What is TSNE?
Overview
Highdimensional singlecell technologies, such as multicolor flow cytometry, mass cytometry, and image cytometry, can measure dozens of parameters at the singlecell level. FCS Express integrates tDistributed Stochastic Neighbor Embedding, otherwise known as tSNE, which is a tool that allows you to map highdimensional cytometry data onto a two dimension plot while conserving the original highdimensional structure to help you visualize and analyze highdimensional data.
Using tSNE Transformations in FCS Express
The final result of the algorithm in FCS Express is a 2D plot in which the positions of cells reflect their proximity in their original highdimensional space. Plots can further be colored with density or heat mapping of each parameter, allowing for easy visualization of populations.
Given that tSNE is an highly demanding algorithm, the De Novo Software 's team made a great effort to improve its speed within FCS Express.
To give you an idea of how tSNE is performing within FCS Express, we have run some speed tests to show how the two methods that are used to calculate tSNE compare with each other in version 6.
The table below shows the elapsed time (in seconds) for tSNE calculation using the BarnesHut Approximation (Amount of Approximation = 0.5)with a different combination of number of considered events and number of considered parameters. In these tests, tSNE was not estimated for unsampled events. Please refer to the Defining a tSNE Transformation section in the software reference manual for more information about the methods and options that are available in FCS Express.
We then repeated the previous tests by enabling the Estimate tSNE for Unsampled Events option. By enabling this option, the events that did not participate in the tSNE calculation will be mapped to the nearest point that did participate in the tSNE mapping. With the file used for this test, the estimation had been performed on almost half a million of events. The result of these tests can be seen in the table and scatter plots below.
The chart below shows how tSNE in FCS Express compares to tSNE as performed in R:
 tSNE stands for TDistributed Stochastic Neighbor Embedding.
 tSNE is a nonlinear data reduction algorithm that takes multidimensional data and represents the original data in two dimensions, while preserving the original spacing of the data sets in the original highdimensional space.
 For a better understanding and more effective use of tSNE, please click here for an excellent overview.
 tSNE is a nonlinear method.
 It differs from other methods in that it is focused on unrolling the native data into a three dimensional space and emphasizing the clusters found within the native highdimensional data set.
 Exact tSNE calculates the pairwise distance between every pair of data points.
 The BarnesHut Approximation calculates an approximation of the Exact tSNE method in that the BarnesHut method approximates the distributions. The BarnesHut method calculates the distance between each data point and its closest neighboring points only.
 If the Amount of Approximation is set to 0, the BarnesHut method is virtually identical to the Exact tSNE method.
 Because the data can be randomly initialized, the data set can produce different results with each analysis.
 However, FCS Express introduced a method by which the user can specify a Seed Value.
 The Seed Value is a numerical constant that is used to standardize the initialization.
 This is the amount of approximation that is performed with the BarnesHut method.
 This value can range from 0 to 1.0, where 0 is No Approximation, and 1 is the maximum amount of approximation. If the amount of approximation is set to 0, the BarnesHut method will be virtually identical to the Exact tSNE method.
 In FCS Express, the default Amount of Approximation is 0.5.
 Perplexity is a measure for information. In tSNE, the perplexity is used to sets the number of nearest neighbors considered.
 In tSNE, typical values for the perplexity range between 5 and 50.
 By default, FCS Express uses a sample size of 3000. However, a user can specify any number for the subset of cells to be included in the transformation.
 However, the larger the sample size, the more time it will take to calculate the transformation.
 The maximum number for the sample size will be the total number of cells within the specified gate of the transformation.
 tSNE is a resourceintensive algorithm because it inspects every single data point and measures the distances between every pair of points.
 The BarnesHut method measures the distance between every point and a subset of points.
 Currently, the result of a tSNE transformation is not saved within the layout or with the .FCS file itself and instead is calculated automatically or manually on demand when opening a layout.
 If your tSNE transformation is computation intensive we suggest to export the tSNE X and tSNE Y parameters with your original data file as a new FCS file. The resulting file can then be accessed and examined quickly and easily without recalculating the transformation. The new tSNE X and tSNE Y parameters may be easily exported via the Export Data dialog. We also recommend to saving the original layout in which the transformation was created so you can easily review the settings used in the transformation at any time.
 Github by Laurens van der Maaten, coauthor of original implementation.
 Distill article discussing the effective use of tSNE
Learn more about tSNE in FCS Express via a recorded webinar.

Installation

Licenses

 Can I get more information regarding the AddOns that can be purchased with a license?
 Can I lock my template based on an electronic signature?
 Does FCS Express have any features to help meet 21 CFR Part 11 compliance?
 Does FCS Express have Quality Control features?
 Does FCS Express offer Single Sign On capability?
 How do I configure SQL Server to host a database for FCS Express?
 What database options are available when I purchase the Security option?
 What is the difference between the different types of Users that are available with a Security and Logging license?
 What is the difference between the Logging option and System Level Audit Trails?
 What SQL Server permissions are needed?


