Powerful, intuitive software
The Attune Cytometric Software is designed to facilitate powerful data acquisition and analysis using an intuitive user-friendly interface. Experiment settings can easily be set up, optimized, customized, and saved for future studies. Voltration and compensation are automated and can be set up using a guide. The software is designed to maximize efficiency in performing data analysis, with fast refresh rates for large data sets (up to 20 million events per sample) with the ability to immediately visualize changes on data plots as you make adjustments.
The software has exceptional tools to simplify experimental setup, including reagent selection using the filter configuration manager. This allows guidance for matching the right reagent to the optimized channel on the instrument by selecting reagents from a drop-down menu of prepopulated or customized reagents, which is then applied to plot labels.
Intuitive software interface
The user interface is divided into four panels. Ribbons and tabs in the top panel enable easy selection of key functions. Collection Panel is where you can easily set parameters to acquire data and see acquisition status. The Experiment Workspace is where you can preview your panel and choose from a variety of plot types and a range of statistics to illustrate your analysis. This workspace is also where you can set Smart gate labeling. The Experiment Explorer makes it easy to manage samples and data. This panel is where you can set up batch processing and instrument and compensation settings for new experiments.
Image processing with the Attune Cytometric Software version 7.1
The Attune Cytometric Software version 7.1 enables high throughput automated image processing of brightfield images and innovative AI data analysis solutions from data generated on the Attune CytPix flow cytometer. High resolution brightfield images show more descriptive features about cells or particles of interest such as size, complexity, particle interaction, and shape, that have previously not been available in tandem with fluorescence and scatter parameters. The software employs machine learning models to automate image processing and generate large measurement datasets rapidly from thousands of Attune CytPix cytometer images. These datasets are provided to the user as distinct image-derived parameters for plotting against fluorescence or scatter data and back-gating with images. You can choose to gate your samples using these parameters to improve gating strategy, verify sample quality, delineate complex samples and develop new applications. These morphology parameters can be utilized for image similarity and dimensional reduction tools for improved resolution into your populations of interest. See table “Image processing parameters” below for more details.
| Feature | Description | PnR[1] |
|---|---|---|
| Co-occurrence features | ||
| AngularSecondMomentCoOccurrenceIntensity | Angular second moment of co-occurrence intensity distribution of pixels within the mask[2] | 21 |
| ContrastCoOccurrenceIntensity | Contrast of co-occurrence intensity distribution of pixels within the mask[3] | 210 |
| EntropyCoOccurrenceIntensity | Entropy of co-occurrence intensity distribution of pixels within the mask[4] | 210 |
| MaximumCoOccurrenceIntensity | Maximum probability of co-occurrence intensity distribution of all pixels within the mask[2] | 21 |
| Intensity and texture features | ||
| AverageIntensity | Average intensity of all pixels within an object | 210 |
| AverageNormIntensity | 100 × AverageIntensity / (0.5 × 2(BitsPerPixel - 1)) | — |
| CVIntensity | 100 × StandardDeviationIntensity / AverageIntensity | — |
| CVNormIntensity | 100 × StandardDeviationNormIntensity / AverageNormIntensity | — |
| EntropyIntensity | Entropy of intensity distribution of all pixels within an object | — |
| KurtosisIntensity | Kurtosis of intensity distribution of all pixels within an object[5] | — |
| MaxIntensity | Maximum intensity of all pixels within an object | 210 |
| MinIntensity | Minimum intensity of all pixels within an object | 210 |
| SkewnessIntensity | Skewness of intensity distribution of all pixels within an object[6] | — |
| StandardDeviationIntensity | Standard deviation of intensity of all pixels within an object | 210 |
| StandardDeviationNormIntensity | 100 × StandardDeviationIntensity / (0.5 × 2(BitsPerPixel - 1)) | — |
| TotalIntensity | Total intensity of all pixels within an object | — |
| Moment-weighted features | ||
| CoherencyWeighted | Measure of the alignment of the substructures within an object | 24 |
| GyrationRadiusWeightedMicrons | Gyration radius (in microns) along the Z axis | 27 |
| MajorRadiusWeightedMicrons | Half the distance (in microns) across an ellipse along its long axis | 27 |
| MinorRadiusWeightedMicrons | Half the distance (in microns) across an ellipse along its short axis | 27 |
| Object features | ||
| ParticleCount | Number of cells within the identified object | 23 |
| Particle interaction features | ||
| ClumpIndexMax | Maximum number of particles among clumps | 26 |
| ObjectCount | Number of objects, where each object is encompassed by an outer mask | 26 |
