High-speed camera option and automated image-processing transform flow cytometry

  • Acoustic focusing fluidics enable the high-sensitivity of the Attune Flow Cytometers even at very fast sample flow-rates (up to 1000 µL/min)
  • The Attune CytPix model adds a high-speed brightfield camera to capture images of cells as they pass through the flow cell, even at the fastest flow rates
  • Image analysis software implements simultaneous measurement of cell morphology parameters in addition to standard flow cytometric parameters

High-speed camera

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.

Consistent image quality even at high flow rates

Acoustic focusing and a high-speed camera combine to image these CAR T cells consistently at low or high flow rates. Foucs and camera settings are easily adjusted to meet experimental requirements.

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.

High-level image gallery view. Early log phase Jurkat cells were acquired unfiltered on the Attune CytPix Flow Cytometer at 200 µL/minute, >105 cells/mL. Image gallery view was used to rapidly scan cellular events.

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.

Morphological characteristics of apoptotic cells.

Jurkat cells were incubated with 10 µM camptothecin for 4 hours at 37ºC to induce apoptosis. Samples were stained with Annexin V and PI and acquired on the Attune CytPix Flow Cytometer at 100 µL/minute. From the singlet population, gating strategies identified three cell subpopulations. About 50% of apoptotic live cells (Annexin V⁺PI-, bottom right) showed some form of apoptotic body such as blebs. About 25% of apoptotic dead cells (Annexin V⁺PI⁺, top right) showed increased cell surface granularity, and there were more partial cells. About 10% of healthy cells (Annexin V-, bottom left) showed apoptotic bodies (though not as severe as those observed among Annexin V⁺ cells). These healthy cells were also morphologically diverse and included some doublets despite upstream singlet gating. Morphological features in the images are indicated by black arrows.

“Traditional antibacterial sensitivity testing can take 1–3 days depending on how difficult the bacteria are to grow, isolate, and then test for sensitivity. The Attune NxT allows us, within 1 hour, to tell whether or not the drug will effectively treat the infection. Within 3 hours, accurately assay the minimum drug concentration needed to inhibit bacterial growth.”

Kieran Mulroney,

Biomedical Sciences Researcher

Harry Perkins Medical Research Institute

University of Western Australia

Acoustic focusing for sensitivity

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

Acoustic focusing (left) precisely positions cells in tight alignment even at higher sample rates resulting in less signal variation and better data quality. Traditional hydrodynamic focusing (right) widens the sample stream core at high sample rates that results in signal variation and compromised data quality.

“When all is said and done, it outperforms [the rest] in that it can analyze 35,000 cells per second with acoustic focusing technology. Its accuracy is not diminished even if you conduct analysis at a high flow rate of 100–1,000 µL/min, which drastically shortens the time required for an experiment.”

Dr. Takashi Satoh,

IFReC Assistant Professor

Osaka University, Japan

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. 

Fast, accurate acquisition

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.

Rapid data acquisition

 

Compares the time required to acquire 1,000,000 events over three competitor instruments running at maximum sample rates.

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.

Minimal data variation

 

Consistent results were achieved at high sample rates using Jurkat cells fixed and stained with propidium iodide, treated with RNase and analyzed at a concentration of 1 x 106 cells/mL. The coefficient of variation (CV) of cells in the G0/G1 and G2/M phases remain consistent, even at the highest sample rate of 1,000 μL/min.

Rare event detection

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.

 

 

Collecting more than 1 million live cells and detecting a rare population of dendritic cells

Plasmacytoid dendritic cells (pDCs) are identified using immunophenotype CD19–/B220high/CD317+. Four-color staining of mouse splenocytes included CD19-Pacific Blue, CD317-Alexa Fluor 488, CD45R/B220-PE direct conjugates, and SYTOX AADvanced Dead Cell Stain. A gate was made on live cells using SYTOX AADvanced Dead Cell Stain, followed by gating on CD19– cells. A two-parameter plot of CD45R/B220 vs. CD317 was used to identify pDCs. A collection rate of 500 μL/min was used to acquire 1.3 million total cells with a cell concentration of 7.5 x 107 cells/mL. Plasmacytoid dendritic cells were identified as dual B220+/CD317+ (upper right quadrant) and constitute 0.851% of live CD19– cells, which is 0.194% of total splenocytes.

