 1.23 e-4; "e-4" means 10 raised to the power of -4).
• Revert. You may now select "Revert" under the file menu to discard changes to the workspace and go back to the last saved version.
• Layout display. The "path name" for a population (e.g., "Lymphs/T cells/CD4") is now displayed (optionally) below the graph in a layout.
• Default gate names. When you create a new gate, the default name in the dialog box is made unique (by appending "-2", etc., if necessary). FlowJo will not allow you to enter gate names that are extant in the subpopulation being gated.
• Rotated axes labels. FlowJo now displays the Y axis label in rotated format in the view window. Likewise, applications which cannot display rotated PICT will display the rotated Y axis label properly (v2.5.1).
• Exporting compensated FCS data. When you export multiple samples (e.g., by selecting a group and choosing "Export"), you now have the option of exporting only the compensated parameters. This saves disk space and simplifies the exported files (v2.5.1).

Bug fixes:

• The 10th parameter in a sample can now be compensated.
• A rare crash could be encountered when saving workspaces in which the "re-open graph windows" was set, and samples had been added to the workspace at different times. This has been fixed.
• The first time a parameter pop-up menu is clicked in a graph window, the check mark appears in the correct location.
• Delete a statistic node will remove its value from an open graph window.
• Choosing Page Setup... from the layout editor sometimes caused windows to be unselectable until FlowJo was switched out of the foreground and back. This has been fixed (v2.5.1).

December 15, 1997: Version 2.4 released.

Special note: With version 2.4, the workspace file format has been modified. Therefore, any workspaces that you save with FlowJo v2.4 can not be read by previous versions of FlowJo. Of course, version 2.4 can read old workspace files.

Major new features:

• Multiple & resizeable graph window styles. FlowJo now provides you with preferences to set four different styles of graph windows. You may select "large" (the same as in previous versions), medium, or small--each window is somewhat smaller in size and displays less information (like statistics). In addition, you may select "resizeable" to create windows that you can arbitrarily resize to any aspect ratio and nearly any absolute size. The settings are selected via the preferences window graph window settings.
• Workspace sample list information. FlowJo can now show you a wealth of information about the each sample in the workspace itself. This information is displayed in resizeable columns. In addition to the sample name and number of events, you may also show the value of any of the keywords in the FCS header, the compensation matrix used on the data, etc. This means that you can display the staining reagents used for any of the fluorescence parameters, allowing you to easily identify the sample. In addition, if you have defined specific keywords for your data files, their values can be chosen to be displayed in the workspace. To change the displayed information, you can either select the new menu item or double click on the divider bar.
• Workspace sample list sorting. You may also sort the samples in the sample list to be sorted on any combination of keys. You may sort from keys such as the fluorescence stains, the number of events in a file, the compensation matrix you used, etc. The sample list is then sorted and displayed. To sort the sample list, either select the new menu item or double click on the divider bar in the area by the word "Sort...".
• Channel statistics. The default unit for statistics output has been the scaled fluorescence value (also known as RFI). FlowJo now allows you to select that statistics be output in terms of channel numbers, ignoring the scaling information supplied by the FCS file. These statistics are selected in the statistics dialog window.

Minor new features:

• Preferences. A new interface that groups similar types of preferences together is now used--it should be much more easily interpreted.
• Adding statistics to multiple nodes. If you select more than one node (subset) before clicking on the "Statistics button" in the workspace window, then any statistics you select will be added individually to each of the selected nodes.
• Mode statistic. A new statistic, the mode (most frequent intensity), is now available.
• Keywords added to export files. If you export data files as FCS format, then FlowJo will copy any relevant keywords in the FCS header to the new file.
• Smaller default workspace window. New workspace windows are created smaller than in previous versions.
• Layout graphs no longer have the sample name listed above the graph as well as below the graph.
• New subset windows are created slightly offset from the "parent" graph window so that they do not completely overlap the original window.

Bug fixes:

• Blank graphs are shown with a text message denoting the graph style.
• The sample name associated with exported graphs will reflect any renaming you have done in the workspace.

October 25, 1997: Version 2.3 released.