| Pixel features | ||
| NumPixels | Number of pixels contained within identified objects | 216 |
| Shape features | ||
| AreaSquareMicrons | Area of the object measured within mask based on pixel count = NumPixels × (PixelSize.MicronsX × PixelSize.MicronsY) | 213 |
| CircularityPercent | Percent circularity of an object = 100 / PerimeterToArea (100 for a circular object) | 29 |
| EccentricityPercent | Eccentricity of an ellipse = 100 × Sqrt(1 − ShortAxisMicrons2/ LongAxisMicrons2) | 0 to 100 |
| MajorDiameterMicrons | Distance across an ellipse along its long axis = MajorRadius × 2 × PixelSize.MicronsX | 27 |
| MinorDiameterMicrons | Distance across an ellipse along its short axis = MajorRadius × 2 × PixelSize.MicronsX | 27 |
| PerimeterMicrons | Perimeter of an object | 215 |
| PseudoDiameterMicrons | Diameter of a circle with an area equal to the area of the object = 2 × Sqrt(Object.Area in µm2/ pi) | 215 |
| MinorMajorRatioPercent | Short to long axes ratio of an object as a percentage = 100 × ShortAxisMicrons / LongAxisMicrons | 0 to 100 |
| System features | ||
| ConfidenceScore | Indicates that one or more objects intersects with the FOV of the image | — |
| IsOnBorder | Indicates that one or more objects intersects with the FOV of the image | — |
| IsProcessable | Indicates that the image is processable | — |
| IsProcessed | Indicates that the image was processed. This is generated when the image processing FCS file is loaded and merged with the raw FCS file data | — |
| [1] $PnR is the range for the selected parameter n. [2] May be most valuable as a dimensionality reduction parameter as meaningful influence may be difficult to detect in a 1D/2D space. [3] Contrast co-occurrence intensity is used to quantify the intensity contrast between a pixel and its neighbor. [4] Entropy co-occurrence intensity is used to quantify the complexity of pixel intensity distributions in an image. [5] Kurtosis of intensity measures the peakedness of the distribution of all pixels within an object. [6] Skewness of intensity measures the degree of asymmetry in the pixel data of an object. Table is taken from Attune Cytometric Software Image Processing Workflow Quick Reference |
||
Our suite of image data analysis tools is the main feature of the Attune Cytometric Software version 7.1. We provide workflow wizards that help users with many levels of experience to access the AI machine learning tools. The Image processing feature is intuitive to use and can be incorporated into user-defined scenarios. You can choose a vendor-supplied model that is pre-trained on leukocytes and beads or refine a model to your specifications using the Train Model feature. The system can process images at speeds up to 1,000 images/second, depending on the size and complexity of images. Image-derived data can be exported into the FCS files. Our Model Explorer allows you to visualize stored models, export to share, and import for later use.
Screenshot of new features in the Attune Cytometric Software interface.
Software features
Core capabilities
- Compensation—fully automated and manual compensation
- Baseline Functional Response (BFR)—automates the detector optimization process
- Instrument tracking—automated baseline and performance test for all channels and linearity
- Traceability—21 CFR part 11 option for flow cytometry data security, auditing, and electronic signature capabilities
- File formats—FCS 3.0 and FCS 3.1
Maintenance
- Automated maintenance—startup (<15 min), shutdown, rinse, wash, debubble, deep clean, monthly decontamination
- User account maintenance—administrative and individual accounts with user log
Visualization
- Image gallery view of imaged events
- Back-gating of images onto flow plots
- Visualize images from specific gate
- Gates—standard, customizable, and derived gates
- Plot previews—easy workspace setup
- Zoom filmstrip viewer—easy visualization and navigation of plots to set gates and plot attributes
- Overlay plot builder
- Heatmap view with color-coding and statistics
- Graphics resolution—publication-quality images (customizable)
Image processing with ACS 7.1 & Attune CytPix flow cytometer
- Automated image processing using 36 morphology parameters
- Choice of 3 processing pre-trained models and unlimited user trained models with Train Model Wizard
- Correlate morphology with standard flow cytometric fluorescence and scatter parameters
Data analysis with ACS 7.1 & Attune CytPix flow cytometer
- Dimensionality reduction wizard that integrates morphology and fluorescence data
- Image similarity calculations to quickly find events of similar morphology
Compensation tools
Compensation for spectral overlap between fluorescence channels is both rapid and accurate using a guided software system. The software streamlines the compensation process by performing automated voltration (Baseline Functional Response) with Attune Performance Tracking beads, providing starting points for voltages that can still be adjusted if desired. The system adjusts the compensation and applies it to the samples so that you only need to prepare the proper controls and adjust the voltage gates.
