University of Pittsburgh Cancer Institute

Amnis ImageStream© 100

The Amnis ImageStream 100 is the first commercially available imaging flow cytometer. It combines advantages of flow cytometry (ability to interrogate large numbers of cells in suspension, well-developed color compensation, analysis and display methods) with those of image analysis (digital imagery of each individual cell, calculation of morphological features from digital images, localization of fluorescence to morphologic features, co-localization of fluorescent probes).

In the simplest terms, the ImageStream may be thought of as a flow cytometer in which the photomultiplier tubes have been replaced by an array of sensitive charge coupled device (CCD) cameras (Figure 1). Cells in the sample are hydrodynamically focused within a quartz cuvette, where they are interrogated by a 480 nm solid-state laser. Scattered incident light and emitted fluorescence are collected at 90 degrees to the excitation beam and broken into 6 images of distinct bandwidth by a dichroic filter stack that directs the resulting spectral bands at different angles. These images are captured by adjacent segments of the camera chip. The images include brightfield (transmitted light), 488nm side-scatter, and 4 emitted fluorescence channels corresponding to those used on many 4-color flow cytometers. Individual objects are identified and distinguished from background in real time in a process analogous to live gating, and the images are stored to a raw image file (RIF). In post-acquisition processing, the 6 images of each object are put into register, color is compensated pixel by pixel, and features such as area and intensity are calculated for each parameter and each image (Table 1). The results are stored in a compensated image file (CIF) for analysis.

The parameters include those familiar to flow cytometry, such as Intensity, defined as the total pixel intensity of an object in a given fluorescence channel (minus background), and Side Scatter, the Intensity of the 488nm image. Cell size can be measured directly as the brightfield area. A wealth of additional parameters (Table 1), drawn from the repertoire of image analysis but foreign to most flow cytometrists, allows characterization of cellular features and localization of fluorescent probes.

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Ten Technical Questions and Answers

When we saw our first ImageStream demonstration we had many questions concerning its design and operation. Our laboratory has now logged about 30 hours on this instrument. Here are our top ten Questions, and the Answers as we understand them now.

Q1: Even the best CCD camera is orders of magnitude less sensitive than a PMT. How does the ImageStream manage to collect enough photons to approach the sensitivity of a flow cytometer?

A1: Several factors are responsible for the remarkable sensitivity and dynamic range. The most important is time delay integration (TDI). TDI was originally designed to increase the sensitivity of cameras used for scanning x-ray imaging applications. The application to image moving cells is novel and depends on a remarkable velocity-sensing technology. Briefly, rather than taking a single image of a cell as it passes before the camera, photocharges are successively shifted across the length of the chip as the cell traverses the 512 pixels of the camera’s field. Conceptually, TDI is equivalent to panning on the moving cell to hold it in the camera’s field of view. Since each pixel maps to 0.5 microns of the imaged cell and the flow rate is approximately 25 mm/second, each image is captured over a period of about 10msec. Successful application of TDI to cytometry requires knowing the location of a cell at any given time with submicron resolution. This is accomplished with a separate infrared laser, which, in conjunction with 2 PMTs, measures object velocity and focus in real time for closed-loop process control. Other factors that increase the amount of collected light include, 1) Slow speed. The flow rate, 30mm/sec, is approximately 1/30 that of a conventional flow cytometer, and 1/300 that of a high-speed instrument; 2) High laser power. The ImageStream uses a 200 mw 488nm solid state laser for excitation (Cf. 15-20 mw in most cuvette-based flow cytometers); 3) A sensitive 6-channel CCD TDI camera specifically designed for this application.

Q2: Doesn’t the cell rotate or wobble as it crosses the camera field?

A2: No. We were fully convinced of the wonders of hydrodynamic focusing when we collected razor sharp images of red blood cells.

Q3: Why doesn’t the brightfield lamp interfere with the measurement of emitted light?

A3: A filter wheel allows a selectable filter to be interposed. Violet (450 nm) light is used when all 4 fluorescent probes are used. Near red (630 nm) gives the best brightfield resolution.

Q4: How does the resolution compare to conventional fluorescence microscopy?

A4: The numeric aperture of the objective is 0.75. When perfectly focused, the images are comparable to that of conventional fluorescence microscopy using a 40X objective. In our hands focus is acceptable for the great majority of events (sharp enough for calculation of cellular features), and really tight about 20% of the time.

Q5: What about the depth of field?

A5: Comparable to conventional microscopy with a 40X objective and a long working distance condenser.

