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The Importance of Cell Tracking and What It Teaches Us

How do we track cellular heterogeneity?

Cellular heterogeneity essentially observes all dimensions of a single cell. Heterogeneity tracking helps us to measure the functionality of each cell. An example of its common use is to track cancer patients’ cell or tumor growth or decrease over time.

Investigating various types of cells and their complex physiological functions has led us to some important insights. To decipher the mechanisms and functions of heterogeneous cell types, monitoring them is key.

By monitoring cells in real-time (known as the cell tracking method), we track the cell’s progress to be able to treat it successfully.

How has cell imaging changed over time?

The first in vivo imaging’ (non-invasive imaging) was reported way back in 18391. Skip forward over 180 years, and imaging such as microscopy has evolved vastly and it has never been more high-resolution.

Both confocal and multi-photon microscopy is widely used for imaging and cell tracking. The evolution and development of these imaging forms have meant that light is no longer limiting our results. This enables us to focus on important areas of cells and is known as diffraction.

Using cell tracking in cancer treatment

Cell tracking and live imaging allow us to obtain real-time dynamic data to capture the evolution of cell-to-cell differences. This technique then enables us to see the living cell’s cellular process over time.


Looking for an automated live-cell microscope? The CytoSMART Omni allows for multi-well imaging for hours up to weeks at a time.


The imaging of live cells and tissues is now a common technique used in research. Measurements of viable cells over time ensures that we can provide a much more complete analysis of the biological process of cells2-5.

During cell tracking, cells are:

  • Kept in focus
  • Continually measured for several days
  • Maintained at 37°C and 5% CO26
  • Proliferation
  • Apoptosis
  • Invasiveness
  • Angiogenesis
  • Drug response
  • Drug metabolism
  • Ameboid-like
  • Mesenchymal mode of single-cell migration
  • Collective migration8,10,11
  • New biomarkers of disease
  • Drug targets
  • Drug mechanism of activity at the cellular to the molecular level12
  • Endogenous reporters - representing fluorescent proteins that are produced by the cells that have to be followed
  • Exogenous probes - interact with the targeted cells14

Where cancerous cells are present, we can monitor behavior or cell population and reconstruct trajectories to treat it in the best way.

In this instance, cell tracking has a key role in investigating cancer cells' potential and invasiveness. Once these are identified, we then have the opportunity to control the disease. It also allows us to monitor whether environmental conditions affect cell migration.

The tracking of cancer cells can also give us more accurate answers about molecular mechanisms of:

The benefits of monitoring single-cell behavior

Monitoring the behavior of a single cancer cell allows us to follow and analyze individual cells. This gives us the possibility to determine the sub-population of the cells with different migratory or proliferative potential. This, in turn, allows us to determine different responses to any given treatment.

Bolgioni et al. developed the live-cell imaging protocol for monitoring the mitotically arrested cells after treatment with an anti-mitotic drug called Paclitaxel. Their method now allows us to decide whether cells die or slip back into interphase.

It is important to detect if the cells evade mitotic death by slipping into interphase because interphase cells can execute apoptosis, undergo cell cycle arrest, and even re-enter the cell cycle. Since mitotic cells are tetraploid, they can drive tumor relapse if they slip into interphase because of gained chromosome instability.

With live-cell imaging, we can detect factors and mechanisms or even control the cell fate post-treatment, which is critical for optimizing the patient's therapies.

Investigating cell changes with cell tracking

By tracking cells, we can monitor the changes in morphological features, which happen during different processes or conditions.

During metastasis, cancer cells experience morphological changes that are characteristic of different types of migration patterns7. These include:

Using both two-dimensional (2D) and three-dimensional (3D) spheroids and organoids, we can investigate changes associated with the cell’s environment.

This tracking of 2D and 3D cultures can give us important information about drug activity mechanisms to measure its success or failures8.

The importance of labelling in cell tracking

When tracking live imagine, cells can be targeted by direct labelling, or transfection with a conjugated (the transfer of DNA between cells, usually bacteria, by cell-to-cell contact) marker gene.

High-resolution imaging and genetically encoded fluorescent probes allow us to monitor and understand cell metabolism and signalling interactions. It also enables us to visualize single molecules in living cells9.

As well as this, we can identify:

The combination of the molecule-specific contrast and live-cell imaging technique makes fluorescent microscopy the most popular imaging approach in cell biology research13.

Fluorophores that can be used for fluorescence-based microscopy are divided into two categories:

The two fluorophores have in common that they should be non-toxic, photostable, and sufficiently bright.

Long-term and short-term cell tracking

Experimental cells can also be labelled with dyes that provide long-term or short-term cell tracking.

Long-term cell tracking allows for prolonged monitoring of the same cell and its behavior. The main limitation of long-term tracking is that fluorescent proteins or reporters often fail to give a strong signal.

Long-term cell tracking also often has non-efficient detection, which can be overcome using other techniques, such as transfection or transduction techniques and ex vivo manipulations14,15.

For short-term cell tracking, cytoplasmic and nuclear dyes are usually used. These dyes then penetrate cells, metabolizing to the products that bind components from the cytoplasm16,17.

The downfalls of short-term dyes mean they usually have high cytotoxicity, and their fluorescence also decays rapidly18.

Labelled cell monitoring can sometimes limit the length of time you can conduct cell tracking19,20. With these findings, there was an increased need to develop a label-free quantitative method of cell tracking21.

This inspired Digital Holographic Microscopy (DHM) - a method that represents the non-phytotoxic technique, which allows long-term imaging22. Images obtained from DHM have pixel intensities proportional to the absolute phase shifts of the cell sample23,24,25,26.

Processing your image analysis results

As we know, image analysis is a multi-step process that begins with low-level pre-processing. A large number of images can be summarised into a few numerical descriptors.

Although cell tracking is an extremely powerful method for investigating cell behavior on different levels, it must be combined with other methods and approaches to thoroughly and successfully characterize and investigate the process.

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