Flow cytometry is an extremely common laboratory technique that is used to measure phenotype at a single-cellular level. It’s a high-throughput technique that can quantitatively assess multiple parameters in a population of cells within a short time. The catch is, regular flow cytometry does not provide any image data. As a result, it is incapable of providing insights into cellular morphology and the localization of cellular components.
To get this kind of data, immunofluorescence microscopy can be used instead of flow cytometry. But microscopy is much lower throughput, and the analysis and classification of images can be subjective and error-prone. In addition, the analysis of rare or non-adherent cells can be challenging. For questions that require ample image data from a large population of heterogeneous cells, microscopy is insufficient.
For many experiments, regular flow cytometry is all you need. Identifying specific cellular markers or quantifying their levels can sometimes be enough to distinguish between cell types in a population or to answer other biological questions. However, this isn’t always the case. Some research questions can only be answered with location data. Take, for example, two cell populations for which the difference between them is not the presence of the marker but whether that marker is inside of or outside of the nucleus. Regular flow cytometry could not distinguish between these two populations, but imaging flow cytometry can.
This article will look at three specific applications of imaging flow cytometry (that were the focus of a recent webinar) performed by Ziv Porat of the Weizmann Institute of Science and his colleagues. Using imaging flow cytometry, these researchers were able to investigate Golgi fragmentation in response to physiological and pathological cellular processes, senescent cell presence and characteristics in aging and disease, and the kinetics of the Mimivirus infection stages.
Golgi fragmentation
The Golgi apparatus is a key cellular organelle for protein modification, vesicular transport, and lipid biosynthesis. In the past, electron microscopy has suggested that the Golgi is static, but we now know that this is not true. In fact, Golgi morphology changes in response to normal physiological processes such as mitosis, apoptosis, and migration as well as pathological processes such as cancer and neurological diseases. Unfortunately, high-throughput study of Golgi morphology has been difficult with the currently available methods.
“The goal of this study was to develop a method to rapidly and efficiently quantify changes in Golgi morphology in a high-throughput, unbiased, and automated manner,” Porat says. “In regular flow cytometry, the Golgi morphology cannot really be quantified, and in immunofluorescence, the number of fluorescent channels is more limited. Additionally, immunofluorescence takes more time to collect cells, is more complicated to quantify, and is difficult to use with rare populations and difficult-to-image cells.”
Using imaging flow cytometry, the researchers were able to detect and quantify changes in the Golgi structure under various circumstances in a non-subjective manner at a rate of up to 5,000 cells per second.
Senescent cell quantification
Cellular senescence is a cell state that occurs in response to persistent DNA damage. It’s important in such processes as development, aging, cancer, and wound healing. However, the identification, quantification, and characterization of these cells have proven to be difficult because the currently available methods are laborious, expensive, and unable to give a quantitative evaluation of senescent cells.
“The reagent normally used to identify senescent cells—X-gal—is not fluorescent; hence, it cannot be used by flow cytometry,” Porat says. “And the fluorescent reagent that can be used for flow cytometry can only be used in intact cells, which does not allow for intracellular staining. In immunofluorescence, this reagent is difficult to quantify and cannot be used with additional markers. Additionally, it is much more difficult to image and quantify non-adherent cells or rare populations with this reagent, and the image analysis is complicated and takes a lot of time.”
According to Porat, their goal for this experiment was to develop a method that would “quantify the senescent phenotype of cells and combine it with additional features and markers in vitro and in vivo.” They used imaging flow cytometry to combine the high-throughput and quantitative abilities of flow cytometry with the high-content image analysis possible with immunofluorescence. By modifying the traditional SA-β-gal assay to work with their ImageStreamX imaging flow cytometer, they were able to effectively identify, quantify, and characterize senescence cells in various tissue types.
Stages of Mimivirus infection
Viral infection can be tricky to study given the heterogeneity of both viruses and their hosts. In order to achieve a more comprehensive view of the process, it is necessary to analyze a large population of cells. Mimivirus is a genus of large DNA viruses that form viral factories in the host cytoplasm for viral replication and assembly. Their DNA encodes over 1,000 proteins.
The aim of this study was to develop a high-throughput method to quantify in detail the infection cycle of Mimivirus. “Flow cytometry cannot quantify morphological features, and immunofluorescence takes a huge amount of time and effort to achieve a large enough magnitude of data,” Porat says. “Imaging flow cytometry, on the other hand, allows robust, high-throughput, automated, and unbiased quantification of morphological features.”
Using imaging flow cytometry, the researchers were able to monitor virally induced processes such as the generation of virus factories, viral transport, viral progeny accumulation, changes in cellular morphology, etc. They determined time frames for each under normal circumstances and also under circumstances such as cytoskeletal disruption and oxidative stress.
The takeaway
Imaging flow cytometry is a powerful tool that combines the detailed imaging data from immunofluorescence with the high-throughput quantitative abilities of flow cytometry. It has made it possible to acquire data for research questions requiring morphological information in a high-throughput fashion. While this article looked specifically at Golgi fragmentation, senescent cell quantification, and stages of viral infection, there are countless other research questions that can now be pursued using imaging flow cytometry.