High Content Imaging and Cell Cycle Analysis

by Raymond Samuel

What is High Content Imaging? 

High content imaging, or HCI, is microscopy that is efficient, objective, and quantitative. 

It is the combination of technologies that allows efficient, objective acquisition of images, automated identification of objects in the images, extraction of information about the objects, and statistical analysis of that information. High content imaging is the convergence of light microscopy, robotics, quantitative digital image analysis, and data analysis. 

What It Lets Us Do 

High content imaging can be used for screening for non-toxic compounds or effective drug candidates. Potency can be determined by assessing toxicity point of departure or pharmaceutical effectiveness. High content imaging can also help determine a mechanism of action for hazard ID and drug classification. All these can be obtained with a high throughput, automated process. 

High Content Cell Cycle Analysis 

At ScitoVation, one application of high content imaging is cell cycle analysis. Proliferating cells cycle through G1, S, G2, and M phases, and this cycle is highly regulated by growth factors, stress responses, and perturbations to the machinery of DNA replication and chromosome segregation. 

Cell cycle arrest can explain reductions in live cell counts in the absence of toxicity.  It can be a sensitive indicator of perturbations to cell health.  The specific nature of the arrest, for example in which phase cells arrest, may shed light on the nature of the perturbation. 

Cells actively replicate their DNA in S-phase.  EdU is a thymidine analog that can be spiked into cell culture medium and which will be incorporated into the DNA during replication.  It can be fluorescently labeled through “click” chemistry after fixation and permeabilization of cells.  Cells that are positive for EdU labeling can be classified as S-phase. 

In cells in M-phase (mitosis), Histone H3 is phosphorylated at serine 10 by the Aurora-B mitotic kinase.  This post-translational modification may be detected by immunofluorescence.  Cells positive for phospho-Histone H3 labeling can be classified as M-phase.

Cells not in M-phase or S-phase can be classified into G1 or G2 phases by DNA content, which is indicated by the intensity of DAPI staining of the DNA. 

At ScitoVation, we are using new approaches and technologies to provide more sound answers. Cell cycle analysis using high content imaging provides insight into perturbations to cell health in an efficient and specific manner. This method, along with many other innovations are what makes ScitoVation pioneers in chemical safety assessment.  

Raymond Samuel: rsamuel@scitovation.com