Amnis® imaging flow cytometers are available on two platforms: the FlowSight® Imaging Flow Cytometer, and the ImageStream®X Mk II Imaging Flow Cytometer. Our cellular specialists are here to help determine the best instrument for your research and lab needs. Click here to contact a specialist
|Product Specifications||FlowSight||ImageStreamX Mk II|
|Description||Compact, high-sensitivity, multi-color imaging flow cytometer. Produces multiple images per cell for quantitative, statistically robust, image-based data at 20X magnification.||High-resolution, high-sensitivity microscope produces detailed brightfield, darkfield, and fluorescence imagery, as well as fluorescence intensity, for a wide range of quantitative, statistically robust cellular analysis.|
|Magnification||20X Fixed||60X / 40X / 20X|
|Pixel Area||1.0 square microns||0.1 / 0.25 / 1.0 square microns|
|Number of Channels||12||6 or 12 high resolution|
|Max Field of View Width||64 microns||128 microns|
|Quantitative Image Analysis||Standard||Standard|
|Brightfield Illumination||10 Channels||10 Channels|
|488 nm Excitation Laser||60 mW||200 mW Standard, 400 mW Optional|
|Laser Power Doubler||Standard||Standard|
|375 nm Excitation Laser||N/A||70 mW|
|405 nm Excitation Laser||100 mW||120 mW|
|561 nm Excitation Laser||50 mW||200 mW|
|592 nm Excitation Laser||N/A||300 mW|
|642 nm Excitation Laser||100 mW||150 mW|
|730 nm Excitation Laser||N/A||50 mW|
|785 nm Side Scatter Laser||70 mW||70 mW|
|Sample Format||Microcentrifuge tube||Microcentrifuge tube|
|Extended Depth of Field||N/A||Optional|
|Sensitivity||10 MESF||5 MESF|
The ImageStreamX and the FlowSight instruments acquire up to 12 images simultaneously of each cell or object including brightfield, scatter, and multiple fluorescent images at rates of up to 5,000 objects per second with high photonic sensitivity.
Detailed analysis of intensity, location, and co-location of probes is achieved using IDEAS® (Image Data Exploration and Analysis Software). IDEAS offers powerful tools for high content, statistically robust analysis of images, as well as standard flow cytometry graphing tools and statistics for hundreds of morphological features in addition to intensity.
The multispectral imaging system acquires up to 12 images per cell in three different imaging modes: brightfield, darkfield, and fluorescence. Light is collected from the cells flowing in the cuvette with custom objective lenses and relayed to the spectral decomposition element, which consists of a custom set of longpass filters in an angular array. The filters direct different spectral bands to laterally distinct channels on the charge-coupled device (CCD) detector. The cell images are collected using patented time-delay integration (TDI) technology, which yields fluorescence sensitivity comparable to the best flow cytometers. With this technique, an image is optically decomposed into a set of sub-images, each corresponding to a different color component and spatially isolated from the remaining sub-images.
Hydrodynamically-focused cells are trans-illuminated by a brightfield light source and orthogonally by laser(s). A high numerical aperture (NA) objective lens collects fluorescence emissions, scattered and transmitted light from the cells. The collected light in optical space intersects with the spectral decomposition element. Light of different spectral bands leaves the decomposition element at different angles such that each band is focused onto different physical locations of the CCD camera(s). As a result, each cell image is decomposed into separate sub-images on each CCD chip based on a range of spectral wavelengths. The FlowSight collects up to 12 images on one CCD and the ImageStreamX collects up to 12 higher resolution images, six per CCD. The images are in spatial registry to enable the detailed analysis of localization and co-localization of probes on, in or between, cells.
Principles of Operation
Amnis cytometers employ a CCD camera. This is the same technology used in medical radiography and military applications to capture images of moving objects.
The custom CCD camera operates in TDI mode that electronically tracks moving objects by integrating pixel content from row to row down the rows of pixels in synchrony with the velocity of the object (cell) in flow as measured by the velocity detection system. Aggregate pixel content is collected off the last row of pixels. Imaging in this mode allows for the collection of cell images without streaking and with a high degree of fluorescence sensitivity. TDI imaging, combined with spectral decomposition, allows for the simultaneous acquisition of multiple spectral images (Multispectral Imaging) of each cell in flow.
Fast imaging is required to obtain enough events for statistical significance. Traditional flash on the fly photography sacrifices sensitivity in order to image rapidly moving objects. Amnis employs a custom camera which is operated using a technique called TDI, a specialized detector readout mode that preserves sensitivity and image quality, even with fast relative movement between the detector and the objects being imaged. The TDI detection technology of the CCD camera allows up to 1,000 times more signal to be acquired from cells in flow than from conventional frame imaging approaches. Velocity detection and autofocus systems maintain proper camera synchronization and focus during the process of image acquisition.
As with any CCD, image photons are converted to photocharges in an array of pixels. During TDI operation, the photocharges are continuously shifted from pixel to pixel down the detector, parallel to the axis of flow, integrating the signal over multiple pixels. By synchronizing the photocharge shift rate with the velocity of the flowing cell, the effect is similar to physically panning a camera. With TDI, image streaking is avoided, despite signal integration times that are orders of magnitude longer than those of conventional flow cytometry. Each pixel is digitized with 12 bits of intensity resolution, providing a minimum dynamic range of three decades per pixel.
Sophisticated Amnis instrument platforms are driven by the powerful INSPIRE® and IDEAS Software packages. Thoughtfully designed user interfaces make the software easy to learn, whether you are familiar with microscopy or flow cytometry.
IDEAS offers powerful, familiar tools for creating histograms and scatter plots. Click on any dot in a scatter plot to see the corresponding cell’s image. Numerical scoring of parameters such as size, shape, texture, colocalization, and intensity allow for quantification of visual data.
The IDEAS Analysis Software Package is extraordinarily robust, providing hundreds of features for every cell. IDEAS Software also allows you to create almost any new feature you may need from the basic feature set.
In IDEAS Software, population statistics and imagery are completely integrated. Every dot on a scatter plot links directly to a cell’s images – click on the dot and you’ll see the corresponding cell. Click on an image to link to the feature values and points in the scatter plot. With its virtual sorting capability, IDEAS Software will show you all the images of a cell population you define.
The IDEAS interface integrates image data, plots, and statistics. The gallery shows you images of every cell, while the workspace gives you graphing tools to define and analyze cell populations and statistics tables to view population stats, as well as individual feature values.
Once you’ve created an analysis scheme in IDEAS Software, you can save it as a template for batch processing future experiments, automated data analysis for high throughput experiments, or to share with colleagues.
Amnis imaging flow cytometry data files (.DAF/.CIF) are directly compatible with FCS Express Image Cytometry software (from DeNovo Software) for further data analysis.
Pre-configured and optimized wizards are provided for many common applications.
Amnis imaging flow cytometers are equipped with INSPIRE Software that simplifies fundamental instrument operations. Instrument setup, calibration, and spectral compensation are straightforward and elementary. An intuitive interface makes data acquisition effortless.
An intuitive user interface provides easy-to-use instrument controls, real-time plotting and graphical gating, and images of every cell. The easy-to-use compensation wizard easily guides you through multi-color compensation.
Luminex flow cytometers and cellular analysis instruments give you instant access to all facets of cellular phenotypes and morphology.