Frequently Asked Questions

MilliporeSigma’s Amnis® instruments are designed to perform any standard flow cytometry assay as well as a wide range of quantitative imaging assays. The ImageStream®^X Mark II can be used to study:

• Nuclear translocation in mixed populations of cells
• Internalization of ligands, phagocytosis, pinocytosis
• Shape change (chemokine-induced, etc.)
• Cell-Cell interactions, including immune synapse studies
• Apoptosis and autophagy, including LC3 spot counting
• Radiation repair, including γ-H2A.X spot counting
• Aneuploidy via FISH spot counting
• Co-localization of markers within cells or on cell surfaces
• Intracellular trafficking and organelle co-localization
• Cell cycle and mitosis in heterogeneous populations
• Morphology-based cell classification
• Small particle detection

With Quantitative Imaging, the FlowSight® can also perform most of the quantitative image-based assays listed above at a lower resolution than the ImageStream®.

The FlowSight® operates at a fixed 20X magnification, can accept up to four excitation lasers, and is optimized for standard flow cytometry assays with visual verification of all events and populations for improved gating and artifact detection. The FlowSight® also has a smaller footprint and lower cost.

The ImageStream®^X Mark II operates at 40X magnification, can accept up to seven lasers, and can perform quantitative image-based assays in its base configuration. With its higher magnification, the ImageStream®^X Mark II will detect more subtle changes in cell morphology than the FlowSight®, particularly for smaller cells, while the FlowSight® is well-suited for characterizing larger cells.

The imaging capabilities of the ImageStream®^X Mark II can be enhanced with the MultiMag option, which adds 60X and 20X objectives, as well as the Extended Depth of Field (EDF) option. The 60X objective enhances the effectiveness of many image-based assays, even in bacteria and yeast, while the 20X objective increases the width of the field of view for the analysis of larger cells. The EDF option significantly augments the resolution of spot counting assays.

The number of peer reviewed publications is approximately 600 as of August 2015, and includes publications in Nature, Science, Blood, and the Journal of Immunology. Please see the Amnis® publication list for the most significant recent papers.

The ImageStream®^X Mark II and FlowSight® systems are the only platforms capable of sensitive fluorescence imaging of cells in flow and are covered by over 45 US and international patents. Though the capacity to image in flow confers clear advantages over nonimaging flow cytometry, the most important characteristics of the Amnis® systems are their speed, high information content per cell, ability to quantitate morphology, and high fluorescence sensitivity. High speed allows the imaging and analysis of large populations of cells for statistically robust assay results even with rare sub-populations in heterogeneous samples. The multiple images of each cell help ensure that different cell populations can be distinguished based on their morphology, granularity (darkfield imagery), fluorescence brightness, and sub-cellular distribution. High fluorescence sensitivity ensures detection of even faint markers and facilitates experiment design by allowing the use of red dyes with low abundance markers.

The primary differences include:

• Faster data acquisition of up to 5,000 cells/sec
• Easier operation via a simplified user interface with graphical gating, the ability to immediately view gated populations during data acquisition, real-time fluorescence intensity crosstalk compensation, and more
• More flexible operation with sample volumes of 20-200 µl, up to seven lasers, and ultra-low laser power settings for marine microorganisms and other very bright samples
• Enhanced capacity for rare cell detection with up to 95% sample utilization efficiency
• Double the fluorescence sensitivity with a 200 mW 488 nm laser standard 
• Linux-based design for more robust operation
• Lower base price

MilliporeSigma has a comprehensive trade-in program for owners of older Amnis® or Guava® cytometers who wish to upgrade to newer instruments. Please contact a sales representative for details.

Both systems can produce up to 12 images per cell simultaneously, including darkfield, two brightfield images, and nine fluorescence images. A tenth fluorescence image can be produced if darkfield is turned off. In addition to fluorescent intensity per cell, over 80 parameters can be calculated for each image that quantitate morphology, fluorescence strength, and fluorescence localization by default with the ability to create additional features as required. Both systems are compatible with a wide variety of fluorescent dyes commonly used in both flow cytometry and microscopy.

