Imaging-enhanced flow cytometer, for simultaneous morphology and flow data analysis

  • The Invitrogen Attune CytPix Flow Cytometer flow cytometer combines acoustic focusing with a high-speed brightfield camera for simultaneous high throughput flow cytometry and high resolution brightfield imaging
  • Automated image analysis translates event features into distinct extended parameters that can be combined with standard flow parameters

Features

The Attune CytPix Flow Cytometer is an advanced cell analyzer that shares common features with the Attune NxT model.

Its distinguishing feature is a high-speed brightfield camera that records images of individual events as they pass through the flow cell. The camera and Attune Cytometric Software help to ensure that the events you analyze 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 help researchers understand the morphology of each cell population identified for analysis. The images can also aid in identifying debris and optimizing protocols.

How it works

As samples are acquired on the Attune CytPix Flow Cytometer, the high-speed camera captures and stores images of a sampling of events. For greater flexibility, Attune Cytometric Software lets you adjust the image capture frequency as needed, and can capture up to 6,000 images per second depending on the flow rate and image size. Acoustic focusing helps to position the cells so that a sharp image is obtained.

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.

Consistent image quality even at high throughput

Acoustic focusing and a high-speed camera combine to image these CAR-T cells consistently at low or high flow rates. Easily adjust focus and camera settings to meet experimental requirements.

Optical focus is maintained regardless of the sample flow rate. You can image events at flow rates up to 1,000 µL/minute, and adjust the focus, imaging window, and illumination settings to your target cell type.

Image analysis for improved data accuracy

The Attune Cytometric Software on the CytPix Flow Cytometer analyzes and interprets cell features from images into quantitative parameters using models pre-trained on leukocytes and beads.  Combined with captured images and flow data, these parameters enable researchers to gain novel insights into their samples. Gates can be adjusted to exclude aggregates, coincident events, debris and unwanted cells.

 

The software automates image analysis at a rate of up to 1,000 images/second and can be managed by users in a processing queue that runs in the background of the software. These extended image-based parameters provide data to confirm singlets with cell count (Particle Count) and morphology features such as roundness (Circularity), size (Area Square), shape (Eccentricity) and complexity (Entropy).  Gating on these extended parameters allows you to quickly and accurately identify populations of interest to confirm gating strategy with little or no manual review. You can use back-gating to scan the panel of full resolution images and correlate what you see with scatter, fluorescence or image-based parameters to any population on the dot plot. Enhance the quality of your data and feel confident when gating with powerful data-driven cell analysis.

Imaging-enhanced flow cytometry applications

Imaging-enhanced flow cytometer applications include almost any study that can benefit from understanding the morphology of each cell population identified. The imaging capability lets you look more deeply into results to: 

Confirm gate accuracy in real time

Use images to adjust gates and camera settings, and exclude atypical cells and debris for more robust gating

Further characterize cell populations

Document morphologically distinct populations in existing protocols such as apoptosis detection

Capture cell-to-cell interactions

Visualize and differentiate coincident events

Visualize structural features of large populations

Use high-throughput, detailed photographic evidence

Discover opportunities for analysis

Based on fluorescence, scatter, and morphological features

 

Measure derived image parameters

Using the image analysis feature of the Attune Cytometric Software 6.0

High-throughput quality control

Detect quality issues quickly by adding rapid imaging to cell culture QC workflows, and monitor changes in cell morphology as the plate is processed

Optimize gating strategy. Even robust manual singlet gating is error-prone and remains a subjective decision point in almost all flow cytometry assays. Imaging can be used to confirm and adjust gates to include only single cells of interest.

 

Here, an experienced user has gated singlets confidently. After evaluating the manual singlet gate, the CytPix image-derived parameter ParticleCount reveals this gate contains more than 4% aggregates.

 

Perhaps most importantly, these events contain cells of clearly different phenotypes which could lead to incorrect conclusions regarding double positive events (especially in rare populations).

Aged whole human blood (AllCells) lysed with ammonium chloride lysis buffer. Image processing was done using the Cells Model_Speed Optimized model. Statistics shown are % gated.

Characterize cell populations. Morphological information from images can add to the richness of flow cytometry data. For example, the figure shows an otherwise conventional apoptosis assay using Annexin V and PI, adding cell imaging to characterize cells in each population to reveal morphologically distinct features. These insights could not have been gained from multiplex staining 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).

"A picture isn't quite worth 1000 dots, but it really helps to see images. It is great to see images that correlate with staining. For example, AnnexinV-positive cells that are morphologically different than healthy cells, or CD14+ monocytes that are larger and more textured than CD3+/CD4+/CD14- lymphocytes. Additionally, it is illuminating to see how many doublets slip through traditional singlet gates, and great to verify the status of unexpected double-positive events in my analysis."

