The Daily Dongle

new way to set number of decades on log plots

In an upcoming Mac 8.9 release, this dialog (FlowJo > Prefs > Workspace > Define..)  has new clicks:

Specifically in the middle section called "Standard Log Conversion Settings" :

The Setting-Formerly-Known-as-Starting Linear Channel Number is now simply called "lowest". This setting, in the units of channels, controls the left edge of the scale. If you type in 1000, you will chop the first 1000 channels from your data, and it will start at 10^3 instead of the usual 10^0.

The new setting, "highest", is in the units of decades. If you leave it at 0, the max value of the scale stays at what it was originally (for example, Accuri files go to 7.2 decades, diva - 5.4, etc.

Notice that the two settings are mutually exclusive.

The second control after you pick your left or right edge, is the number of decades in the "pulse area/height" text fields.

In the screenshot shown, I'm setting my prefs for Accuri data. I want to shave 0.3 off the top (assuming my fluorescence range usually doesn't hit the max value) and show what's left as 5 logs. Which would look like this:

Which looks more manageable than the full scale:

Look for the 8.9 beta, out now. 

8.8.7b4 - introducing Vertex Gating

Way cool new feature: you can now link gates' vertex coordinates to a summary stat of another gate!

here's a quicktime demo, ~4MB

oh, and you need this new beta build: Download FlowJo

Finally, if you need a refresher in FJML, it follows: 

8.8.5 and all the "transform" preferences..

Since 8.8.5 introduced yet another transformation preference, while I still haven't grasped all the existing ones, it's time to write it up in one place.

There are six prefs, and they are so named and located:

(P1) Allow custom visualizations ("Preferences->Workspace")
(P2) Transformation settings ("Preferences->Workspace")
(P3) Enable Transformation for uncompensated area parameters ("Preferences->Workspace->Define 32 bit")
(P4) Transformation settings for Uncompensated parameters ("Preferences->Workspace->Define 32 bit")
(P5) Transform height parameters too ("Preferences->Workspace->Define 32 bit")
(P6) Transform all compensated parameters ("Preferences->Workspace")

Note: P6 is the new preference.

(A) If P1 is not set, no transformation is done on any parameters. If you motion to Platforms/comp/define biexponential.. operation, you'll first be prompted to change this preference.

For uncompensated parameters:

(B) If incoming data is floating point (DiVa), then transform the uncompensated parameters if P3 is set.  Use Settings in P4.  Do this for all Area parameters, and if P5, all parameters.

(C) If incoming data is not floating point, then no transformation is done.

For compensated parameters.

(D) If the uncompensated parameter was transformed then use its settings (this is ONLY scenario (B), above).  Therefore, these parameters will be transformed using the settings in P4.

(E) If the uncompensated parameters was not transformed, and P6 is off, then compensated parameters will not be transformed.  If P6 is on, then the compensated parameters will be transformed using settings P2.

For manual transformation of compensated parameters:

(F) Use settings in the dialog window, which are copied from P2 (and if changed, are copied back into P2).

Final notes: 

P2 does not allow specification of the width parameter.   P2 uses the same width parameter as defined in P4.

Most users will usually use either the P4 settings (DiVa) or the P2 settings (non DiVa).  Only users which analyze data from both kinds of machines will end up using both sets of preferences.

CXP data in FlowJo-Mac

Team Mac wins the gold ribbon for being the first to decipher the CXP file format and extract the FCS3 information. Please note -  that this is a new feature, and you can benefit from it by moving to version 8.8.5 - however, there is no way to retroactively apply this to your existing analyses, therefore, it's something you can choose to take advantage of from now on.

Below, see the fluorescent parameter scaling in the same FCS file before (left) and after (right).

We are now able to read the raw 20 bit data and convert the coulter-specific keywords for comping data to get the matrix. So the graphic on the right, above, shows the same data as on the left, except in full resolution and with biexponential transforms enabled.

Notice the CD3+/CD19- cluster has shifted from a position of about 20 to about 20,000 - so summary statistics are shifted up by approximately 1024 for bright cells. Because of the randomization and binning done in "dim" areas, there is no way to predict exactly the shift of cells' fluorescences, so don't compare before/after results. Naturally all gates made on in the old workspaces WILL NOT WORK properly in the new version's scaling.