The ImageStream®^X Mark II comes standard with both 488 nm (blue) and 642 nm (red) solid state lasers and a 785 nm laser dedicated for scatter. Four additional laser lines are currently available, including 375 nm, 405 nm, 561 nm, and 592 nm, all of which are solid state and variable power. The standard 200 mW 488 nm laser can also be upgraded to 400 mW for samples requiring extremely high sensitivity, such as cells with dimly-expressed markers and sub-micron particulates.

The FlowSight® comes standard with a 488 nm (blue) laser and 785 nm scatter laser and can be outfitted with up to three additional lasers, including 405 nm, 561 nm, and 642 nm lines.

The FlowSight® can image up to approximately 4,000 cells per second and the ImageStream®^X Mark II up to 5,000 cells per second at 20X magnification, independent of the number of images acquired per cell. The imaging rate is proportional to the sample concentration and assumes a cell concentration of ~2 * 10⁷ cells/ml. The ImageStream®^X Mark II can be run at 40X and 60X magnification with proportionately lower imaging rates. The total sample-to-sample cycle time is approximately 1.5 minutes, including a thorough automated flush between samples.

Both the ImageStream®^X Mark II and the FlowSight® have fluorescence sensitivity equal to or better than conventional flow cytometers, particularly for smaller objects like bacteria, yeast, or sub-cellular organelles. The ImageStream®^X Mark II has a measured sensitivity of less than 5 MESF and the FlowSight® achieves less than 10 MESF. Though MESF is an incomplete measure of sensitivity, more comprehensive sensitivity assessment techniques demonstrate favorable results compared to conventional flow cytometers.

The standard magnification of the ImageStream®^X Mark II is 40X. The optical system employs a 0.75 numerical Aperture (NA) 40X objective lens and is diffraction-limited. The image pixel size is 0.5 x 0.5 microns, so a 10 micron diameter cell covers approximately 300 pixels. The MultiMag option includes additional 60X/0.9 NA and 20X/0.5 NA objectives. The FlowSight® operates at a fixed magnification of 20X with a 1.0 x 1.0 micron pixel size.

EDF stands for Extended Depth of Field and it allows the ImageStream®^X Mark II to keep the entire cell in focus, rather than just a single plane of focus as in conventional microscopy. EDF is broadly useful for a range of assays but is particularly helpful for applications that rely on the absolute counting of spots, such as fluorescence in situ hybridization (FISH). Though neither the FlowSight® nor the ImageStream®^X Mark II are confocal imaging systems, EDF allows the ImageStream®^X Mark II to produce imagery similar to a maximum projection of a confocal image stack, but with speed and sensitivity orders of magnitude faster than a confocal microscope. EDF also allows the ImageStream®^X Mark II to operate faster than instruments without EDF by running a wider fluid core, and improves data analysis and consistency by ensuring uniform focus for virtually all the cells in the data file. EDF is not available for the FlowSight®.

We typically recommend acquiring a minimum of 500-1000 cells or events in order to ensure statistical robustness. Both the ImageStream®^X Mark II and FlowSight® can also return any unused sample following data acquisition.

Yes. Both the FlowSight® and the ImageStream®^X Mark II use syringe pumps to drive the sample, so the volume consumed during data acquisition is well-defined. Since both systems also image every object in the fluid core, both the absolute count and the sample concentration are derived with high accuracy without the need for calibration beads.

Flow forces tend to orient long objects vertically, so the principal constraint on maximum size is the width of the object. The ImageStream®^X Mark II running at 40X magnification has a field of view 60 microns wide. At 60X magnification, the field width is 40 microns wide and at 20X magnification the field width is 120 microns. The FlowSight® operates at a fixed 20X magnification with a field width of 60 microns.

The smallest object that can be detected is a function of its brightness due to fluorescence labeling and/or side scatter characteristics. Both the ImageStream®^X Mark II and the FlowSight® can detect polystyrene microspheres as small as 100 nm in diameter via side scatter or fluorescence, and can resolve mixed populations with 50 nm size resolution.

