Alan Waggoner, Ph.D., professor and PI of the Technology Center for Networks and Pathways (TCNP) at Carnegie Mellon University, is part of a team that has developed a new technology that allows scientists to tune the fluorescent signal of a new class of genetically encoded biosensors. This biosensor technology can enable researchers to obtain new information about pathway regulation, and has the potential to lower background fluorescence and improve overall sensitivity compared to traditional fluorescent protein biosensors.
The approach involves single-chain antibodies, known as FAPs, or fluorescence activating proteins. FAPs are able to bind to nonfluorescent fluorogen dyes and switch them “on,” displaying a large increase in fluorescence. Researchers can use membrane-permeant or impermeant fluorogens and both spatially and temporally control the fluorescent signal. These “smart” biosensors are amenable to high-content imaging, in addition to high-throughput analyses such as flow cytometry and plate reader assays.
In cells, the process of placing and removing a membrane protein from the cell surface is complex and tightly regulated, and is important for cell function, Dr. Waggoner says. Dr. Waggoner’s research has focused on the study of GPCRs and other cell-surface molecules that can be tagged with a FAP.
Diseased cells often exhibit altered levels of cell surface proteins that may play a role in disease progression. For example, researchers have found that the transport of a surface protein, CFTR, is altered in cells from cystic fibrosis patients compared to healthy cells.
Dr. Waggoner and colleagues in the Pittsburgh TCNP and the University of New Mexico have attached a FAP to the CFTR protein and are now performing high-throughput screening assays to identify compounds that help restore normal protein transport of the CFTR protein to the cell surface. These compounds could serve as potential drug candidates to treat the disease.
Dr. Waggoner says he and his TCNP colleagues are always interested in speaking with researchers in both academia and industry to learn what the key questions are in the field that the FAP technology may be useful for addressing.
“It’s going to take years and years to figure out all the assays that people can use so that they can in detail understand the kinetics of different pathways,” Dr. Waggoner says. This detailed understanding of how cells are regulated is the first key step to being able “to understand and change disease states.”
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.