With flow cytometry, a scientist can analyze thousands of particles or cells. Add imaging to that analysis, and the samples can be visually identified. That combination makes up imaging flow cytometry (iFC).
“Most technologies for cytometry only provide fluorescence from cells,” says Kent Peterson, president and CEO at Fluid Imaging Technologies. “iFC provides for the identification via digital imaging at the single-cell level, as well as fluorescence data.”
Wojciech Wojciechowski, scientific manager of the University of Rochester Medical Center Flow Cytometry Shared Resource Laboratory, performs flow cytometry with an Amnis Imaging Flow Cytometer, Image Stream-X, from MilliporeSigma. “Whenever an experiment requires analysis of both a high number of analytic data points—mostly cells—processed in a reasonably short time and with statistically quantifiable accuracy, and at the same time needs some kind of spatial distribution of the measured parameters—for example, where in the cell a given target is present—imaging flow cytometry is the technique of choice,” he says.
That covers a lot of possible applications. For example, Sébastien Coquery, technology manager at the University of North Carolina’s Flow Cytometry Core Facility, notes that iFC can be “used to look at probe co-localization, internalization such as drug uptake or phagocytosis, cell-to-cell interaction, cell cycle and cell death, morphology/shape changes, and more.”
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This technology can also be used to search through samples. As David Basiji, affiliate professor in the department of bioengineering at the University of Washington, notes, iFC can be used in “finding and identifying very rare cells with a low false-positive rate.”
Other experts point out additional benefits of iFC. “iFC is the best platform when you want to know not just how much of a protein is being expressed but if it has a significant localization feature that impacts its function or the function of a treatment condition or drug,” says Kelly Lundsten, business segment manager for advanced cytometry at BioLegend.
As an example, she says: “Say your protein is expressing green fluorescent protein and localizes to a lipid raft but when a transporter or channel is blocked, the expression level doesn’t change but the localization does, maybe gets diffusely cytoplasmic. That is significant and would’ve required both microscopy and flow experiments to be done sequentially, when it could instead be seen simultaneously using iFC.”
So, much of iFC’s value comes from its versatility and its ability to provide multiple capabilities in one platform.
Analysis options
Like most lab hardware, the value of iFC depends on the analytical capabilities, which involve the software. Depending on the iFC platform and the associated computational tools, many aspects of a sample can be determined automatically.
“With proper software, such as Fluid Imaging’s VisualSpreadsheet, image analysis can be used for classification of different types of cells,” Peterson explains. “For example, pattern-recognition algorithms can differentiate, classify, and match particles based on user-defined parameters.”
Some experimental situations, though, don’t require iFC. “When analyzing a sample that contains mostly homogenous cells or particles, an iFC is not required since morphology is not essential to the analysis,” Peterson explains. “Morphology data are not necessary when you know that all the particles are the same.” In such a situation, the important data involve particle or cell size, distribution, and fluorescence.
“If tissue context—that is, the pattern of cellular distribution within the tissue—is useful information, a more traditional microscopy approach is advised since getting cells into solution sacrifices contextual information,” Basiji adds. “If you need to see morphology at a sub-micron level and are willing to give up speed and statistical significance, confocal or super-resolution microscopy is advised.”
The key is fitting the technology to the scientific questions.
The key is fitting the technology to the scientific questions. “All platforms and assays should be selected by what is required for the biology and the experimental question and constraints and not vice versa,” Lundsten says. “You never make your scientific question fit a specific platform.”
One application that Wojciechowski mentions is cell-cell interactions. “This kind of assay would be employed when studying different kinds of intercellular synapses,” he explains. An example is the connection between a cytotoxic lymphocyte and a cancer cell. “With iFC, it is possible to establish if given cells are connected and form an actual synapse or if it’s just a random coincidence where cells are simply close to each other,” Wojciechowski explains. “This kind of application can also be used for studying interaction of various cells of immune system with platelets or other small vesicles.”
Image: The immune synapse between T cells and antigen-presenting cells. Image courtesy of David Basiji.
Platform-purchasing points
Anyone in the market for this technology should keep a list of must-have features. “When considering the purchase of an iFC platform, the most important elements include accuracy, repeatability and ease of use,” Peterson suggests. “It is also especially important that the instrument is paired with appropriate image-analysis algorithms.”
In thinking of features that a buyer should consider, Coquery picks out a few:
- hardware features: number of lasers, number of detectors, speed of image capture, computer power
- software features: user-friendliness, statistical features available, frequency of updates by vendor
- price
- size
Peterson adds two other key things to keep in mind. For laser wavelengths, he encourages buyers to note the scope and selection of excitation wavelengths. He says that it’s useful to have interchangeability of multiple magnifications. The method of analyzing a sample also matters.
Making the most of iFC’s versatility should also be considered. Lundsten mentions that a customer should get a platform that delivers any needed multiplexing. “You still want to be able to use all the same fluors or parameters as a normal flow cytometer,” she says. Plus, she notes that sensitivity and subcellular resolution limits should be considered.
An avalanche of advances
So many things keep changing in iFC technology that it could be nearly a full-time job for scientists to keep up with what is possible. Nonetheless, the sources for this article point out a few interesting improvements.
“Our newly patented oil-immersion Nano-Flow Imaging Cytometer offers the ability to detect particles as small as 50 nanometers, and image as low as 300 nanometers,” Peterson says. “Through the unique combination of oil-immersion microscopy, a blue LED, along with a higher power objective, FlowCam Nano is able to detect and image particles in the nanoparticle range.”
Ongoing interest in nanoparticles for research and applications drives the technological advances of iFC in this area. “FlowCam Nano represents an important step forward with its unique ability to image and characterize nanoparticles in real time,” Peterson explains. “The combination of fluorescent detection with the identification of nanoparticles through imaging and particle/cell morphology will provide researchers and scientists with new tools for product development, process monitoring, and quality control.”
Even with so many improvements in iFC, the technology can still improve. For instance, Basiji says, “The biggest advance that remains to be made is to produce an imaging flow sorter.”
The range of advances that keep impacting iFC makes it a challenge for scientists to keep up and know what would work best in a specific research environment. Still, it is clear when iFC comes in handy. “When you need identification and classification with morphology, you should utilize flow-imaging microscopy,” Peterson explains.