The distinguishing feature of the Attune CytPix Flow Cytometer is a high-speed brightfield camera that records images of individual cells coming through the flow cell. The camera and Attune Cytometric Software enables users to visually verify that events recorded by the detector are single cells as opposed to doublets, clumps, or debris. This is crucial in cell and gene therapy research applications, but is useful in almost any flow cytometry experiment to assist users assess sample quality, optimize protocols, and record the morphology of each cell population identified for analysis.
Imaging can be added without changing existing flow cytometry protocols or reducing throughput. The Attune CytPix Flow Cytometer can capture clear images, even while maintaining standard flow cytometry acquisition speeds. Imaging also benefits from acoustic focusing, which helps to position the cells so that a sharp, centered image is obtained.
By using the image gallery view, users can scan cell populations rapidly for outliers. In this view, doublets and other aggregates stand out, even in cursory review. With the Attune CytPix Flow Cytometer, users can highlight structural features of large populations in record time. This allows adjustment of gates to include cells of interest while excluding aggregates, unwanted cells, and debris.
Morphological information from images adds to the richness of flow cytometry data over multiplexed staining alone. For example, the figure shows an otherwise conventional apoptosis assay using Annexin V and propidium iodide (PI), with added cell imaging to characterize cells in each population, revealing morphologically distinct features. These insights could not have been gained from flow cytometry data alone.
Attune Flow Cytometers combine ultrasonic waves like those used in medical imaging with hydrodynamic forces to precisely position cells into a single, focused line in the central axis. Enabling cells to be tightly focused at the point of laser interrogation allows the system to collect more photons, helping to ensure data quality regardless of the sample-to-sheath ratio.
Acoustic focusing is useful for cellular imaging on the Attune CytPix instrument, helping to position the cells precisely so that a sharp image is obtained. As demonstrated in these videos, cells imaged without acoustic focusing were fuzzy and out of position, while cells imaged with acoustic focusing were clear and centered in the field of view.
Acoustic focusing positions cells for optimal imaging
Without acoustic focusing (left), beads appear off-center and often blurry. Acoustic focusing (right) reduces lateral position variation, temporal variations, and depth of field limitations to obtain a sharp image.
Acoustic focusing also lets your lab rapidly acquire high-quality data. Users can achieve sample throughput rates of 12.5 µL/min or 1,000 µL/min, up to 10 times faster than traditional hydrodynamic focusing systems and acquisition speeds of 35,000 events per second. This means processing all samples—including low-concentration and precious samples—more quickly and accurately with minimal loss in quality.
Acoustic focusing minimizes variation regardless of the sample rate, so you don't have to make the tradeoff between throughput and sensitivity. This is demonstrated in the cell cycle analysis example below, where it's critical to precisely detect differences in fluorescence intensity between multiple cell populations.
Detection of rare events requires acquisition of high numbers of cells to attain a reliable measure of accuracy. Attune Flow Cytometers allow dilute samples to be processed quickly at sample input speeds of up to 1 mL/min, significantly faster than conventional cytometers that support maximum sample input rates of 60–100 µL/min. Acoustic focusing thus offers a unique combination of speed and quality, cutting the time to collect rare events significantly over long acquisition times.
Attune Flow Cytometers support both high speed of acquisition and high sensitivity to enable easy detection and phenotypic and functional characterization of rare cells. This step-by-step strategy for detecting rare events in our Flow Cytometry Learning Center will help you think through the best approach for pre-analytical, analytical, and data analysis phases of your research.
Attune Flow Cytometers are configurable with up to 4 spatially separated lasers and 16 parameters. Spatial separation provides flexibility for multicolor panel design and streamlines compensation. The system offers superior speed with acquisition rates of up to 35,000 events per/second with high sensitivity to meet a range of research requirements.
High sensitivity distinguishes between dim signals and background, resulting in less variation and better signal separation. Fluorescent resolution coefficient variation is less than 3% for a single peak, and predicted MESF is ≤80 (FITC), ≤30 (PE), ≤70 (APC). Sensitivity in comparison to competitive systems is described below.
The compact size of the Attune Flow Cytometer also provides the flexibility of using it within a biosafety hood. This helps avoid contamination or infection when working with hazardous or unknown samples.
Attune Flow Cytometers feature a novel optical design that delivers first-class reliability and superior performance over time. The flat-top beam profile of the solid-state lasers minimizes the effects of changes in fluidics or optics, which in turn can lead to instability or alignment issues and instrument downtime.
Laser misalignment is a major concern with users of conventional flow cytometers. The flat-top lasers used in the Attune Flow Cytometers have an intensity profile that allows a wider window of alignment over Gaussian lasers used in traditional systems. The flat-top lasers also have a higher tolerance for misalignment that allows them to maintain high sensitivity and low CVs.
To prevent clogging and allow for volumetric analysis, the Attune Flow Cytometers use a positive displacement syringe pump to control sample volume. The system can perform volumetric cell counts in a known volume (gated or total events) and can easily gate out dead cells to count live cells only in a live/dead analysis. Unlike traditional cytometers that operate at a maximum pressure of 15 PSI, Attune Flow Cytometers control pressure at 75 PSI, reducing the likelihood of cell-cell interactions and resulting in clog resistance.
The Attune Cytometric Software is designed to provide powerful data acquisition and analysis using an intuitive user-friendly interface. Experiments can easily be set up, customized, and saved for future studies. Compensation is 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 unique tools to simplify experimental setup, including reagent selection using the filter configuration manager. This provides 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.
The Attune Cytometric Software version 6.0 enables high throughput automated image processing of brightfield images generated on the Attune CytPix flow cytometer. High resolution brightfield images provide more descriptive features about cells or particles of interest such as size, complexity, texture, and shape, that have previously not been available in tandem with fluorescence and scatter parameters. The software employs machine learning models that are pre-trained using leukocytes and beads to automate image processing and generate large measurement datasets rapidly from thousands of Attune CytPix 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.
|Intensity Standard Deviation|
|Perimeter Area (µm)|
|Pseudo Diameter (µm)|
|Major Axis (µm)|
|Minor Axis (µm)|
|Minor to Major Axis Ratio (%)|
The image analysis feature of the Attune Cytometric Software 6.0 is intuitive to use, and can be managed in a user defined queue. The system can process images at speeds up to 1,000 images/second,depending on size and complexity of images. Image-derived data can be exported into the FCS files.
Image processing with ACS 6.0 & Attune CytPix flow cytometer
Compensation for spectral overlap between fluorescence channels is both rapid and accurate using a guided software system. 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 unstained parameters and 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 controls allow for fine tuning, and compensation can be subsequently modified should you need to add or remove parameters after setup.
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 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.|
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:
For research use only, not intended for use in diagnostic procedures.