Frequently Asked Questions

What are the primary applications for imaging flow cytometry?

Amnis’ instruments are designed to perform any standard flow cytometry assay as well as a wide range of quantitative imaging assays.
The ImageStreamX 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-H2AX 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 studies in heterogeneous populations
  • Morphology-based cell classification

The FlowSight, when equipped with the Quantitative Imaging option, can also perform many of the quantitative image-based assays listed above. 

What are the main differences between the FlowSight and the ImageStreamX Mark II?

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 ImageStreamX Mark II operates at 40X magnification (double that of the FlowSight), can accept up to seven lasers, and can perform quantitative image-based assays in its base configuration. With its higher magnification the ImageStreamX Mark II will detect more subtle changes in cell morphology than the FlowSight, particularly for smaller cells.
The imaging capabilities of the ImageStreamX Mark II can be further enhanced with the MultiMag option, which adds 60X and 20X objectives, as well as the Extended Depth of Field (EDF) option, neither of which are available for the FlowSight. 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 greatly enhances the effectiveness of spot counting assays.

How many studies have been published by Amnis users?

There are over 250 publications in Nature, Science, Blood, the Journal of Immunology, and numerous others. Please see the Amnis publication list for the most significant recent papers. Reprints are available upon request.

Is Amnis technology unique?

The ImageStreamX 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 imaging in flow is novel, 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 (brightfield imagery), granularity (darkfield imagery), and fluorescence brightness and sub-cellular distribution. High fluorescence sensitivity ensures detection of even faint markers and eases experiment design by allowing the use of red dyes with low abundance markers.

What is included with the Quantitative Imaging option for the FlowSight?

The Quantitative Imaging (QI) option includes hardware and software modifications to double image resolution, increase system sensitivity, and provide greater experimental flexibility. Hardware modifications include a laser retro-reflection system that doubles fluorescence sensitivity for dyes excited by the 488 nm laser, as well as a multi-channel brightfield illuminator that allows brightfield imagery to be directed to unused fluorescence channels. The IDEAS data analysis software included with every FlowSight automatically recognizes QI data files and enables a full suite of image processing tools. 

What are the main differences between the earlier ImageStreamX and the new ImageStreamX Mark II?

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
• More efficient 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
• More affordable with two laser configurations starting at $199,000

Can I upgrade an ImageStreamX to an ImageStreamX Mark II?

Yes. Customers who took delivery of an ImageStreamX any time in 2012 are eligible for an upgrade at low or no cost, depending on their instrument configuration. Customers with ImageStreamX orders pending will receive Mark II systems at no additional charge. ImageStreamX systems delivered prior to 2012 can also be upgraded to the Mark II configuration. Amnis will announce the further details and pricing for the upgrade program later in 2012.  

Can I upgrade an older ImageStream to an ImageStreamX Mark II or a FlowSight?

The original ImageStream system has no parts commonality with the ImageStreamX Mark II or the FlowSight and cannot be upgraded. However, EMD/Merck Millipore 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. 

How many colors can the ImageStreamX Mark II and FlowSight systems image?

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. Each image is used to calculate over 80 parameters that quantitate morphology, fluorescence strength, and fluorescence localization. Both systems are compatible with a wide variety of fluorescent dyes commonly used in both flow cytometry and microscopy.

How many excitation lasers can be fitted to the ImageStreamX Mark II and FlowSight?

The ImageStreamX Mark II comes standard with both 488 nm (blue) and 642 nm (red) solid state lasers and can be outfitted with up to five additional lasers. Available lines currently include 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 can be outfitted with up to three additional lasers, including 405 nm, 561 nm, and 642 nm lines.

How fast are Amnis’ imaging flow cytometers?

The FlowSight can image up to approximately 2,000 cells per second and the ImageStreamX Mark II up to approximately 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 cell/ml. The ImageStreamX Mark II can be run at 40X and 60X magnification with proportionately lower imaging rates. The total sample-to-sample cycle time is approximately one minute, including a thorough automated flush between samples.

What is the minimum number of cells per sample?

The ImageStreamX analyzes more than half the 50 microliter sample volume, so samples with as few as 10,000 cells will still yield images of over 5,000 cells.

How sensitive are the Amnis instruments?

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

How detailed are the images?

The standard magnification of the ImageStreamX Mark II is 40X. The optical system employs a 0.75 numerical aperture 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 60X objective provides increased resolution and sensitivity while the 20X objective provides increased speed and a wider field of view for large cells. The FlowSight operates at a fixed magnification of 20X with a 2.0 x 1.0 micron pixel size. If equipped with the Quantitative Imaging option, the pixel size is reduced to 1.0 x 1.0 microns.