Automated compensation supports both negative and universal negative (unstained) controls. The workflow also allows you to set up and collect compensation controls directly from a plate if needed. This automation eliminates trial and error and delivers compensation rapidly and accurately. In addition, on-plot compensation adjustment allows for fine tuning, and compensation can be subsequently modified.
.jpg "Overview of negative gating compensation")
How to use negative gating compensation
(1) Negative gating is useful for heterogeneous samples that are often found in tumor and blood samples. (2) Choose fluorophore-labeled antibodies based on the emitted emission spectrum. Dyes should have minimally overlapping spectra. (3) Prepare single-stained controls. An unstained control is not required. (4) Fluorescent compensation beads can also be used to set compensation parameters when there is not enough sample. (5) Use the guided system and select for negative gate. (6) Run sample and adjust the voltages to see the fluorescent label on the plot. Press Apply Compensation.
Baseline Functional Response
One of the key barriers to increased adoption of flow cytometry is its inherent complication—the myriad of instrument settings users must expertly adjust before acquiring data. To obtain high-quality fluorescent flow cytometer data, well-optimized instrument settings are required. Baseline Functional Response automatically sets the optimal voltage for each detector, enabling users to optimize their experiments quickly by suggesting the minimum voltage required for the best signal.
Schematic demonstrating Baseline Functional Response software feature on instrument set-up steps.
Optional 21 CFR Part 11 Software
The FDA released the Electronic Records and Signatures Rule, known as 21 CFR Part 11 in August 1997.* This rule defines the requirements for use of electronic documents in place of paper documents. The law specifies the system elements, controls, and procedures that are necessary to help ensure the reliability of electronically stored records.
This software license for the Attune acoustic focusing cytometers supports compliance with 21 CFR Part 11 FDA guidelines for Security, Auditing, and Electronic Signatures using a SAE console. The Attune 21 CFR software is available for use only for flow cytometry data on Windows 10 computers.
* https://www.fda.gov/regulatory-information/search-fda-guidance-documents/part-11-electronic-records-electronic-signatures-scope-and-application#i (accessed February 27, 2020)
The software enables the administrator to restrict access to authorized personnel via user ID and password.
The software allows the administrator to set expiration duration for both passwords and user IDs. In addition, the system will log users out after established duration of inactivity.
ID and password must be re-entered to sign or modify a record.
Event notification automatically records and reports any forgery attempts in the administrator dashboard.
The software lets the administrator set access and manipulation controls at a granular level over various software functions and options using user role definitions.
Auditing features track actions performed by users and changes to the Security Auditing Electronic signatures (SAE) module settings. The SAE module automatically audits some actions silently. This allows the administrator to audit specific user actions and specify the audit mode, as well as generate reports of audited user actions, SAE module changes, software, and instrument activity.
An electronic signature determines if users are required to fulfill signature requirements before performing specific functions. The software allows administrators to configure an e-signature so that a given user can start a run only if the associated data are signed. In addition, settings allow for an e-signature to be set so that multiple signatures are required and that only specified roles can sign.
A Sign Record feature allows records to be signed on demand.
An e-signature sign record is prepared by creating a PDF preview.
Subsequently the Sign Report is prepared for signature.
The experiment is signed and user is notified that subsequent edits will obsolesce the e-signature.
Request Signature allows the user to request an e-signature.
E-signature request alerts ensure that signatures are assigned to your documents.
The approver is notified of e-signature request at login and may view pending signatures. The approver may sign the report and designate the reasons for signature.
Advanced data analysis software
In collaboration with De Novo Software, Thermo Fisher Scientific offers FCS Express Flow Cytometry Analysis Software. FCS Express software gets you from raw data to easily understandable, beautifully formatted, presentation-ready results more easily and in less time than any other flow cytometry software. This product consists of a one-year license for FCS Express software obtained via digital download.
FCS Express software features include:
- Fully integrated analysis, statistics, graphing, and reporting
- Microsoft Office inspired interface that makes learning easy, while integrated spreadsheets and direct high-resolution exports save you from using multiple software tools to achieve a final, publication-ready result
- Full support for Attune instruments that helps ensure a seamless transition from instrument acquisition to final result
- Dedicated and knowledgeable support team with less than 24-hour response times
Additional software licenses
For Research Use Only. Not for use in diagnostic procedures.