Special note: With version 2.3, the workspace file format has been modified. Therefore, any workspaces that you save with FlowJo v2.3 can not be read by previous versions of FlowJo. Of course, version 2.3 can read old workspace files.

Major new features:

• Quadrant gates. FlowJo now lets you create quadrants to perform quadrant statistics. FlowJo shows you the percentages of each quadrant "live" while you track the quadrant crosshairs. See the information on how FlowJo implements quadrants.
• Floating graph specification window. You can now display a floating window which completely controls the drawing of graphs and gates. From this window, you can select gating tools (such as quadrants, polygons, rectangles, ellipses, etc.), you can change the X or Y parameters, the graph type & options, and you can select new gating options.
• Preferences. FlowJo now gives you several new preferences that control the default behavior of the program. For instance, you can now specify what sort of graph to display when you create a gate on a graph: should FlowJo show you the same parameters or choose new ones? and, should the resulting graph type be the same as the one on which the gate was drawn, the same as the preference, or a "blank" graph that allows you to fully define the graph before FlowJo computes it?
• Layouts. You can choose to view the output of a layout in a window (in addition to copying it to the clipboard, saving to disk, or printing). From this view, you can still choose to save or copy the graphic! You can change the magnification of this view in order to view large, complex layouts all at once.
• Dongles. You can now purchase a "dongle" to run FlowJo. This allows you to run FlowJo on any PowerPC Macintosh in your laboratory rather than purchasing user licenses for each Macintosh or running it only on a single machine. In addition, "dongles" allow you to run FlowJo on PowerBooks! (For more information, go to the FlowJo home page).
• KeyServers. For laboratories with several Macintoshes, you can now purchase a KeyServer option with any number of user licenses. This allows up to a specified number of simultaneous users of FlowJo, anywhere on your AppleTalk network. These users do not have to have their own serial numbers or dongles; rather, FlowJo will query the one Server Macintosh you set up to see if it can execute. (For more information, go to the FlowJo home page).

Minor new features:

• Changing graph options in layout items. The layout item information dialog (a window that appears when you double click on an item in a layout) now lets you change the graph axes, style, and options for that item.
• Cellquest titles are displayed in the workspace. Files created by CellQuest have titles in a special FCS keyword. These values are now displayed for the sample node in the workspace.
• Exporting histogram data. When you "copy" a histogram graph, FlowJo puts two items on the clipboard: the graph, and a text item containing the histogram data. By pasting into a spreadsheet, you will can get the gated histogram, for analyses such as DNA cell-cycle, etc.
• Selecting overlapped gates. By pressing the command key (apple key) while clicking in an area of the graph, you can iterate through overlapping gates to select the gate below the top-most gate. Command-clicking again will select the next gate in the same area. You can simultaneously press the shift-key to simultaneously select several overlapping gates.
• Colorized gates. You can select the color with which any gate is drawn; you can also specify that the gate is "tinted" when it is drawn.
• Exclusionary gates. Gates can now be "exclusionary", i.e., a gate selects all events which fall outside of the polygon rather than those which fall inside.
• Ellipse gates. You can draw horizontal or vertical ellipses to uses as gates.
• Workspace window: You can option-click on a disclosure triangle (to the left of any population node, controlling the display of subsets) and FlowJo will open (or close) all of the disclosure triangles of all subsets of that node--in other words, you can easily open or collapse an entire analysis tree by holding the command key while clicking on the triangle. The command-key performs the open/close on all "siblings" of the selected node as well. (In previous versions, option-click performed the "sibling" operations).
• Help text in tables & layouts. Help text is now shown automatically for many of the controls in tables and layouts, as it was for workspaces and graph windows. Simply move the mouse over a button or text item, and FlowJo tells you more about that item!
• Finding moved data files. When you use a workspace that references data files which have been moved, FlowJo asks you to find the new location for the data files. It now does this in more intelligent way, looking for subsequent data files first in the new location and then in the old location before asking again.
• Cursor tracking. The graph coordinates are shown during all cursor tracking operations, including creating new gates.
• Show memory & time since last save in workspace. New preferences allow you to show the amount of free memory available to FlowJo, or to show the time elapsed since you last saved the workspace.
• Menus. The menu items to select the graph style or options are now heirarchical.
• Sample information dialog. By doing a "Get Info..." (menu command, or cmd-i) on any sample, you will see a dialog that shows you information about the sample, including a list of all of the FCS keywords and values, and which compensation matrix was used if the sample has been compensated by FlowJo.

