At PerkinElmer, Louise Armstrong, Ph.D., market development director, is part of a team that is working on the development of improved imaging technologies to meet researchers’ live-cell imaging needs.
Photobleaching has long been a problem for the field of fluorescence imaging. During image acquisition, the same light that is used to cause fluorescence also causes fluorophores to become bleached and no longer emit a signal.
There are two primary directions that researchers have turned in an effort to minimize photobleaching, Dr. Armstrong says.
Some choose to forgo the higher quality images obtained with laser scanning confocal microscopy, and instead acquire images and movies with a standard fluorescence microscope, which uses a less intense light source and reduces the rate of photobleaching.
Others have looked to spinning disk confocal microscopy, which combines a confocal microscope with a pair of spinning disks that splits the laser beam into many points across the specimen. By splitting the beam, researchers can obtain a sharp image across the entire field of view, instead of building up the image point-by-point as with traditional confocal microscopy.
The spinning disk approach significantly reduces and can even eliminate photobleaching, Dr. Armstrong explains. It also reduces the time to acquire images, which makes it possible to perform imaging at a fast rate over short time scales, or have longer time intervals between images over a time scale of multiple days. Prior to multipoint confocal microscopy, imaging over multiple days was not feasible because the fluorophores would become entirely bleached.
The key to performing live-cell imaging is optimizing experimental conditions such that the minimum amount of light is used, Dr. Armstrong says. “It’s all about getting as much signal as possible without photobleaching.”
While real-time two-dimensional live-cell imaging is a step up over simple snapshots of cellular events, Dr. Armstrong says that three-dimensional live-cell imaging will be the way of the future.
“In the last few years, technology has advanced enough that it’s now possible to generate 3D images and to manage the large amounts of data that this creates,” Dr. Armstrong notes. The cameras are fast enough, photobleaching is managed, and with the right software, two-dimensional image slices can be reconstructed to give quantifiable three-dimensional models of cellular processes changing over time.
These advancements, in conjunction with new developments in the field of three-dimensional cell culture, make 3D live-cell imaging an expanding field with lots of room for growth, Dr. Armstrong says.