GEN What are the gaps in cellular imaging reagents, and are label-free methods making an impact?
Evans: There is still a lot of room for improvement in the live-cell area. There are companies that are engineering cell lines with reporters or markers of interest that are expressed stably, so you don’t have to add antibodies or live-cell dyes, which helps a lot, as long as the cell line has been validated.
There is definitely room for improvement in the performance and variety of available live cell dyes. With image analysis, there’s some catching up to do to deal with that multiplicity of labels. So it’s sort of a chicken-and-egg situation where the multispectral aspects of the reagents haven’t been pushed because the analysis capability hasn’t been there to cope with it.
The live-cell dyes or the cells that are engineered with fluorescent proteins are the biggest gap that I see. There’s also some development of multifunctional reporters for not just protein species, but also forces, things that will actually report traction by the cell and look at interactions between cells, with a matrix or with their neighbors, and actually have a readout. There are also calcium sensors, of course, which are the most prevalent nonprotein analyses that have been used primarily in industry.
von Leoprechting: Live-cell imaging will be mainly driven by label-free approaches and innovations in FRET-type reagents for live-cell imaging. The big problem with fluorescent probes is the phototoxicity over longer periods of exposure. This is where, from the reagent development side, new dyes that have higher quantum yields and improved linkers are of interest. For the same reason, spinning disk confocal systems already allow you to minimize the toxic impact of the light energy the cell is exposed to. In HCS our observation is that for live-cell assays label-free techniques like texture analysis as well as 3-D/4-D features become more important.