Q6: Has Amnis done its homework and taken advantage of recent advances in flow cytometry automated color compensation?

A6: IDEAS analysis software provides automated calculation of compensation matrices using the inverted matrix method. Unlike any flow cytometry software that I have seen, the user can visually gate out autofluorescent events from bivariate scatter plots prior to calculation of spillover coefficients, improving compensation.

Q7: What about data display?

A7: All parameters can be displayed on linear or "hyperlog" scales, similar to those used in flow cytometry. The hyperlog function is linear as it passes through zero and approaches logarithmic within the first decade (or within a user defined range). Boolean gates of a variety of shapes can be placed on populations. Gated regions can be "color-evented." IDEAS has a long way to go before it has all of the functionality of the best commercial flow cytometry software, but it has tackled the most important issues first.

Q8: Can flow plots be correlated with photographic images of cells?

A8: Yes! Clicking on individual points in a bivariate scatter plot brings up the actual image of the cell. When a gate is created around a population, a gallery of all of the images within that gate can be displayed, the equivalent of "virtual cell sorting."

Q9: Six digital images are collected and stored for each cell. How long does it take to acquire and display the data?

A9: A typical blood mononuclear cell maps to about 400 pixels. There are 1024 channels of resolution per pixel and 6 images per cell. A great deal of processing is performed by the INSPIRE acquisition software in real time by the built-in dual Xeon processor server running 4 simultaneous threads. Instrument control is managed by a separate internal Linux box. Since color compensation must be performed on each individual pixel, this is done post-acquisition using IDEAS software. This computation-intensive task is best done in batch mode on a separate workstation. Both the size of the data files and the complexity of the data guarantee that exploratory analysis will be time consuming. Once the analytical strategy has been developed for a particular test, analysis templates and batch processing take some of the pain out of analysis. We have requested a state of the art Windows-based workstation to free the ImageStream's dedicated computer from the task of data analysis.

Q10: How large are the data files?

A10: This imaging system creates 100-200 composite images per second. A 50,000-event raw image file (RIF) occupies just over 1 GB of file space, and this requirement is doubled when a compensated image file (CIF) is created for analysis. We are currently establishing a separate server with 3.6TB of RAID5 disk space on a gigabit network to support the ImageStream.

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Advantages of Imaging Flow Cytometry over Conventional Flow Cytometry

• Ability to localize fluorescence within cells and quantitatively determine fluorescence in defined cellular features (e.g. nucleus versus cytoplasm, endosomes versus lysosomes)
• Ability to co-localize fluorescent signals in the entire cell or within selected cellular features (e.g. quantifying tagged proteins within organelles)
• Ability to localize fluorescent signals during cellular interactions (e.g. adhesion molecules in the immune synapse)
• Availability of 35 intensity-based and morphologic parameters per channel with ability to create user-defined parameters based on Boolean and algebraic calculations (e.g. nucleus-to-cytoplasm ratio).
• Ability to view images (single or composite fluorescent images, or brightfield) associated with a single point or a region on a bivariate scatter plot
• Morphologic parameters can serve as surrogates for one or more fluorescent parameters, freeing up fluorescent channels (e.g. detection of apoptosis cells by comparing nuclear area and peak nuclear pixel intensity, ability to distinguish prophase, metaphase and anaphase on the basis of nuclear texture, size and aspect ratio)

Advantages over Fluorescence Microscopy Based Image Analysis

• Higher throughput by 2-3 orders of magnitude gives better statistical analysis
• Choice of objects and subpopulations for analysis is objective rather than subjective

Advantages over Fluorescence Activated Cell Sorting Followed by Image Analysis

• Can "sort" on calculated morphologic features and by location of fluorescence within a cell
• Does not require two complex instruments which are usually not in the same location (1.8 miles apart in our case)
• No loss of cells or viability issues
• Can perform kinetic experiments
• Advantages over Image Scanning Cytometry
• Better suited to cells in suspension (simplified sample handling)
• Gives light scatter measurements
• Findings more easily translated to flow cytometry (e.g. for polychromatic multiparameter analysis or cell sorting)
• Cells in suspension are not altered in function or appearance by adherence to a solid substrate

Advantages over Image Scanning Cytometry

• Better suited to cells in suspension (simplified sample handling)
• Gives light scatter measurements
• Findings more easily translated to flow cytometry (e.g. for polychromatic multiparameter analysis or cell sorting)
• Cells in suspension are not altered in function or appearance by adherence to a solid substrate

View Flourochromes for the ImageStream (requires Adobe Acrobat)