Label-Free Assays in High Content Imaging

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.