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.

Designed for flexibility

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.

Sensitivity measurements across flow rates

Fluorescent microspheres (Spherotech Rainbow 3.2 μm) were run on a high-end conventional flow cytometer (A) and on the Attune NxT Flow Cytometer (B and C) using a 561 nm laser and 610/20 (A) or 610/15 (B and C) emission filters. The conventional cytometer was run using the highest sensitivity setting (~12.5 μL/min). The Attune NxT Flow Cytometer was run at 12.5 μL/min (B), which is equivalent to the traditional flow cytometer and 500 μL/min (C; 40x more sample). The Attune NxT Flow Cytometer results were equal to or better than those from the conventional flow cytometer, even at the highest flow rate.

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.

Novel optical design

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.

Emission profile of lasers 

 

Gaussian laser used in traditional cytometers (left) with misalignment and flat-top laser used in the Attune Flow Cytometers (right) showing proper alignment if shifted.

Clog-resistant technology

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.

 

“One of the problems that everyone is familiar with who works in flow cytometry is clogging. Clogging is a thing of the past with this instrument. You have so many samples that you have to run, you can’t wait between samples to clear everything out.”

Bruno Sainz,

CLIP Investigator

Universidad Autónoma de Madrid

Madrid, Spain

Volumetric sample entry 

 

Using positive displacement, the syringe pump works like a pipette. Tension is created with the plunger pushing down (A). The tension is released as the plunger rises with the sample (B). Clogging is less likely to occur under this high and controlled pressure environment.

“We needed to have a flow cytometer that would allow us to take samples from tumors that had tended to clog other machines. Therefore the Attune NxT is the flow cytometer of choice for our application. The Attune NxT has a number of characteristics that are critical for our research… program. Not only does it have acoustic focusing, which will allow us to go at higher sample throughput, but it also has a larger flow cell, so when we’re looking at tumor cells and isolating cells from those tumors we don’t have to worry about clogging and having to spend a lot of downtime getting the instrument up and running again.”

Charles Prussak,

PharmD, PhD

Director of the Cell Therapy Translational Laboratory (CTTL)

University of California San Diego

Powerful, intuitive software

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.

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.

“The Attune NxT software is very user friendly and intuitive, significantly reducing training time required to get new users up-and-running.”

James Hemphill,

Ph.D

Senior Scientist

Kytopen

Image processing with the Attune Cytometric Software version 6.0

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 features
Maximum Intensity
Minimum Intensity
Total Intensity
Average Intensity
Intensity Standard Deviation
Intensity %CV
Intensity Skewness
Intensity Kurtosis
Intensity Entropy
Shape features
Area (µm2)
Perimeter Area (µm)
Circularity (%)
Pseudo Diameter (µm)
Major Axis (µm)
Minor Axis (µm)
Minor to Major Axis Ratio (%)
Eccentricity (%)
System Features
On Border
Confidence Score
Processed
Processable
Object Features
Particle/Cell Count

 

Pixel Features
Pixel Count

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.

Software features

 

Core capabilities

  • Compensation—fully automated and manual compensation
  • 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 and customizable gates
  • Plot previews—easy workspace setup
  • Zoom filmstrip viewer—easy visualization and navigation of plots to set gates and plot attributes
  • Graphics resolution—publication-quality images (customizable)


Image processing with ACS 6.0 & Attune CytPix flow cytometer

  • Automated image processing of more than 25 image parameters
  • Choice of 3 processing models
  • Correlate image parameters with standard flow cytometric fluorescence and scatter parameters

 

Compensation tools

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.

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.

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 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 intended for use in diagnostic procedures.