Painting Cells Like an Artist: Rendering Color to the Cellular Realm
Gaining novel insights into cellular morphology and mechanics
Cell imaging involves the use of microscopy to better understand cellular dynamics and the role they play in biological function. A wealth of information can be gleaned about pathogen biology, physiological processes, and conditions by monitoring and observing cells under a microscope. Various microscopy techniques have been developed over the last several decades enabling researchers to study living cells and cellular processes in great detail. Phenotypic screening and morphological profiling are two such methods.
Phenotypic screening is a powerful tool utilized to identify novel small molecules or genetic perturbations that have potential as probes, therapeutic agents, or genetic regulators for various biological processes. High-throughput microscopy is commonly used in phenotypic screening. It is also referred to as high-content analysis (HCA), a process where automated microscopy is combined with multi-parametric analysis to provide rich and detailed data of cellular phenomena. Unlike traditional imaging techniques such as fluorescence imaging which is time-consuming and laborious when it comes to quantifying essential cellular processes, HCA platforms provide an automated workflow that is not only affordable but far more efficient for researchers as a tool to handle large sample sizes while delivering robust and reproducible experimental results. However, large-scale imaging experiments as observed in HCA typically focus on the extraction of only a handful of cellular features, leaving behind a large amount of quantitative data unexplored.
Morphological profiling offers a solution to this dilemma by quantifying a very large set of features, in the hundreds to thousands range, for a given sample in an unbiased approach. This makes way for the characterization of a sample’s condition through the identification of significant change within a subset of profiled features. In this manner, profiling avoids selective customization in the imaging of a sample and is far more generalizable as a method where only a single experimental run is necessary to identify various biological processes or pathogens of interest. With single-cell resolution, morphological profiling has been used successfully to characterize genes and compounds in numerous studies.
Cell painting assay
Cell painting utilizes morphological profiling in action in what is a high-content, multiplexed-image-based assay for cytological profiling. A generalizable method, cell painting helps researchers access information on the various features of a cell. Six fluorescent dyes are used to “paint” the different components of the cell including the nucleus, endoplasmic reticulum, mitochondria, cytoskeleton, Golgi apparatus, cytoplasm/plasma membrane, and RNA in an effort to capture a representative phenotypic state of the whole cell. Automated image analysis software is then utilized to extract measurements such as intensity, texture, shape, size, and proximity of an object to its neighboring structure, an indication of the location of organelles relative to one another, from 100 to 2000+ measurements per cell. Altogether, these measurements help reveal the biological state, or phenotypic profile, of the cell in a single assay that is compatible with standard high-throughput microscopes.
Although there are various other methods to generate high-resolution profiles of biological samples, cell painting, as a morphological profiling method, provides profiles at the levels of individual cells with the ability to resolve changes in subpopulations of cells. The choice of dyes, as opposed to antibodies, to “paint” the cell also makes the assay ideal for large-scale experiments especially considering factors such as cost and complexity. The adaptability of the cell painting assay to image living cells by using live-cell compatible stains or fluorescently tagged proteins adds to the method’s versatility.
This is further supported by the myriad applications where the cell painting assay has been a powerful resource. For example, matching unannotated genes to known genes based on similar phenotypic profiles as characterized by the assay can help in identifying their biological functions via profile similarities among genetic perturbations. The opposing limit of overexpressing variant alleles helps distinguish the functional impact of genetic variants by comparing profiles that are induced by wild-type and variant versions of the original gene. Cell painting profiles also help in identifying diseases according to their phenotypic signature, can serve as a screening tool to revert said signature back to “wild-type”, and also help cluster small molecules and identify which of them yield similar phenotypic effects. In the case of the latter, morphological profiles generated via cell painting pave the way toward a more efficient screening technique with minimal phenotypic redundancy.
Advancing image analysis—The Thermo Fisher Scientific story
Cell painting is fast emerging as a valuable tool for many important life-science applications, particularly, drug discovery. Moving forward, it has become imperative to make this tool as accessible as possible to the scientific community. On this front, there is a broad platform of tools and technology from Thermo Fisher Scientific that provide researchers with what they require for their cell imaging needs. In an effort to democratize cell painting, there is a rich history in cell imaging at Thermo Fisher Scientific evidenced by the development of innovative cell imaging platforms, software, and reagent kits.
Via this platform, the goal has been to enhance cell painting research, achieve greater clarity, and assist in groundbreaking discovery. This is enabled by the availability of ready-made cell painting reagents kits, such as the Image-iT Cell Painting kit, and high-content imaging systems in CellInsight systems from Thermo Fisher Scientific that help characterize the intricate morphologies of cells. The ready-made cell painting kits simplify cell painting workflows and remove the hassle of staining protocols. Dyes such as the Alexa Fluor line provide fluorescence of great brightness and photostability thus allowing high-contrast discrimination of stained samples, while overall being superior to antibody staining. In what is a convolution of multi-parameter microscopy with multiplexable fluorescent reagents, and supplementary software such as Studio 5.0 Cell Analysis, cell imaging technologies and their adaptation for cell painting assays from Thermo Fisher Scientific have set the standard, and can be used to identify and measure the individual features of cells within the field of view.
Beyond the qualitative mix of art and science that is cell painting, HCS and HCA systems from Thermo Fisher Scientific also enable quantitative analysis of cell painting images further facilitated by a one-click cell painting mode/module in the supplementary software. The CellInsight systems alongside Studio 5.0 Cell Analysis Software and the Image-iT Cell painting kit provide for multiparameter cell painting to promote greater insights and results in experiments surrounding drug discovery and understanding phenotypic change at the cellular level. The ultra-fast time-to-data and the possibility to conduct imaging and analysis in parallel provide researchers with a simplified cell painting workflow that is both efficient and rigorous. Together, these tools from Thermo Fisher Scientific make cell painting more accessible than it has ever been in the past, lending further to democratizing cell painting research techniques.
Cellular imaging in the form of cell painting combines the fields of computational analysis and cell biology toward gaining a deeper understanding of cellular processes in efforts toward novel developments in drugs, identifying potential therapeutics, investigating gene function, and assessing environmental toxicants. Since its inception, cell painting has become the standard for phenotypic drug discovery and safety application. The assay, in and of itself, is not easy and comes with its fair share of challenges and they are met by the range of tools from appropriate labeling reagents, to optimized instrumentation for detection, and supplementary analysis software from Thermo Fisher Scientific in an automated and simplified workflow process that helps make sense of the thousands of features generated during profile analysis. This central library of resources from Thermo Fisher Scientific helps researchers access what they require for their cell painting assays, the resultant data analysis, and the acceleration of groundbreaking research.
Take advantage of our 40+ years of experience and expertise with end-to-end cell images products and resources, including relevant tools, protocols, selection guides, inspiration, and more at your fingertips. To learn more about cellular imaging and related technologies, visit the Thermo Fisher Scientific imaging resource center: www.thermofisher.com/cellularimaging.