Kathryn Fox,

University of Wisconsin-Madison School of Medicine and Public Health

Analyze cell-to-cell interactions. Imaging can even show interactions between cells. In the figure, engineered CAR-T immunotherapy cells were co-incubated with Ramos (lymphoma) cells and stained, acquired, and imaged on the Attune CytPix Flow Cytometer. Images from quadrant Q2 (positive for both stains, acquired as a single event) show the CAR-T cells visibly targeting the Ramos cells, clear evidence of engineered cell potency.

Visualization of CAR-T cells targeting lymphoma cells.

CAR-T and Ramos cells were labeled with CellTrace Far Red and Violet respectively and incubated at a 1:1 ratio for 1 hour at 37°C. Unfiltered samples were acquired on the Attune CytPix Flow Cytometer at 200 µL/minute, >8 x 105 cells/mL. Images of quadrants Q1 (top center), Q4 (bottom right), and Q3 (bottom left) show individual Ramos cells, CAR-T cells, and debris, respectively. Images from quadrant Q2 (positive for both stains, top right) reveal both cell types fused together, acquired as a single event as the CAR-T cells engulf the Ramos cells.

We previously demonstrated the power of imaging CAR-T/Ramos cell interactions. Let’s look at just the population of greatest interest, the double positive events, to learn more. We can now use extended image-derived parameters (circularity vs skewness of intensity) to further examine the features of these populations and refine gating on these events, increasing data robustness. Here, we show that by gating strategies using image-based quantitative parameters can distinguish interacting cells from coincident events to more accurately analyze interacting cells.

Visualization of CAR-T cells targeting lymphoma cells.

CAR-T and Ramos cells were labeled with CellTrace Far Red and Violet respectively and incubated at a 1:1 ratio for 1 hour at 37°C. Unfiltered samples were acquired on the Attune CytPix flow cytometer at 200 µL/minute, >8 x 105 cells/mL. Images of quadrants Q1 (top left), Q3 (bottom right), and Q4 (bottom left) show individual Ramos cells, CAR-T cells, and debris, respectively. Images from quadrant Q2 (positive for both stains, top right) reveal both cell types fused together, acquired as a single event as the CAR-T cells engulf the Ramos cells. Percentages are % gated.

In the cell image galleries, annotated events are outlined in black with yellow dots indicating center positioning. Image processing was done using the Cells_Half_Resolution_v22 model Using circularity vs skewness of intensity, we are able to differentiate between attached (cell interactions between CAR-T and Ramos cells) and detached where cells are in same field of view but not showing cell-to-cell interactions.

Discover analysis opportunities

Backgating imaged cells on the Attune CytPix Flow Cytometer also allows you to use morphological features to discover interesting subpopulations that would not be apparent from flow cytometry data alone.

 

For example, E. coli cells incubated over time develop into two types of colony-forming units (CFUs): short CFUs that resemble single cells, and elongated structures with incomplete fission rings, representing incomplete constriction at each approximate cell length. Neither a traditional singlet gate (SSC-A vs SSC-H) nor a fluorescence gate (SSC vs nucleated stain) sufficiently separates these populations. But with the Attune CytPix imaging-enhanced flow cytometer, you can view and group the images and gate the CFU types based on their morphological characteristics.

 

Discrimination of two E. coli CFU types. E. coli cells were incubated overnight at 37ºC followed by 3 days at 4ºC. Samples were acquired on the Attune CytPix Flow Cytometer at 100 µL/minute. From the images, two types of CFUs were identified: (A) short colonies resembling single cells and (B) elongated structures with incomplete fission rings. Representative images from each population are shown. Backgating on the selected images demonstrated that the two populations are distinct on FSC vs SSC dot plots (orange dots, left).

Cell culture QC. Adding rapid imaging to quality control (QC) workflows can detect and track down cell culture issues early in the process. In one lab, for example, a routine passage check of a Ramos (lymphoma) cell culture observed reduced cell counts and survival despite appearing confluent. Further investigation revealed substantial microbial contamination, but when and where did it begin?

 

Because the cell line had previously been analyzed on the Attune CytPix Flow Cytometer, the researchers went back to the images and were able to document the microbial infection at least five days earlier. At that time, the early signs were dismissed as debris, but the retrospective evaluation demonstrated shared characteristics with the problematic cells in culture. Tracing the infection helped the lab establish additional laboratory procedures for screening and protection of assay-critical cell lines.

Investigating the contamination of a Ramos cell culture.

Ramos (lymphoma) cells in culture showed reduced cell counts and survival during a routine passage quality check, despite appearing confluent. Further evaluation showed microbial contamination, confirmed by imaging and backgating on the Attune CytPix Flow Cytometer. Early signs of this contamination had initially been dismissed as debris. 

