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| There are a number of default behaviors of FlowJo which you can alter. These are modified through the Preferences dialog, shown below. To get to this dialog, select "Preferences" under the "Edit" menu on OS9 computers or under the "FlowJo" menu on OSX computers. Preferences are grouped by topic. To select a set of preferences, clicking on the appropriate tab at the top of the dialog.
For more information about each type of preference, click on the topic: With the release of FlowJo version 4.4, the program will use a different preferences file. After you launch FlowJo, you will need to enter your serial number (if you are not using a dongle) and then set all of your desired preferences. The new file is named “FlowJo Preferences.plist” under OSX, and “FlowJo v4 OS9 Preferences” under OS9 (instead of “FlowJo v4 Preferences”). On OSX, FlowJo now users Apple’s built-in Preference Manager for better performance and consistency. This Preferences file can now be directly edited just by double-clicking on it; however, we do not recommend this as FlowJo may not be able to recognize the values you change. If the program ever has problems running, even before it tries to open a workspace or view any data, then it is useful to try removing the FlowJo preferences file (search for files containing the name "FlowJo"), restarting and letting FlowJo recreate this file from scratch.
Workspace Preferences The section marked Appearance defines preferences about the how the workspace appears. You can have FlowJo show "Elapsed Time Since Last Save" to show the amount of time that has past since you saved this workspace. The time counter is shown in the tool bar of the workspace window. Further options let you choose to have FlowJo display a light grid over the sample and group list to aid in visualization. The setting "Gate Statistic is Freq. of Parent" allows you to specify that the percentage shown in workspace refers to the portion of the immediate parent population, as opposed to the portion of all cells. In the Sample Identification area, you can choose how FlowJo will display the data file names in the workspace window list. When you choose to display the sample's file name from the "Sample Name Keyword" - FlowJo examines the FCS keyword header information and uses the sample name keyword from the data file as the file name in the workspace. NOTE: Options chosen when acquiring your data can cause all the samples in the workspace to be named identically (i.e., each file name is missing the suffix .001, .002 etc.). To avoid this problem, choose the "Use Data File Name" option. This option displays the name as it is on the system disk (i.e. with the .001 suffix) rather than using the internally-defined keyword. An alternative file naming option is to "Specify a Keyword" from the data file. For instance, displaying Sample ID or Patient ID as the file name may aid in identifying your samples. Annotation such as Sample ID or Patient ID can be entered while acquiring your data or in FlowJo. The final button in the section is to set the order and size of columns visible in the workspace. If you click on "Use As Default", then FlowJo records the columns & spacing that you have set in the current workspace, and uses that for all new workspaces that you create. The right section, Behavior relates to preferences regarding the workspace operations. One option is to "Remind to Save" your work, protecting from loss of (much) data, should the program or computer crash. By entering a non-zero value in the box, you instruct FlowJo to remind you to save the workspace every few minutes. When this amount of time has elapsed, FlowJo puts up a dialog and lets you choose to save or not to save the workspace (note that this dialog has a checkbox that lets you specify to always use the same answer: if you check this box and click "Yes", then FlowJo will automatically save the workspace for you every time period that you select here). "Save with Incrementing Version Number" saves a new workspace every time FlowJo saves your work. The workspaces are named with incrementing version numbers (i.e., name.jo-1, name.jo-2 etc.) when the "always use the same answer" box is checked upon the first save (see above). The popup menu for "Adding Analyses to the Workspace" controls how FlowJo causes recalculation of gates & statistics that are copied between subsets (or samples) to occur. Depending on how large your experiment is, it may become cumbersome to have FlowJo recalculate all populations affected by the action of dragging a population to a group or sample. This setting will permit you to postpone recalculation on some or all samples to maintain the responsiveness of the program. The default behavior is to compute immediately (which may cause FlowJo to need to load the sample data). You can also choose to have the statistics computed "only when needed" or "only if sample loaded". The latter option will cause statistics to be computed only if the sample has already been read into memory (and still resides in memory).. FlowJo will always compute the statistics once they are requested by the Table Editor or Layout Editor... this option is designed only to control the calculation of statistics for display in the workspace window. The next section relates to the Compensation Platform. Often, compensation causes data to be compressed onto the bottom or left axis in displays. This can be un-aesthetic... therefore you can choose to have FlowJo transform data display to improve visualization of the data. The "custom display visualization" allows you to apply the custom transformation from the Compensation menu and to choose the default values for the "Additional Negative Display Size" and the number of "Positive Decades of .Log Display". "Copy $Comment to annotation" will cause the keyword of comments made during acquisition to be turned into an analysis annotation. These settings apply immediately, to all open workspaces and to workspaces you will open in the future. The button at the bottom of this section, marked "Define…" goes to a separate dialog specifically for dealing with the data generated by Becton Dickinson's digital Vantage (Diva) system. This 32 bit linear data does not fit the normal data standards used by other instruments, but FlowJo has special settings to read the DiVa acquisition files.