For this reason, all old workspaces with CXP data will open in the "old scaling" (FCS2 mode). Any time you use this new version to make a new workspace with CXP data, they will be scaled according to the 20 bit FCS3 info.

Also, some time ago we included this preference in the "general" tab:

If enabled, it fixes this problem:

sometimes files from the same experiment will show different ranges in the scatter parameters. To avoid that, check the "ignore.." pref, restart FJ. THIS DOES NOT FIX SCALING IN OLD WORKSPACES.

Here is the  blurb from engineering:

[...] the most important change is that CXP files are now read in as FCS3.0 files.

There is a new preference in "workspace" tab:


that we elected to set "ON" by default, which will automatically assign a default standard transformation to all compensated parameters. The parameters for this transformation are the ones in the preferences.

For CXP files that are 20 bit, the decades that are shown is the default from the 32 bit settings -- i.e., height parameters.  If you set it to 4 decades, you get an identical display to CXP's FCS2.0 data embedded in the same file.  We find it looks better for many of the files we looked at with a setting of 4.5 or 5 -- but that's up to users.

Note that the compensated parameter display will only look identical to the FCS 2.0 data if you set the new preference to "OFF" before you create the workspace...

The biexponential transform looks best with a 4 decade range and a extra negative decades value of 0 (these are the defaults), but you might want to increase the extra to 0.5 or 1, as usual.

Download FlowJo Mac Beta 8.8.5b2

Workaround for 8.8 File Export Bug

For those of you who would like to export your files out of FlowJo (for Mac) as new .fcs files, you may have come across a nagging little bug: your file names ($FIL) get cut short and no longer make sense. A workaround is presented here to ease the headache.

The key? Assign a separate set of keywords that you can use after exporting to orient yourself to the proper sequencing of your sample set. To save time add these keywords at acquisition, but in case this step was passed up, the example below will show how to add the the keywords in FlowJo.

To add the keywords in FlowJo (for this example, Tube Name and Tube Number), go to Workspace=>Add Keyword. A new Workspace column will appear with each keyword added. Type the keyword into the first cell and then copy/paste that word into each cell in the column (alternatively, use a spreadsheet to copy/paste an entire column of cells into FlowJo).

If you are going to use keywords you assigned at acquistion, throw them into the Workspace from by adding columns from the Edit button.

Then create a value series (in this case, 1 to 5) by typing in 1 (or any other number) in the top cell of the Tube Number column. With your cursor still in that column, go to the Workspace menu, hold down the Option key and select Create Value Series from the options.  You can then create a numerical value series however you would like.  1-16, 100-150, etc.

After doing these steps, my workspace has the two columns I added: 

 

Change the name that FlowJo displays in the workspace "Name" column in preferences (go to Workspace=>Workspace Preferences). If you already set up keywords during acquisition (best practice), it is very easy to select a combination of keywords that you would rather have displayed instead of the $FIL keyword (aka data file name). In my workspace, I made two new keywords with named Tube Name and Tube Number.  I can then export everything with the file name Tube_1 through Tube_5.

After exporting as a new .fcs file (Workspace=>Export) I added the samples back into FlowJo and here is what is displayed in the Name column:

 

Granted this isn't the most elegant solution, but the "frustration savings" should more than offset the cost.

For more on Exporting, go here: http://www.flowjo.com/v8/html/export.html

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content credit: Nicholas Ostrout
editing credit: Ryan Farmer

What's new in FJ Macintosh

Lots of development has been ongoing on both fronts (mac and PC).

First, For our loyal Mac users: announcing 8.8b5

you can now bend quad gates (hold down option key while you drag the center point):

->->  

hint: once "bent", the quads will become stand-alone gates. To make sure the gate boundaries don't drift, use "alt+drag" to move the center point from now on.

you can also align multiple gates and "mold" them together (cmd+a, cmd+opt+m):
 ->->

now you can drag the "molded" vertices together when you hold down the "option" key while dragging them:

->->

8.7.3 and recalculation frequency

So, you miss the old "immediate" recalculations from 8.7.1 version? From an email I sent to one of our Users earlier today asking about this -

we changed this behavior to address certain flowjo crashes. The most stability we've achieved in the program, is in this version, because of this change. The crashes caused by instant live recalc affected all speeds and models of macs, not just the older ones. If you haven't hit this, you're one of our more careful users who probably waits until "tasks" are finished before you queue up more gate movements, batches, etc.. ;)

To get the old behavior of recalc with a keystroke, click on All Samples and hit [cmd]+[=], or batch, or make a table, all values will be recalculated as needed. We cannot post version 8.7.1 and previous because of fairly major stability concerns on all Macintosh platforms (and various other bugfixes.)