Videos and demos

Laser and detector configurations

Lasers Laser configuration Number of detection channels for included lasers Total detection channels* Catalog number
Violet, 405nm Blue, 488nm Yellow, 561nm Red, 637nm
2 Blue/yellow
✝ 3 4 ✝ 9 A51842
Blue/red
✝ 4 ✝ 3 9 A51840
Blue/violet
4 4 ✝ ✝ 10 A51841
Blue/violet 6
6 3 ✝ ✝ 11 A51843
3 Blue/red/yellow
✝ 3 4 3 12 A51845
Blue/violet/yellow
4 3 4 ✝ 13 A51846
Blue/red/violet
4 4 ✝ 3 13 A51844
Blue/red/violet 6
6 3 ✝ 3 14 A51847
4 Blue/red/yellow/violet
4 3 4 3 16 A51848
Blue/red/yellow/violet 6
6 2 3 3 16 A51849

*Number of detection channels includes all fluorescence channels as well as a forward scatter and a side scatter channel.

✝Laser upgrade available; green laser not available on Attune CytPix

Specifications

The Attune CytPix flow cytometer is 9 cm (3 in) taller than the Attune NxT flow cytometer due to the addition of the high-speed brightfield camera, preserving its small footprint and biosafety hood compatibility, thus saving bench space.

Optics: Fluorescence detection

Laser power

Laser

Wavelength (nm)

Beam-shaping optics (BSO)* (mW)

Diode power** (mW)

Violet

405

50

100

Blue

488

50

100

Yellow

561

50

100

Red

637

100

140

* Amount of measured usable laser power after light has gone through the beam optics and shaping filters.

** Vendor-specified theoretical maximum.

Laser excitation

Optimized excitation for minimized stray laser-line noise and losses to reflection

Laser profile

10 x 50 μm flat-top laser providing robust alignment

Emission filters

Up to 14 color channels with wavelength-tuned photomultiplier tubes (PMTs); user-changeable, keyed filters

Laser separation

100 μm

Optical alignment

Fixed alignment with prealigned welded fiber; no user maintenance required

Onboard thermoelectric cooler

No warm-up delay; fiber isn’t affected by “on/off”

Simmer mode

Instant “on/off” reduces usage and/or aging by 10x; only turns on when acquiring samples; reports hours of usage

Flat-top laser specified at the flow cell

Coefficient of variation (CV) <3% over the width of the flat-top laser

Upgradable

Convenient field changes

Optics: Imaging

Laser excitation

405 nm

Pulse width

< 50 nanoseconds

Fluidics

Flow cell

Quartz cuvette gel coupled to 1.2 numerical aperture (NA) collection lens, 200 x 200 μm

Sample analysis volume

20 μL–4 mL

Custom sample flow rates

12.5–1,000 μL/min

Sample delivery

Positive-displacement syringe pump for volumetric analysis

Sample tubes

Accommodates tubes from 17 x 100 mm to 8.5 x 45 mm

Fluid-level sensing

Active

Standard fluid reservoirs

1.8 L focusing fluid tank, 1.8 L waste tank, 175 mL shutdown solution tank, and 175 mL wash solution tank

Fluid storage

All fluids stored within instrument

Extended fluidics option

Configuration for 10 L fluid

Nominal fluid consumption

1.8 L/day

Automated maintenance cycles

≤15 min start-up and shutdown—deep clean, sanitize, and debubble modes

Performance: Fluorescence detection

Fluorescence sensitivity

≤80 molecules of equivalent soluble fluorochrome (MESF) for FITC, ≤30 MESF for PE, ≤70 MESF for APC

Fluorescence resolution

CV <3% for the singlet peak of propidium iodide–stained chicken erythrocyte nuclei (CEN)

Data acquisition rate

Up to 35,000 events/sec, 34 parameters, based on a 10% coincidence rate per Poisson statistics

Maximum electronic speed

65,000 events/sec with all parameters

Carryover

Single-tube format: <1%

Forward and side scatter sensitivity

Able to discriminate platelets from noise

Forward and side scatter resolution

Optimized to resolve lymphocytes, monocytes, and granulocytes in lysed whole blood

Forward scatter

Photodiode detector with 488/10 nm bandpass filter

Side scatter

PMT with default 488/10 nm bandpass filter; optional 405/10+OD2 nm bandpass filter

Fluorescence detectors

14 individual detectors

Electronic pulse

Measured area, height and width pulse for all detectors

Violet side scatter resolution

Can be configured for violet side scatter to better resolve particles from noise

Minimum particle size

0.2 μm on side scatter using submicron bead calibration kit from Bangs Laboratories—0.1 μm on side scatter under following conditions: Using an Attune NxT Flow Cytometer with standard 0.5 mm blocking configuration, an Invitrogen Attune NxT 488/10 Filter (Cat. No. 100083194), and Attune Focusing Fluid (Cat. No. 4488621, 4449791, or A24904) that has been passed through a 0.025 µm filter