The narrowest pathway in the system is 250 microns and all fluids are pumped via motor-driven syringe pumps, so clogging problems are rare. To ensure trouble-free operation, Amnis® recommends that clumpy samples be filtered through a 70 micron nylon mesh before running on the instrument.

Yes. The fluid pressures and shear forces within both systems are very low compared to conventional flow cytometers. Please keep in mind that the systems currently have no provision for temperature-controlled incubation.

Most sample preparation protocols for flow cytometry, including those for adherent cells, can be adapted to imaging flow cytometry with relatively minor adjustments in cell concentration, sample volume, and probe titration.

Amnis® recommends the use of either DI water or standard phosphate-buffered saline as sheath fluid. Our systems’ fluidics are conceptually similar to those of a flow cytometer but the actual design is quite different and will not necessarily function properly with commercially available sheath fluids intended for conventional flow cytometers. Our systems also use standard cleanser, debubbler, and sterilizing solutions which can be purchased from a variety of sources at nominal cost.

Both the ImageStream®^X Mark II and the FlowSight® can store data in Amnis®’ own image data file format, in FCS format, or both. The Amnis IDEAS® data analysis package can also export calculated image feature values in plain text format or to an FCS file.

IDEAS® can export the raw image data for user-specified cell populations in TIFF format.

Yes, for some experiments results can be optimized using masking tools, which define a specific region of interest within an image. New parameters can then be created by calculating features using custom masks, or through combining values from more than one feature using mathematical operators. These approaches have been used to address challenging data sets including morphometric analysis of sperm health, detection of asymmetric cell division, micronuclei formation, and more. For example, if a cell is counterstained with a fluorescent DNA binding dye, the nucleus will be imaged along with a brightfield image of the cell as a whole. The cytoplasmic area can then be calculated by subtracting the nuclear image area from the cellular image area. The nuclear to cytoplasmic area ratio could also be calculated by dividing the nuclear area by the cytoplasmic area calculated previously. Once defined, these user parameters are automatically calculated for all cells and can be plotted on histograms and dot plots just like any other standard parameter.

IDEAS® will run on most modern PCs running Windows 7 or more recent versions (32 or 64 bits). At least four gigabytes of RAM are recommended to facilitate the analysis of larger data files (more than 10,000 cells) and/or the analysis of multiple data files at the same time. IDEAS® can also take advantage of higher video resolution settings to display more graphs and images at a time. The best way to transfer data is via gigabit Ethernet.

A typical 10,000 event file requires roughly 500 MB for both raw and processed data file storage. The file size can be reduced by reducing the number of collected channels.

The ImageStream®^X Mark II and FlowSight® are available with a dedicated high speed workstation that incorporates a 1.5 terabyte RAID and can be connected to the instrument via a gigabit Ethernet link. The workstation’s RAID is typically sufficient for at least the first year of data storage. Data can be backed up to DVD, external hard drives, or a storage server via a network.

IDEAS® is a Windows application but a number of our users run it on Intel-based Apple hardware using Parallels, VMware, and other Windows environments. IDEAS® is not officially supported by Amnis® on Apple hardware.

No. Amnis® recommends that each site have one or two advanced users, but these users can readily train others. Both the ImageStream®^X Mark II and the FlowSight® are intuitive to operate and highly automated, including one-button calibration at the beginning of the day and one-button sterilization at the end of the day. Instrument operation can be learned in a matter of hours and users can become functional with the IDEAS® data analysis software in a day or two. Amnis® offers numerous training and support options including telephone and web conferencing, on-site training, web site knowledgebase, and training classes offered at our facility.

Instead of using photomultiplier tubes, the ImageStream®^X Mark II and FlowSight® employ a custom CCD camera to image the cells. TDI is a method of CCD camera operation that greatly increases sensitivity, even at very high imaging rates. Essentially, TDI is a means of electronically panning the camera to track the cells in the flow stream. As the cells move down the field of view, the photoelectrons on the CCD chip are shifted down the chip. This process is synchronized by measuring the velocity of the cells and constantly adjusting the shift rate of the photoelectrons to match. The end result is that light from each cell can be collected ~1000X longer than standard imaging techniques without blurring. With TDI operation, all objects in the flow core are imaged without the need for a triggering system.