The first section holds preferences that define how a graph window should look when you first open it. The "Graph type and options" are identical to those you can specify when you open the graph options disclosure triangle on the graph itself. This will be the default graph type displayed whenever you open a sample for the first time. To draw large dots, uncheck the High Resolution display option-these dots are twice as large in each dimension, and may be easier to see on slides or publications. "Forward scatter on X...", when checked, specifies that FlowJo shows Side scatter vs. Forward scatter the first time you open a sample's data (otherwise, it shows Forward vs. Side scatter). Checking "Use Scientific Notation for Log Axis labels" with direct the program to draw axis labels as 100, 101, 102, etc. If unchecked the labels would be drawn 1, 10, 100, etc. The "Add event number parameter..." option adds an additional parameter to the axes pulldown menus. The event number parameter allows you to display the cells on a plot in the order they were run through the machine. For instance, if you collect 10,000 events, and display the Event Number parameter on the X-axis, the first cell will be at the left edge of the graph and the 10,000th cell will be at the right edge of the graph. This is useful to display vs. another parameter such as scatter in order to determine if changes occurred during collection. Checking "Show uncompensated parameters in menus" causes FlowJo to display both the uncompensated (without brackets) and compensated (bracketed) parameters in the axes menu pulldown lists. If you use Dot Plots (and in general, you shouldn't!), and you have more than 10,000 events in a file, you may wish to limit the number of dots drawn to speed up display or to keep it from "blacking out" completely. This can either be an absolute number, or a percentage of the total number of events in the population. If you enter 0 in this box, regardless of the radio buttons' settings, all of the events will be drawn. "If more than this percent of events is on the axis", FlowJo will open a warning dialog with an explanation or it will simply show a warning on the graph window. If you enter 100 in this box, the warnings will not be displayed. The Gating section groups preferences together that determine the behavior of gates (line weight and gate color) and gated populations. "Show gate frequencies on plots" specifies that FlowJo should draw the frequency (within the parent gated population) of any gate drawn on a graph. The frequency is drawn in percent. The frequency is drawn on exported graphs whenever the gate is drawn, if this preference is selected. "Also Show gate name" displays the gate name inside gate on the graph window. "Show live quadrant statistics" specifies that FlowJo should calculate quadrant statistics whenever the quadrant tool is selected. If unchecked, the statistics are not displayed in the graph window while you track a quadrant gate. "Auto-select gating tool when clicking in a graph" speeds the process of creating gates, because the mouse is automatically ready to start creating a polygon or other gate. If this is off, then you need to select the tool at the top of the graph window before gating. Checking "Always ask for Quadrant gate name prefix" brings up a dialog every time you create a quad gate. This dialog asks for a prefix to append to the quad gates - useful if you create multiple quad gates and want them to be listed together in the workspace window." When the "Auto-select gating tool..." option is checked, a polygon gate is started by simply clicking in the graph window. If this option is unchecked, a gating tool must be chosen from the top of the graph window before a gate will be drawn. "Choose rectangle gate by default" determines whether the polygon or rectangle is the default gating tool. "Auto-set tinting for new gates" if checked will set the attribute to draw the gates with a colored tint. (Be forewarned that sometimes when exporting tinted gates, the color will become opaque and hide the data underneath the gate. Test with your presentation or graphics editing software before using tinted gates too freely.) The set of preferences in the box marked "Graph options for new subsets" specifies what will happen when you draw a gate and create a new population. The graph for the new population will either have the same parameters as the graph on which you drew the gate; or, if the box is checked, FlowJo tries to intelligently select a new pair of parameters to display. The graph type itself is selected by the pop-up menu. Here you can select that the new graph type is either (1) the same as the graph on which you drew the gate; (2) the same as you specified in the preferences section above; or (3) a blank graph. The latter is useful when you are working with enormous data files, where you would like to specify the graph before FlowJo takes the time to calculate it for you. Finally, you can add a custom list of gate or "Population names..." that you use frequently. If this list is defined, the gate name dialog will show a popup menu listing all of the names you have defined. You can either choose a name from this list or if you start typing the name of the population - FlowJo will automatically fill in the rest of the name. This will facilitate more consistant naming conventions, and reduce redundant typing.
