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The Cytek® Amnis® ImageStream®X Mk II imaging flow cytometer combines the high throughput performance of flow cytometry with the imagery and functional insights of microscopy. This unique combination enables a broad range of applications that would be impossible using either technique alone.
The ImageStreamX Mk II system is a benchtop, multispectral, imaging flow cytometer designed for the rapid acquisition of millions of cells with up to 12 channels of cellular imagery. The instrument collects large numbers of digital images, performs spectral compensation and provides high quality images of every cell in your sample. Combined with our advanced image analysis software where each object is measured for hundreds of parameters using not only fluorescence intensity but the fluorescence spatial location as well, the ImageStreamX system offers an unprecedented level of cellular information.
The Complete Spectrum of Imaging Applications
The high resolution images and high throughput of the ImageStreamX system, combined with pixel-by-pixel spectral compensation and hundreds of quantitative features, makes the ImageStreamX system the most capable imaging flow cytometer to address an unlimited number of applications. The ImageStreamX system allows researchers to perform novel applications such as measuring protein colocalization, nuclear translocation, spot count, cellular shape change, and cell-cell interactions to name a few. The flexibility in system configuration makes the platform ideal for labs that need to accommodate a diverse array of applications. The unique quality of the image data makes it perfectly suited to capture that critical piece of information to get the science right and win that high-impact factor journal publication. Learn more about the complete spectrum of imaging applications by reviewing our publications or downloading the Application Notes.
Automated Workflows and the Image Analysis Software Suite
Data analysis on the ImageStreamX system is as simple as clicking on “Start Analysis” and following the automated workflows for any of the core applications in the IDEAS® image analysis software.
To customize analysis, the software has 86 features per channel, 22 function masks, and a machine learning module that creates a novel algorithm to classify your specific cell populations. The “Feature Finder” wizard can then be used to identify the feature that best identifies the cells of interest.
With the Amnis® AI software option, users gain access to the most advanced image analysis tools available, seamlessly integrating the ImageStreamX data set with deep learning using convolutional neural networks.
These powerful tools make image analysis easy and enables a whole new level of sample exploration. Learn more about the ImageStream data analysis software suite.
See How Imaging in Flow Has Changed Research at Roswell Park Comprehensive Cancer Center (RPCCC)
At the RPCCC Flow and Image Cytometry facility, Dr. Paul Wallace, Dr. Hans Minderman, and their staff offer users the best tools available for increasing statistical power and throughput for applications like measuring NF-kB translocation, previously performed using laborious microscopy. Watch to learn how Amnis protocols have helped RPCCC researchers detect rare events, such as circulating tumor cells.
ImageStream®X Mk II Optical Layout
Cells are fluorescently labeled in suspension and then loaded into the system using a syringe pump. Samples are hydrodynamically focused in a flow stream, and fluorescence is excited using multiple laser lines. The brightfield, SSC, and fluorescent images are captured by a 20x, 40x, or 60x imaging objective and transferred to the filter stack where each color is projected to a spatially discrete channel on the cameras. Up to 10 colors of fluorescence are collected in spatial registry, and each probe is measured for intensity, cellular morphology, and relative location to other probes.
Time Delay Integration Camera Technology
The Cytek® Amnis® ImageStream®X Mk II uses a charge coupled device (CCD) operated in time delay integration (TDI) mode. This mode captures images of moving objects with very high image quality and exceptional photonic sensitivity. In TDI mode, the photon charge is transferred down each pixel row in precise synchrony with the transit of the cell, allowing for increased light integration time. This effect is similar to physically panning a camera with the object. Each pixel row is then read off the bottom of the detector and is used to reconstruct the image of the cell for investigation using image analysis software tools.
