Microfluidic Imaging Flow Cytometry – Challenges and Developments

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Flow cytometry (FC) is an indispensable technique for studying cells derived from tissues and cell cultures. FC uses optical measurements to count and distinguish different cells from a population, thus enabling studies on cell differentiation and growth of cells in a mixed culture.

flow cytometryImage Credit: Jarun Ontakrai / Shutterstock.com

While standard FC devices combine already multiple optical measurements for cell characterization, recent advancements in microfluidic imaging flow cytometry (IFC) offer wider analyzes by combining standard FC with microscopy.

These developments have recently been reviewed by Swiss and South Korean scientists, led by Professors Andrew deMello and Jaebum Choo, in the journal Current Opinion in Biotechnology.

Cells within a laboratory cell culture usually show considerable heterogeneity in terms of cellular differentiation, cell cycle state, and metabolic activity, even if a culture is derived from a single cell type. This heterogeneity is important in scientific studies as it bears the potential that cells might, for instance, react differently to drugs or toxins.

Furthermore, in whole tissues, multiple cell types are found, but a drug will only target a particular cell type. To thus analyze a single cell type in a mixture of cells, cytometric methods are a prerequisite.

Principles of flow cytometry

Flow cytometry has, therefore, become a common technique in cell culture analysis. In FC, a population of cells is passed one cell after the other through a nozzle, so that individual cells can be analyzed. After coming from the nozzle, cells pass through an optical measurement system that firstly determines their forward scatter (i.e., light absorbance) and side scatter (i.e., light scattering).

The scattering intensity relates to the cell’s size and composition. In order to identify different cell types, common FC approaches include immunofluorescent cell labeling, where antibodies linked to a fluorescent molecule may bind to cell-type-specific surface proteins. The resulting fluorescence can then be read out by a suitable optical system in the FC device.

While these sophisticated, established FC applications already collect multiparametric information, they are error-prone as unspecific aggregates and contaminations can produce scatter- and fluorescence-signals that are indistinguishable from actual cells.

Microscopy-integration in imaging flow cytometry

In order to facilitate the differentiation of actual cells from debris, microscope systems have been used in microfluidic imaging flow cytometry (IFC). By using wide-field cameras, a whole focal plane with multiple, parallel channels containing individualized cells can be monitored simultaneously.

With modern computational image analysis, IFC provides information of the cell shape and structure as cytometry parameter, in addition to all scattering and fluorescence information provided in conventional FC. However, these microfluidic setups have challenges associated.

Challenges in IFC

In microfluidic devices, efficient and robust flow paths are key. For instance, a microfluidic flow cell shall enable the distribution of a cell mixture into individual analysis channels, without destroying cells by shear forces or blocking the flow path with cell aggregates.

The review authors summarize that this has been achieved with several, very different flow path designs. Whether there is one optimal design or different flow paths for cell individualization might have advantages for different cell types, something which might only be revealed in the future.

A second key aspect for IFC is the selection of imaging cameras, which may differ in speed of image collection, spatial resolution, and wavelength sensitivity. However, tremendous improvements in microscope camera technologies have been achieved in the past, and IFC as a microscopy technique can integrate these.

With parallel flow channels for cell analysis, scientists have been reporting analysis rates beyond the 50,000 cells per second in conventional FC. Thus, IFC is already competitive with FC in terms of analysis rate but provides superior depth of information on the analyzed cells.

Future advancements in IFC – what to expect

As the review authors note, computational image analysis and machine learning are developing at a high pace, and will certainly drive IFC by accelerating computational image analysis. On the optical side, first studies have used volumetric confocal microscopy, where not only a single focal image plane is recorded by microscopy, but multiple focal slices through a whole volume of a sample.

The application of volumetric microscopy poses additional demands to the camera speed but also increases the amount of data recorded per cell, making fast computational image analysis even more important. After all, these developments make it likely that future flow cytometry will be able to assess individual cells from a mixed population in a comprehensive way.


Stavrakis S et al. High-throughput microfluidic imaging flow cytometry. Current Opinion in Biotechnology 2019, 55, 36-43; DOI: 10.1016/j.copbio.2018.08.002.

Further Reading

Last Updated: Nov 13, 2019

Dr. Christian Zerfaß

Written by

Dr. Christian Zerfaß

Christian is an enthusiastic life scientist who wants to understand the world around us. He was awarded a Ph.D. in Protein Biochemistry from Johannes Gutenberg University in Mainz, Germany, in 2015, after which he moved to Warwick University in the UK to become a post-doctoral researcher in Synthetic Biology.


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