Performance: Imaging

Pixel Resolution

0.3 microns/pixel

Objective Magnification

20x

Objective Numerical Aperture (NA)

0.45

Theoretical Resolution

0.6 micron

Detection Limit

Visually detect 800 nanometer particles

Image Capture Rate

Up to 6,000 images/second depending on image size and event rate

Image Size

96 x 96 pixels–248 x 248 pixels

Field of View

29 x 29 μm–74 x 74 µm

Software

Compensation

Full matrix—automated and manual modes, on-plot compensation tools for fine adjustment; use of tubes and wells

Flow rate

Precise flow rate control via software; no hardware adjustments

Live streaming

Live update of statistics during acquisition of events up to 35,000 events/sec

Overlays

Comparative analysis between samples; 3D view

Sample recovery

System able to return unused samples

Concentration

Direct concentration measurement without use of counting beads

Software layout

Fully customizable for each user account

Bubble detection technology

Stops automated run to preserve sample integrity

Maximum single-event file

20 million with option to append

Heat map

Set up for definition of plate layout; screening view for analysis for tubes and plates

Threshold

Up to 4 individual thresholds with user option to apply Boolean logic

Gating

Hierarchical gating with the ability to derive gates

Voltage

User adjustable

Window extensions

User adjustable

Area scaling factor (ASF)

User adjustable

Acquisition settings

Documented in FCS files and maintained upon import

Templates

Create from existing experiments—instrument settings, workspaces, run protocols, heat map settings, and compensation settings optimized and defined previously

Tube-to-plate conversion

One-click transition from tubes to plates and vice versa; no disassembly, no additional QC, no reboot required for conversion between plates and tubes

Graphics resolution

Publication-quality data plots: support for TIF, PNG, BMP, JPG, GIF, and EMF; quickly copy and paste data plots to any external application (e.g., Microsoft™ PowerPoint™ software)

User account administration

Administrative creation of individual user accounts with designated roles, advanced setting permissions, management of individual accounts, user time tracking, and sample count

Imaging software

Image Capture settings

Set total number of recorded images, image frequency, image capture gate, image size and position, focus, and illumination for control over experiment design and data footprint

Image view

Image view option allows overview of the gallery of images combined with Cell image option to view individual images on the workspace for any cell population

Image backgating

Correlate images to flow cytometry data by backgating either all or selected images onto supported workspace plots

Image Measurement tool

Elliptical tool to measure the area of events in images in µm2

Image Export options

Export images as 8-bit TIF, PNG, GIF, BMP, JPG, or EMF

Image analysis

Image analysis models

leukocytes; beads

Parameters /features

>25 measured image parameters in 5 categories: shape features, intensity features, object features, pixel features, and system features

Sample size range of models

5-20 μm

Image processing speed

Fast: 1,000 images/second
Standard: (varies by image size)

Pixels

16-bit image (248 x 248)

File type

extended FCS

File type 

<10% error on shape features

Quality and regulatory

Instrument tracking

Automated daily baseline and performance test with Levey-Jennings plots

Warranty

1 year

Production verification testing

Each instrument is tested and verified for assembly integrity and performance to specifications

Quality management system

Manufacturing standards comply with the requirements of ISO 13485:2003

Robust installation specifications

Units installed by engineer; preplanning checklist, delivery, and installation; and performance validation compliance with standardized procedure

Regulatory status

For Research Use Only

Computer

Software requirements

Invitrogen Attune Cytometric Software

Monitor

27 in. flat panel (1,920 x 1,080 resolution); dual-monitor capability

Computer

Minitower desktop

Operating system

Microsoft™ Windows™ 10 64-bit

FCS format

FCS 3.1, 3.0

Processor

Intel™ Core™ i7 processor

RAM

64 GB

Hard drives

2 x 8 TB SSD, 560 MB/sec; controller RAID1, integrated

GPU

NVIDIA Quadro P2200

Installation requirements

Electrical requirements

100–240 VAC, 50/60 Hz, <150 W
Thermo Fisher Scientific certifies that the Attune Flow Cytometers conform to relevant directives to bear the CE mark. The instrument also conforms to the UL and CAN/CSA general requirements (61010.1). The Attune Flow Cytometers are Class I laser products per Center for Devices and Radiological Health (CDRH) regulations and EN/IEC 60825.

Heat dissipation

<150 W

Temperature operating ranges

15–30°C (59–86°F)

Operating humidity

10–80%, noncondensing

Audible noise

<65 dBA at 1.0 m

Instrument size
(H x W x D)

~49 x 58 x 43 cm (19 x 23 x 17 in.), including fluid bottles

Weight

~33 kg (73 lb)

For Research Use Only, not intended for use in diagnostic procedures.