Other Resources
- Oceanography on the Cytek® Amnis® ImageStream®X Mk II System: Classifying Diatoms and Other Phytoplankton
- Single Cell Protein Secretion Assays Using Nanovials and the Cytek® Amnis® ImageStream®X Mk II Imaging Flow Cytometer
- Illuminating T Cell–APC Interactions Using Multispectral Imaging Flow Cytometry
- Detection of Extracellular Vesicles Using the Cytek® Amnis® ImageStream®X Mk II Imaging Flow Cytometer
- Measurement of Protein Aggregates and Silicone Oil Droplets in Protein Formulations Using Cytek® Amnis® Imaging Flow Cytometry
- Quantitating NF-кB Translocation Using the Cytek® Amnis® ImageStream®X System and Optimized Reagent Kit
- The Kinetics of CpG Internalization and Sub-cellular Organelle Co-localization Within Circulating Human Plasmacytoid Dendritic Cells
- Measurement of Immune Cell Function Using Cytek® Amnis® ImageStream®X Imaging Flow Cytometry
- Imaging Flow Cytometry of Mixed Algae Cultures for Biomass Evaluation
- Quantitation of DNA Damage and Repair Using H2AX Spot Counting on the Cytek® Amnis® ImageStream®X Imaging Flow Cytometer
- Cytek® Amnis® ImageStream®X Mk II Flow Cytometer High Gain Mode for Increased Sensitivity in the Detection of Small Particles
- A Rapid and Fully Automated in vitro Micronucleus Assay Using the Cytek® ImageStream®X Mk II and Cytek® Amnis® Artificial Intelligence
- Immuno-flowFISH Assessment of Trisomy 12 in Chronic Lymphocytic Leukemia Using Imaging Flow Cytometry
Publication Lists
So Many Parameters
Exceptional Sensitivity
Novel Applications
High Fluoresence Sensitivity
The fluorescence sensitivity of the ImageStreamX system was measured using Spherotech 8-peak rainbow calibration beads. All 8 peaks are clearly visible with Channel 3 ≤2 MEF (molecules of equivalent fluorescence) and a Stain Index (SI =(Mean2-Mean1)/(2xSD1)) of 92.2 between the first peak and the next dimmest bead. The high quantum efficiency, low noise camera enables the ImageStreamX system to capture images of every particle with very high photonic sensitivity in all 12 channels. This is critically important for identifying subcellular organelles, dim surface signals, or detection of small particles.
Multiple Magnifications Captures the Big Picture
The ImageStreamX system offers multiple magnifications to accommodate a broad range of cell types. The 20x objective at a resolution of 1 µm/px is used for larger 120 µm diameter cells, the 40x objective at 0.5 µm/px is ideal for a balance of speed and resolution, and the 60x objective at 0.3 µm /px resolves smaller primary cells. The high-resolution images enable accurate classification of populations based not only on fluorescence intensity but cellular morphology as well. The cells displayed in the figure are THP-1 cells imaged at each magnification on the ImageStreamX system.
Performance of Morphology-Based Measurements
Nuclear translocation of NF-kB in THP-1 cells or any other signaling molecule can be identified using the Similarity feature. The fully compensated high image quality of the ImageStreamX system enables superior separation between cells that sequester NF-kB in the cytoplasm (A) versus those that localize NF-kB in the nucleus (B). Learn more about nuclear localization.
Internalization and Colocalization in Rare Cell Types
The ImageStreamX system rapidly collects high resolution images of rare cell types to accurately assess internalization and colocalization of particles using the unique image-based features of IDEAS® image analysis software. In this example, CpGB (red) mimics viral particles binding to pDCs (A) and shows how it is internalized (B) and colocalized to the lysosome (green) (C) of the cell. Representative images are displayed for each region. Learn more about internalization and colocalization features.
Small Particle and Extracellular Vesicle (EV) Detection
The high photonic sensitivity and high-quality images of the ImageStreamX system make it possible to track EVs from production and capture by a surrounding hydrogel (A) to free circulating particles (B) and finally the binding (C) and internalization by cells (D). Learn more about particle capture and small particle detection in our application notes.