 Can I track usage of the internet dongle?
 Can I try out the Internet Dongle before I make a purchase?
 Can the administrator log users out?
 Do you have to be connected to the internet at all times with the Internet dongle?
 How can users be added to an internet dongle license?
 How do I activate my dongle?
 How do I change my internet dongle/site license password?
 How many people can be logged in at the same time?
 How many user accounts can I create?
 If a user left the computer running can the user log themselves out from another computer?
 What are the differences between the internet dongle and network licensing options?
 What happens if I lose my internet connection?
 What happens if the user leaves the computer without logging out?
 What happens to the users login in case of an unexpected interruption? For instance, a software crash, power failure, etc.
 Why am I receiving a message that FCS Express cannot connect to De Novo Software servers?


 Can I convert my Cytek license from the countercode licensing option to another licensing option?
 How can I claim my license purchased through BD Accuri Cytometers?
 How can I claim my license purchased through BD Biosciences?
 How can I claim my license purchased through Nexcelom Biosciences?
 How can I claim my license purchased through SysmexPartec GmbH?
 How can I claim the FCS Express license that came with my Cytek instrument purchase?


Data Analysis

 Can FCS Express integrate Python scripts?
 Can I use the FlowAI script in FCS Express?
 Can I use the FlowClean R Script with FCS Express?
 How can I recreate ratiometric data acquired in FACSDiva?
 How do I use R Integration with FCS Express?
 How does FCS Express implement software compensation?
 If my data does not have a Time parameter, can I create one?
 What is compensation?
 What is the compensation workflow in FCS Express?

 Can I customize the display of my data from different instruments?
 Can I disable the live updating feature?
 How can I display all of my detectors for my Cytek data?
 How can I set FCS Express so my FCS 3.0 biexponential data looks the same as it did in the BD FACSDiva software?
 How do I display Summit data in FCS Express as it appears in the Summit Software?
 How do I fix the biexponential axes on a plot?
 How do I rescale CytoFLEX data so it displays as it did at acquisition?
 How do I update my density and contour plots created in Version 4 to use the newest color palette?
 What are resolution options?
 What is Biexponential and Hyperlog Scaling?
 What is the best way to set FCS Express to display FCS 3.0 data from FACSDiva on a 4 decade log scale?
 Where can I get more information regarding DNA analysis using the Multicycle AV?
 Why can’t I change my plot axis labels from the Name keyword to the Stain keyword?
 Why do my dot plots appear sparse and blocky?
 Why is the text on the right most label cut off my plot?

 How are statistics in FCS Express calculated compared to how they are calculated in BD FACSDiva?
 How can I display my statistical data in Scientific Notation?
 How do I calculate EC/IC Anything?
 What is “Stain Index” and how do I calculate it with FCS Express?
 What is MFI (Mean or Median Fluorescence Intensity) and how do I calculate it in FCS Express?
 Why is the Geometric Mean being reported as NaN or ##ERROR##?

 Are Beckman Coulter LMD files unique?
 Can I find a support resource page for the analysis of Cytek data in FCS Express?
 How can I easily create the "filename" column in the "ExtraKeywordsTable.csv" file?
 How can I load data from the BD Accuri C6 Flow Cytometer?
 How do I change the display in my plots from one data file to another data file?
 How do I export .ICE files from Thermo Cellomics HCS Studio?
 How do I tell FCS Express what plate size to use if that information is not included in the data file?
 How do I upload files to the De Novo Software FTP site?
 How do I use BD Accuri CFlow files with Multicycle DNA analysis in FCS Express?
 What is the Elapsed Time setting in the Gallios software and how do I convert it to real time?
 Why are there sometimes access violations when I save and load files?
 Why do I get the message that a data file exported from a FACSDiva™ Experiment is invalid?

Image Cytometry

 How do I adjust the axes to display small particle data from Amnis CellStream?
 How do I choose which images and parameters to view in a Data Grid?
 How do I export/save data from IDEAS software and load it in FCS Express?
 How do I make my images in the data grid larger?
 How do I pseudocolor images in a data grid?
 How do I work with Amnis derived image cytometry data in FCS Express?

 Can I display heat maps with my Image Cytometry data?
 Can I work with data from PerkinElmer Instruments?
 Do you offer 21 CFR Part 11 compliance options for the Image Cytometry Version?
 Do you offer image segmentation or image analysis?
 How do I use CellProfiler Data with FCS Express?
 How do I use ImageJ with FCS Express?
 What file formats are compatible with FCS Express Image Cytometry?
 Where can I find Nexcelom Resources and Applications?


FCS Express on Mac

Upgrading FCS Express

 Can different versions of FCS Express exist on the same computer?
 How can I view and convert my V3 layouts to FCS Express 7?
 How do I import my version 3 security databases into newer versions of FCS Express?
 How do I update Density Plots created in Version 4?
 Is there an upgrade discount from earlier versions of FCS Express?
 Why are my density plots from V3 not displayed correctly in later versions?
 Why are there fewer outlier dots on my FCS Express 5 and later density plots than in V4?