Bug fixes:

• Autosave feature: Does not ask to save multiple times if you leave and come back a long time later--only asks you once!
• Re-opening windows preference: now works.
• You can now specify a compensation matrix for a sample by selecting any node belonging to that sample rather than just the sample node itself.
• You can get help from the gate naming dialog.
• The cursor's "Y coordinate" is properly displayed for histograms and CDFs.
• A rarely-occuring bug causing FlowJo to hang when a node was renamed has been fixed.
• FlowJo was not consistent about allowing identically-named sample nodes; you may now have the same name for different samples in the same workspace without problems. You may not have identically named subsets at the same level within a population.

August 12, 1997: Version 2.2 released.

Major new features:

• High-resolution graphic exports. There is now a preference which can be set that allows all exported graphics (copying from the graph window, or using the layout editor) to be created as high-resolution PICT objects. The default is that the graphic itself is a bitmap ("paint") object. Exporting high resolution PICT objects allows you to set line styles, color, widths, etc., individually for separate contour lines or histograms. It also allows you to easily overlay histograms and to rescale graphs without loss of image quality. The default export is still bitmap object, because for many purposes this is high enough resolution and it is handled much faster by most drawing applications.
• New graph window styles. The look and feel of graph windows has been changed in several ways:
  1. A new preference allows you to specify a reduced graph window style. This makes for less clutter on Macintoshes with smaller monitors, allowing you to view more graph windows easily.
  2. The arrangement of buttons and information has been modified on the graph window to be somewhat easier to understand.
  3. New graph windows are opened to the side of the workspace window, rather than directly on top of it.
• Autosaving workspaces. You may now have FlowJo automatically query you at any desired interval if you want to save any given workspace that is currently open. The interval is specified in the preferences. If you choose not to save, FlowJo will ask again after the same interval expires.

Minor new features:

• The way graphs are represented internally has been changed so that much less memory is required. This allows you to simultaneously open many more graph windows while running FlowJo.
• There is a new preference allowing you to set the default arrangement of forward vs. side scatter when you first open a graph (i.e., forward scatter on the X or Y axis).
• Holding down the "option" key while selecting the menu items "Open other graphs..." or "Change other graphs..." skips the confirmation dialog and executes the command immediately.
• The name of the workspace has been added to the header on printed layouts.
• Histograms are automatically shaded with light gray; the shading is exported with the histogram when you copy (or use the layout editor).
• Cmd-I can be used to bring up the dialog to specify how a graph should look (also a menu item under the Graph menu).
• You can press the help key (usually the top left key in the center key section of your keyboard) at any time in the program to get context-specific help (like clicking in help buttons, a web browser is launched to provide this help).

Bug fixes:

• FlowJo used the configuration data from InternetConfig (IC) to determine which browser to launch for help information. If the computer had not been configured with IC then FlowJo would give the incorrect error that there was not enough memory to launch the browser. If IC has not been configured, FlowJo now tries to launch Netscape; if it can't find Netscape it tries to launch Explorer, before giving an error. If you still get an error after pressing the help key (or clicking a help button), then download InternetConfig from www.Stairways.com and run it to configure your Macintosh.
• FlowJo ignores FCS keyword information in the header for parameters that do not exist in the data. Some Coulter acquisition files have $Pn keywords for parameters beyond that stored in the file; FlowJo assumed that the presence of the keywords indicated the presence of the data.
• A crash could occur when you copied an analysis (gate, statistic, etc.) from one workspace to another, closed the first workspace, and then requested the new analysis. This has been fixed.
• Occassionally, FlowJo would transpose the axes on a gate when copying between different samples. This has been fixed.
• FlowJo would crash when an item from the layout editor was dragged to the trash (in the Finder). This has been fixed and now works properly.
• FlowJo would crash when reading empty workspaces. This has been fixed.
• Dragging items into the table editor now autoscrolls the table properly.

June 23, 1997: Version 2.1 released.