Label-Free Assays in High Content Imaging

Label-free screening relies on acquisition and analysis of bright-field images of live cells having no exogenous labeling. Collecting images of unlabeled cells over time is a powerful tool for the study cellular processes or responses in an unperturbed fashion.
This blog post will provide a brief introduction to label-free, brightfield imaging for live cell analysis, with a spotlight on assays supported by the Hermes imaging system and WiSoft Athena analysis software.

What is label-free screening in high content imaging?

High content imaging relies on a computer-controlled microscope to autonomously acquire images of biological samples treated with a library of test compounds.  High-content screening is the simultaneous collection and quantification of multiple parameters from the acquired images.  The methodology typically employs fluorescence labeling and high-resolution imaging of fluorescently labeled cells.
However, high-content imaging is not limited to the use of fluorescence for cellular imaging and detection. Brightfield images of unstained samples enable inspection of cellular health and behavior that is compatible with assays intended to screen compound libraries.  Label free assays are used when samples being studied need to be free from external genetic or chemical manipulation.

Why is label-free high-content imaging necessary?

When applied in live cell experiments, fluorescence stains incubated over long periods of time can cause cytotoxicity or alter cellular function and homeostasis.  Genetically encoded fluorescent proteins that benefit from specific labelling within cells require transfection or cell-line creation, and must be ensured not to interfere with protein function.  Additionally, overlapping excitation and emission spectra of fluorescent dyes limit the number of channels available for imaging. 7 Moreover, sample labeling prior to imaging can have long-duration protocols and increase experimental costs as some dyes are quite expensive.
Label-free high-content imaging enables researchers to investigate live cells utilizing non-invasive and non-toxic techniques with no need to carry out sample labelling preparations.  Specifically, primary cells obtained from donors are a particular sample type that benefits from the use of label-free methodologies.
In the most straightforward implementation, bright field imaging is performed using transmission illumination with white light. Many standard microscopes use xenon light sources for such imaging, while light-emitting diode (LED) illumination sources are commonly used in compact and enclosed HCS microscopes.
Valuable information can be extracted from the acquired brightfield images, even without contrast enhancement optics, such as DIC or phase contrast microscopy. For instance, they can be used to perform segmentation of cells and colonies, which is useful for common assays such as proliferation or migration assays. However, the segmentation of brightfield images is challenging, especially for detecting single cells, since the overall intensity of the background is approximately the same as the intensity of the cell.
IDEA Bio-Medical offers advanced solutions to overcome this inherent optical challenge and support various label-free screening assays.  We apply digital software-based tools for contrast improvement of images acquired in bright field illumination. Such enhancement alongside unique image analysis algorithms, including deep learning artificial intelligence approaches, to allow accurate identification and segmentation of whole organisms, cells and objects in bright field images in a fully automated fashion.  A few of the label-free assays supported by Hermes high content imaging system and its accompanying Athena image analysis software are described below.

Clonogenic assay / colony formation assay

The Clonogenic assay is an in-vitro cell survival assay based on the ability of a single cell to grow into a colony. This application allows label-free cell colony detection providing true single-cell count per colony. Clonogenic activity is a sensitive indicator of undifferentiated cancer stem cells, with the assay testing every cell in the population for its ability to undergo “unlimited” division.  This assay can also measure effects of compounds requiring cells to undergo several rounds of replication.
Traditionally, the clonogenic assay is an end-point assay, with the unlabeled cells being fixed and stained with crystal violet for visual inspection.  Thus, study of the same colonies over time is not possible.  Analysis is subjective because the number of colonies are counted manually and the plates themselves must be stored if re-analysis is required.
The automated application within the Athena software package identifies individual cells in brightfield images, then merges them to colonies.  The results provide a count of the number of colonies present, while also reporting their morphometric features such as size and shape.  Uniquely, the application provides a true cell density measurement since the detection of colonies relies on identification of individual cells.
Live-cell imaging on the Hermes enables time-lapse acquisition to follow the behavior of individual colonies over time without fixation.  Moreover, because the images of the sample are digitized, there is no need to store the plates for a later time as the data can readily be re-analyzed.
Figure 1: Clonogenic assay / colony formation assay by Athena software

In-Vivo Zebrafish screening

Zebrafish are a model organism whose use in toxicology and other screening studies is steadily increasing.  An application exclusive to IDEA Bio-Medical within Athena automatically detects zebrafish contour and their internal organs in brightfield with no required user input.  The anatomy identified is coupled to fluorescence channels to permit anatomy-specific study of fluorescence changes.
For more information, check our post about Zebrafish to learn more about this up-coming model organism and IDEA Bio-Medical’s affordable, user-friendly Hermes Zebrafish Partner as a unique solution for in vivo, image-based zebrafish screening.
Figure 2: AI-powered Zebrafish Detection. Fish, organs & regions identified automatically by Athena deep-learning based software. Visit https://idea-bio.com/all-applications/application-zebrafish-in-vivo-assays/ to learn more.

Confluency assay

This assay enables quality control of cell culture by analysing the confluency of cell monolayers. It performs automated measurement of confluency using detection of area covered by cells and calculate the fraction of this area from the total scanned area.
Figure 3: Confluency assay demonstrated with Athena software analysis. left image shows 29% confluency while right image shows 68% confluency.

Wound Healing/ Scratch Assay Athena application

Scratch assay, or wound healing assay, is used to study cells motility by measuring their speed and motion while closing a scratch made in a confluent sample. Measuring cell motility properties has implications for cancer and metastasis studies, chemo-taxis, embryonic development and more. This application measures the size of a scratch or a wound in cultured cells at different time points, while the cells are migrating to close the gap of the scratch.
Label-free high imaging is a quick and affordable technology with unrealized promise. Research specific events like drug response, treatment resistance, and differentiation in an undisturbed, natural environment are crucial.

Are you interested in high-content imaging products? Contact a member of the IDEA Bio team today to request a demo of any of our systems.
Discover all of our high content Imaging products and applications.

references

  1. Kasprowicz, R., et al. (2017). Characterizing live cell behaviour: Traditional label-free and quantitative phase imaging approaches. Int J Biochem Cell Biol. doi: 10.1016/j.biocel.2017.01.004.
  2. Wojcik K, Dobrucki JW. (2008). Interaction of a DNA intercalator DRAQ5, and a minor groove binder SYTO17, with chromatin in live cells-influence on chromatin organization and histone-DNA interactions. Cytometry A. doi: 10.1002/cyto.a.20573
  3. Blasi T., et al. (2016) Label-free cell cycle analysis for high-throughput imaging flow cytometry. Nat. Commun. doi: 10.1038/ncomms10256
  4. Kang J., et al. (2016). Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines. Nat. Biotechnol. doi: 10.1038/nbt.3419
  5. Martin, J (2010). Label-free imaging and temporal signature in phenotypic cellular assays: a new approach to high-content screening. Curr Protoc Pharmacol. DOI: 1002/0471141755.ph0913s50
  6. Wang, S., et al. (2020). Label-free optical imaging in developmental biology. Biomed Opt Express. doi: 10.1364/BOE.381359
  7. Jyrki Selinummi et al., 2009, Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images. PLOS ONE https://doi.org/10.1371/journal.pone.0007497