The first section Table Editor contains only options specific to the Table Editor. Tables can optionally include summary statistics as additional rows in the output tables. If this is checked, additional rows for mean and standard deviation will be appended to the table. If the summary statistics are showing, values in the table will also be highlighted. If the value is greater than one standard deviation from the mean, it will be bold and italicized. If the value is greater than two standard deviations from the mean, it is also turns red. The option "Use Short Statistic Names" causes an alternate set of statistic names to be used in the creation of tables. There is a tendency for the first row of tables, which contains the column headers describing the statistics, to become long and unwieldy. This setting will reduce that effect by substituting % for frequency, P for parent, and G for grandparent. Within the Layout Editor section, you can choose the default format of batch layouts. The choices include New (Batch) Layout, Tiled Report, Web Report, Movie or Print Directly. You can learn more about the options here. "Allow Stain Name Mismatch" loosens the strictness on matching gates across samples. Normally a sample must match both the parameter (e.g., FL1) and the stain name (e.g. CD3) in order for its graph to be included in a layout. If this setting is on, then only the parameter name is required to match. When making histogram overlays, it is sometimes useful to separate the curves so that the overlap does not confuse the graphic. This checkbox will offset successive curves in the overlay so that it is easier to read them. The "Use Placeholders" setting will create new layouts so that they only show the box enclosing a graph (and its legend and annotations) instead of actually plotting the data. If the layout is complex or references large data sets, the recalculations in the layout can be quite slow. Using placeholders will speed up of the response time. This option is also available within the layout window itself. This preference will determine its default state. The check boxes in the section "Use Layout Annotation" determine which information is included in the annotation of a graph added to a layout. The title, sample name, population, path, frequency and count can each be individually included in the annotation. The "Legend position" determines where the legend for overlayed graphs is placed. Each of the Layout Editor and Table Editor have buttons that will cause the current output to Under the Layout Options, you can choose to have a "statistics table automatically created" when you drag a special analysis platform such as cell cycle to the Layout Editor. When this box is checked FlowJo places both a graph of the platform analysis and a statistics table. Within the layout editor, double clicking on most items, or choosing the Get Info… command from the menu will edit that item's specific attributes. If multiple items are selected, then a "multiple item settings window" comes up which contains all of the settings available. Setting the check box "Get Info uses Multiple Items Settings Dialog" will cause the larger dialog to be shown all the time. The checkbox "Draw Border" determines whether a line is drawn around the text boxes by default. The "Define Legend Palette" button allows you to define the colors of the overlaid graphs in the Layout Editor. The first color by default is red and the next is blue etc. This can be changed to all black and gray scale if you do not have a color printer. The "Define Printed Layout Information" button allows you to define the headers and footers for printed pages. If you print the workspace window, a table or a layout, information such as the date and workspace name show up as headers and footers on each printed page. Click this button to type in your own text, choose from special strings (such as date and time) and even to place graphics at the footer, header or in the background of each printed page. When HTML Output is produced, either directly from the layout editor, or from the tiled or animated views of the batch window, the file format of the data is controlled by these options. You can choose to "save web graphics as" JPEG, PNG, TIFF, PICT, BMP, Quicktime or Photoshop file formats. You can also choose files that contain additional HTML for the header and footer of the pages you produce. These may contain your logo or link information that you want included in the pages you create.
