By Christian Oberdanner
With “fake news” topping the headlines these days, we’re painfully aware that hearing just part of the whole story can lead to seriously wrong ideas that can have embarrassing or even disastrous consequences. The same is true when analyzing cell populations. Every individual cell has its own story to tell, so population averages and random samples are often misleading. When running assays on a cell imaging system, microplate reader or flow cytometer, can you be sure you are getting the whole truth? If not, it may be time to consider whole-well imaging.
Can you trust your cytometry results? Whole well imaging capability on your microplate reader can help you distinguish fact from fiction.
To get a complete understanding of what’s happing in a cell culture, it makes sense to collect information from all the cells in the sample, without disrupting their behavior and complex interactions. An average intensity measurement made with a microplate reader will not tell you the whole story. Microscope images that capture only a portion of the well will not tell you the whole story. Flow cytometry of individual cells that have been stripped from the culture dish and separated from their neighbors will not tell you the whole story.
Not only can incomplete information be perilously misleading, you may also fail to notice serendipitous occurrences – like a rare interaction between cells that defies current theory, or an emerging clonal population that is expressing unusually high levels of a critical byproduct. Goodbye serendipity, Hello “fake news”!
To get the most from assays performed on adherent cell cultures, you'll want to know what is happening to each and every cell, and where it’s happening—in every well of the plate, and in context with all the other cells in the culture. Otherwise, you are not getting the most out of the sample statistically. Even more importantly, you are likely to miss valuable information about:
The bottom line is that every cell matters, because every cell is telling an important part of the story. If you capture just a few images from an entire well or rely on a population-averaged measurement, you are settling for a smaller effective sample size (read: less robust statistics) and literally throwing away valuable data!
Figure 1. Whole-well fluorescence image from a cell viability assay reveals a discrete patch of dead cells at the well periphery, where a solution containing DMSO was dispensed into the well. Analysis overlays indicate dead cells (magenta overlay) and viable cells (green overlay) detected after staining with propidium iodide and calcein-AM, respectively. Inset shows a close-up of the area demarcated by the white box at the well edge.
The answer? One of the best and least destructive tools for capturing all of the individual cell data from hundreds or even thousands of cells at a time is whole-well imaging (Figure 1). Reliable whole-well imaging, where the entire well area is captured in a single shot, has entered the mainstream only relatively recently. Even more recent is the emergence of whole-well imaging capability for multimode plate readers. Progress has been slow largely because of the technical challenges inherent in achieving sufficient resolution and definition at the well edges, without compromising too much on acquisition speed.
Fortunately, the technical obstacles are being overcome, and there are now more types of systems on the market that offer whole-well imaging. Multimode microplate readers with whole-well imaging capability are particularly powerful because they give you the freedom to use a broad range of probes and detection modalities. Of course, not all cell imaging platforms perform the task as well or as efficiently as you need them to, particularly if your work involves kinetic analysis of living cells (more on that subject in future blogs). It therefore pays to research what is available on the market and know which factors are critical to enabling high quality whole-well imaging for both fixed and live-cell assays.
If you suspect your cell-based assays are telling you only half the story, check out Tecan's new Spark® Cyto, the first live cell imaging microplate reader to offer real-time cytometry.
Learn more about the Spark Cyto live cell imaging microplate reader with real-time cytometry.