No. The fluidic system is designed to be extremely laminar, non-pulsatile, and to prevent cell tumbling.

The SpeedBead® reagent is a suspension of small microspheres that is run at all times in the ImageStream®^X Mark II. It is used during the daily calibration routine (ASSIST) as well as by the real-time velocity detection and autofocus systems. The ASSIST calibration routine is a comprehensive set of automated self-tests that use SpeedBead® imagery to ensure that all the internal systems (illumination, collection, fluidic, electronic, etc.) are functioning within factory specifications. The routine takes approximately 15 minutes to run and requires no user intervention.

The FlowSight® uses a calibration bead reagent for ASSIST but does not employ SpeedBeads for velocity detection or autofocus during sample data acquisition.

The ImageStream®^X Mark II is approximately 90 cm wide by 60 cm deep by 65 cm tall (36” x 24” x 26”) and weighs about 130 Kg (300 lb). The FlowSight® is approximately 45 cm wide by 63 cm deep by 47 cm tall (18” x 18” x 25”) and weighs about 60 Kg (135 lb). Both systems are completely self-contained, requiring no external air connections or tanks. Both systems include a floor-standing computer for data acquisition and a small Windows desktop for instrument operation and data analysis.

The most fundamental difference is that the ImageStream®^X Mark II and the FlowSight® image cells in suspension rather than on slides or plates, so they are much better suited to blood, cells in other bodily fluids, and non-adherent cell cultures. More significant, however, is that HCS and laser scanning systems are optimized to image only a few hundred cells per sample in a semi-quantitative manner. These systems are typically used in pharmaceutical screening environments with cellular assays that employ homogeneous cell cultures, just a few strong fluorescent markers, and that have very distinct readouts. In contrast, Amnis®’ systems are designed to image tens or hundreds of thousands of cells per sample with high fluorescence sensitivity, up to 12 high resolution images per cell, extremely low background, and highly accurate image segmentation. These capabilities enable detailed studies of complex and/or subtle biological phenomena in highly heterogeneous cell samples. Both the FlowSight® and the ImageStream®^X Mark II can be employed for HCS of non-adherent cells with the addition of the AutoSampler option.

While this is certainly possible, the Amnis® systems offer a major advantage to coupling the image of a cell to its position on fluorescence plots. For example, in IDEAS® it is possible to click on any dot in a dot plot and see the associated image, or alternatively click on any image and see the associated position in the dot plots. Not only does this ‘digital’ sorting greatly simplify the workflow compared to flow cytometry followed by manual microscopy, image-guided flow cytometry analysis greatly facilitates positioning of regions and rejection of artifacts, and is especially useful for rare cell analysis. Additionally, microscopy is constrained by sample size, in that the number of cells it is practical to image in a reasonable period of time is limited, which generally precludes the robust statistical analysis of populations/phenotypes that is the clear advantage of flow cytometry. Finally, Amnis® systems include QI software, which automates quantitative morphology, allowing identification and analysis of populations based on morphological characteristics that could not be achieved by cell sorting in combination with microscopy.

Amnis® was founded in 1999 and introduced the first-generation ImageStream® 100 in 2005. Amnis® brought the second-generation ImageStream®^X to market in 2009, offering ten times the speed, twice as many images per cell, and higher image quality at 2/3 the price of its predecessor. In 2011, Amnis® released the low-cost FlowSight®, an imaging flow cytometer with breakthrough ease of use and power at a price comparable to conventional flow cytometers.

Cutting-edge products and rapid growth led to the acquisition of Amnis® by MilliporeSigma (a subsidiary of Merck KGaA, Darmstadt, Germany) in late 2011. In July of 2012, Amnis® released the ImageStream®^X Mark II, an improved version of the ImageStream®^X with greater speed, ease of use, and optimizations for rare cell analysis.