What is the EDF option?

EDF stands for Extended Depth of Field and it allows the ImageStreamX 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. Though neither the FlowSight nor the ImageStreamX Mark II are confocal imaging systems, EDF allows the ImageStreamX 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 ImageStreamX 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.

What is the minimum number of cells per sample?

Both the ImageStreamX Mark II and FlowSight will image ~95% of cells in a 200 µl sample volume, regardless of the number of cells present. Both systems can also return any unused sample following data acquisition.

Can Amnis instruments perform absolute cell counts?

Yes. Both the FlowSight and the ImageStreamX 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 core, both the absolute count and the sample concentration are derived with high accuracy without the need for calibration beads.

What are the largest and smallest objects that can be imaged?

Flow forces tend to orient long objects vertically, so the main constraint on maximum size is the width of the object. The ImageStreamX 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 ImageStreamX Mark II and the FlowSight can detect polystyrene microspheres well below 300 nm in diameter via side scatter and can resolve mixed populations with 50 nm size resolution. 

Is clogging a problem?

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.

Can the ImageStreamX Mark II and FlowSight image live cells or cell conjugates?

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.

Can the ImageStreamX Mark II and FlowSight be used with adherent cells?

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.

Are the ImageStreamX Mark II and FlowSight compatible with the sheath fluids used by conventional flow cytometers?

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. 

Can I analyze the data using other flow cytometry software packages?

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

Can IDEAS export raw image data for analysis by other image analysis packages?

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

Can IDEAS be customized to suit my own needs?

The parameters calculated by IDEAS can be user-customized. Any parameter can be calculated for a specific cellular region of interest. 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. 

Does IDEAS require special computer hardware?

IDEAS will run on most modern PC’s running Windows 7 (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.

How big are the data files?

A typical 10,000 event file requires roughly 500 MB for both raw and processed data file storage.

What do you recommend for data storage and transfer?

The ImageStreamX 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. 

Is IDEAS Apple Macintosh compatible?

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.

Do Amnis instruments require a dedicated operator?

No. Amnis recommends that each site have one or two advanced users, but these users can readily train others. Both the ImageStreamX 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 offer numerous training and support options including telephone and web conferencing, on-site training, web site knowledgebase, and training classes offered at our facility.

What is Time Delay Integration (TDI)?

Instead of using photomultiplier tubes, the ImageStreamX 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. 

Don't the cells tumble in flow?

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

What is the SpeedBead® reagent, how is it used, and how much does it cost?

The SpeedBead reagent is a suspension of small microspheres that is run at all times in the ImageStreamX 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 about 10 minutes to run and requires no user intervention. The SpeedBead reagent is provided free of charge during the initial year of warranty coverage and is also included as part of Amnis’ service contracts.

How big are the instruments?

The ImageStreamX 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.

How do Amnis’ systems differ from high content imaging systems (HCS) and laser scanning microscopes?

The most fundamental difference is that the ImageStreamX 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 ImageStreamX Mark II can be employed for HCS of non-adherent cells with the addition of the AutoSampler option.

Couldn’t I just use a cell sorter followed by microscopy to do what Amnis systems do?

It is easy to conceive of using a cell sorter to isolate rare cells on a slide for subsequent imaging. In practice, this is very difficult to accomplish because the cells of interest must be phenotypically distinct from other cells in the sample in order to be sorted with high purity. In most cases, a mixture of cells is sorted which cannot be differentiated by the microscope due to its limited sensitivity and fewer fluorescent colors relative to the sorter. Even if a pure population is sorted, the microscope is limited in its ability to quantitate multicolor imagery accurately due to fluorescence crosstalk. Even if this limitation is overcome by using a limited number of spectrally distinct colors, the microscope is constrained in the number of cells it can image in a reasonable period of time, so statistical population information will be unavailable. Variation from cell to cell in small analysis populations can mask subtle differences that the analysis of a larger population would reveal clearly using the objective and quantitative morphometric parameters offered by the Amnis systems.

What is the history of Amnis?

Amnis was founded in 1999 and introduced the first-generation ImageStream 100 in 2005. Amnis brought the second-generation ImageStreamX 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. 
Amnis’ breakthrough products and rapid growth led to its acquisition by EMD Millipore (Merck Millipore outside the US) in late 2011. In July of 2012, Amnis released the ImageStreamX Mark II, an improved version of the ImageStreamX with greater speed, ease of use, and optimizations 

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