The first section of this pane General Appearance & Behavior controls miscellaneous settings that affect the program's look-and-feel. Don't automatically look for FlowJo updates is necessary to turn on if your computer is not online. Otherwise, FlowJo will check on the internet to make sure you have the latest version of FlowJo. Those interested in having the latest version may want to turn on the Inform me of Beta versions option. Mute Sounds will suppress the various sounds FlowJo makes when completing actions such as copying to the clipboard or adding a statistic to the workspace. Suppress 'live' window operations turns off the feature support dragging entire images of windows, instead of the outline boxes, as older Macintosh systems supported. If you are running FlowJo on a system older than 8.5, make sure this box is clicked as the 'live windows' will crash your computer. The box to Disable Symmetric multiprocessing prevent FlowJo from taking advantage of the multitasking capabilities of newer Macs. If you have a single processor computer, this preference will be on as some older computers have problems with FlowJo multiprocessing. FlowJo has a "task monitor" window, a small status bar that displays the current number of graphs and calculations it is currently working on. Because its workload can sometimes get quite extreme, the monitor window can be useful in assessing how much longer you have to wait. This popup menu will let you position or hide the Task Monitor Window. Finally, for some sites, FlowJo retrieves data over the internet; in these cases, you can select a maximum cache size that FlowJo uses to keep copies of the data on your Macintosh (and, if you desire, to have FlowJo delete the cache files when you quit). The settings within the box labeled Remote data transfer, are only used by a small number of sites that provide centralized data storage for their FCS files. If files are stored remotely, FlowJo can use FTP or HTTP to download them to your Macintosh as they are needed. To speed up subsequent access to the same files, FlowJo maintains a cache on local hard drive. These settings will determine the maximum size of the cache, and whether the program should delete all of the files when it quits. The top right pane governs preferences on Exporting Graphics. The "Include gates..." option specifies whether gates on a graph are copied into exported graphs by default. When you press the option key while selecting "Copy", this attribute is toggled. "Include text data…", if checked, causes FlowJo to put two distinct items on the Clipboard whenever you select "Copy" from a Histogram window (or a Kinetics window): one is the graphic, the other is a text representation of the processed data. Depending on whether you copy into a graphics program or a spreadsheet, you will get the appropriate item. (Some graphics programs incorrectly prefer text when pasting; if this is the case, select this checkbox to have FlowJo only copy the graphic). You can choose between four common file formats for saved images. If the files will be staying on the Macintosh, PICT is generally a good choice, as it is readable by the widest variety of Mac software. JPEG and GIF are both popular cross platform formats for use in Windows software or in web pages. TIFF is another graphics format, which may be applicable in some contexts. Within the different format, there are type specific options. In most cases, FlowJo only generates graphs with a small number of colors in them. But you can set a maximum, to conserve memory and conform to certain publishing constraints. PICT graphs can be created as vector based graphics, or a condensed bitmap. The axes are always created as vector based, as that improves printability and editability of the output. But creating a dot plot of tens of thousands of dots, each as its own vector definition will bring most graphics packages to their needs. Unless you plan to edit attributes of the dots (such as to emulate the "large dots" function from the Graph pane), it is best to leave this off. GIF files can be written with a couple of fancy options, used frequently in web sites. Interlaced means that the file is written in alternating panels, so the user can get an increasingly good view of the graphic as it's being read in from the network. This is not as necessary as it was in the days of slow modems, but is nonetheless a nice effect. Secondly, the GIF format supports having the white color to be transparent, enabling the overlaying of graphs in a web page. JPEG files use compression to save space. The degree of compression is variable, with the understanding that in most cases, increasing the compression will reduce the quality by limiting the number of colors in the image. JPEG is generally used with photographic images, so the image size and quality are critically important. In the case of FlowJo, the images are very small, use few colors and compress quite well. As a result, these settings are not going to have much effect on the output. "Back & Forth Looping" directs the movie viewer in the layout editor to loop such that they play a movie from beginning to end, and then play it backwards from end to beginning. If this is not set, the movie will play from beginning to end and then jump back to the beginning and play it again. |
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be written to a file and launched by a different program. For the table editor, you can pick a spreadsheet or statistics package as your Helper Application. For the layout editor, you can pick a graphics or publication program. Use the Choose... buttons here to pick the applications you wish these buttons to launch. In response to the Help button in any of the FlowJo's windows, the program will direct the Finder to launch the web Browser you have chosen in your computer's preferences, and display the HTML reference manual page specific for the context.