Concatenation Revamped in FlowJo for Mac Beta

With the new user interface, you have complete control over concatenation.  There are two modes of concatenation:  

1. if you select multiple gates (from the same or multiple samples) when launching the platform, you will be creating a single output data file that is a concatenation of those gates [this is the same as the old platform]. 
2. If you select a single gate from a single sample when launching the platform, you can "batch" concatenate -- create multiple files from sets of concatenations. 
   
   • You can define sets either by concatenating every "n" successive files together (e.g., if you have 20 data files comprising 5 sets of 4 files, you can set it to concatenate every 4 files together to make 5 output files; it concatenates in the order that the files appear in the current group). 
   • Or, you can have FlowJo concatenate files that share the value of a given keyword (e.g., if you have different numbers of tubes that share the keyword "Sample ID", then choose this option; files for every unique value of "Sample ID" will concatenated into a single output file).

Options for concatenation now include the ability to have every single event from each gate in the output file, or, to have the same number of events from each gate in the output file. You would use the latter to ensure that the resulting concatenated file has equal representation from each of the input sources.

You can also select one or more keywords whose values will be used to create new parameters in the output data file.  [This was the new feature in 8.7.2, discussed before].  You can select an option to "spread" these values a little so that they appear more nicely in the output file [see below for example].

There is only a single "Concatenate..." command now in the workspace menu (no more option-command) that handles these operations. 

Get new beta 8.7.2b4 here

ratio parameter range in flowjo

There was a FJ question on PUCL abbreviated and answered below (I've been warned by owner not to post FJ related stuff to the list...)

our Fura red/fluo4 ratio parameter does not have consistent ranges in our experiments - how can I change it manually?

This is currently only possible in the Macintosh version of the program. Windows users will have to wait until 7.5 version ships (we hope to demo it @ ISAC)

On the mac, When you define the ratio parameter, you can specify the min and max values for the range of that new parameter right under the formula portion of the dialog. There, you can pick your custom min/max numbers and re-use them in other experiments to have consistent plots.

Is there a way to make the ratio parameter range uniform for all my experiments?

Yes, one way is described above (pick some min/max values and reuse them.) Another way is to make one derived parameter for one file, then apply it to the rest of the files in your group (assuming those files have the same source parameters to make the ratio.) To do this, click on the file you just derived in the workspace, then click Platforms, Derived Parameters, copy to group's samples.

Can I show statistics in the layout?

Yes, to show kinetics stats, simply drag your kinetics node from the workspace window to the layout. If you want to show summary stats, you can first make them in the workspace window using the "sigma" button, then drag the stat-nodes from the workspace window to the layout window.

Clipping Masks and FlowJo Graphics

This brief is intended for FJ V8 users who:

- have plots in layouts which they copy/paste into illustrator
- set their plots to not have axes labels or numbers, just ticks, ie..

If you copy/paste a label-less, number-less graph into illustrator, you may notice that it's missing axes ticks:

in flowjo:

in AI:

So what happened there? where did the ticks go? Here's a hint.. select the graph in illustrator with the Selection Tool (Black arrow, keyboard shrtct "V"):

Kind of looks like they're there now, doesn't it. Well in fact they are. However, the Clipping Mask we create only extends as far as the 0 point of the graph, which is a few pixels short of encompassing the ticks. There are two ways to fix this:

1. right- or ctrl- click this chart with the Selection Tool, and ungroup. Right click again and release the mask. Done.

2. in FJ, create an object, like a line whose pen/stroke have been set to white (ie, it won't show when printed). Place this object south-west of the lower-left most object in your layout. This will extend the clipping mask in that direction, and thus, include the ticks so you don't have to deal with it ;)