| Performance Characteristics | 40x Magnification | 60x Magnification | 20x Magnification |
|---|---|---|---|
| Numeric Aperture | 0.75 | 0.9 | 0.5 |
| Pixel Size | 0.5 x 0.5 µm | 0.3 x 0.3 µm | 1.0 x 1.0 µm |
| Field of View | 60 x 128 µm | 40 x 170 µm | 120 x 256 µm |
| Imaging Rate | 2,000 Obj/Sec | 1,200 Obj/Sec | 5,000 Obj/Sec |
Sample Characteristics
- Volume: 20-200 ul
- Utilization Efficiency: up to 95%
Physical Characteristics
- 35" W x 26" H x 25" D (889 mm x 660 mm, 635 mm)
- 400 lbs. (182 kg)
Automated Instrument Operations
- Start up and shut down
- Sample load, acquisition, compensation, batch analysis
- Laser alignment, focus, calibration, and test
Illumination
- Excitation: Standard 488 nm; Optional High Power 488 nm, 375 nm, 405 nm, 561 nm, 592 nm, and 642 nm
- Side Scatter: 785 nm standard
- Brightfield: Customizable in any channel
Operational Requirements
- 450 W, 100-240 VAC, 50/60 Hz
- No external air or water required
12 Fluorescent Channels
- Brightfield and SSC
- Fluorescence
Cytek® Amnis® ImageStream®X Mk II Kits
| Product | Category | Part Number |
|---|---|---|
| Amnis® NF-kB Translocation Kit | Cell Pathway | ACS10000 |
| Amnis® Protein Aggregation & Silicone Oil Detection Kit | Drug Discovery | ACS10001 |
| Amnis® SpeedBead® Kit for ImageStream®X System, ISX400041 | Calibration | CN-0440-01 |
Options
The ImageStreamX system offers field upgradeable options for a configuration customized to your experiments.- 6 or 12 image channels
- MultiMag 20x 40x 60x objectives
- High Gain Mode
- Extended Depth of Field (EDF)
- Autosampler 96-well plate
- Up to 6 lasers:
- 375 nm, 405 nm, 561 nm, 592 nm, 642 nm
- High powered: 400 mW, 488 nm
IDEAS® Image Analysis Software with Machine Learning Module
IDEAS® Image Analysis Software: IDEAS software is powerful and easy to use, allowing users to create publication ready figures of ImageStream data. IDEAS software combines familiar tools from flow cytometry and microscopy software including dot plots, histograms, statistic tables, image gallery display optimization, and more.
Features Include:
- Click on a dot in the dot plot, and see the corresponding image
- Wizards for easy-to-follow standard workflows for applications such as Apoptosis, Cell Cycle analysis, Colocalization, Internalization, Nuclear Localization, Shape Change, and Spot Counting. The Feature Finder wizard will find the best feature for each unique experiment
- Compensation wizard that applies spectral crosstalk correction for each pixel in the image
- 86 features per channel measuring both fluorescence intensity and cellular morphology information
- 22 function masks that identify subcellular components and refine the region of interest
- Customizable statistics tables
- Templates that facilitate repeat experiments and standardize analysis for all samples
- Batch processing for automated compensation and processing of each sample
Machine Learning (ML) Module: ML module is a plug-in for the IDEAS software that enables generation of novel features specifically tailored to identify the desired cellular morphology. ML module greatly simplifies analysis by allowing researchers to hand-tag truth populations and use those images to automatically create a unique classifier that will find like images.
Compliance-Enabled Software Solutions: INSPIRE™ data acquisition software and IDEAS® data analysis software are available in 21 CFR Part 11 enabled versions that allow users to meet requirements for regulated settings.
Amnis® AI: Discover New Dimensions in Cell Analysis
Amnis® AI Image Analysis Software: Amnis AI software represents the next generation of image analysis software. This stand-alone software package allows scientists to visually categorize multiple cell classes and train the software to identify those populations in unknown samples. The software produces easy to understand results including a confusion matrix table, probability index, precision, recall, and F1 statistics to ensure accuracy of results. This tool simplifies complex image analysis experiments by allowing the expert to train the model and any researcher to repeat the analysis. Thus, critical expertise is maintained during each analysis session and the analysis is made significantly more consistent and reproducible. Amnis AI features include:
- Computer aided hand-tagging
- Population clustering displayed on object map plots
- Deep Learning using convolutional neural network, random forest, and transfer learning algorithms
- Accuracy analytics to verify that results match the expectation
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Further information on our legacy Cytek® Amnis® FlowSight® and Cytek® Amnis® CellStream® systems can be found using these links.
Please reach out to technicalsupport@cytekbio.com for assistance or if you have any questions about these instruments.