Segall also employs intravitral imaging, specifically multiphoton fluorescence microscopy, to directly witness migration as it happens. Multiphoton microscopy is well suited to intravital imaging because it uses relatively low power excitation and is able to penetrate deeper into tissue than standard confocal microscopy.
In this case, Segall and his collaborators image the tumor surface through a surgical flap or glass “window,” visualizing motion with the help of genetically encoded fluorescent tags — for instance, collecting time-lapse images as red macrophages enter and leave a cyan fluorescent protein-labeled tumor. More recently, they have adopted photoconvertible proteins, fluorescent proteins (such as Dendra) that fluoresce at one wavelength until excited by light of a specific energy, causing them then to emit a different color.
With that tool, Segall and his team can effectively time-stamp proteins to distinguish those that are newly synthesized from those that are not. He hopes to use them to study the trafficking patterns of tumor macrophages in transgenic animals — where they come from, how long they stay, and where they go.
“Now that we have photoconvertible proteins, we'll be able to track the macrophages over days and see what they do,” he says.Imaging Etc.
Time-lapse imaging is key for researchers studying cell migration in vitro, too. Josh Rappoport, co-director of the Birmingham Advanced Light Microscopy facility at the University of Birmingham, UK, uses the technique to study the impact of endocytosis on cell migration in cancer cell lines in the lab.
Several years ago, Rappoport, unsatisfied with the in vitro migration and chemotaxis assays available at the time developed a low-tech solution. Published in BioTechniques in 2010, his approach first spots chemoattractant-laced low-melting-point agarose into tissue culture dishes, after which cells are plated and watched as they “invade” the droplets — or, more accurately, burrow underneath them.
Rappoport describes the event in terms reminiscent of a college student with a bad case of the munchies. “Imagine if there was a giant pile of snow in your backyard, and somebody put a pizza underneath the pile of snow,” he says. “So you lay down on the ground and you crawl underneath the pile of snow to get to the pizza.”
He images that process with a mixture of techniques including brightfield, epifluorescence, confocal, and total internal reflection fluorescence microscopy, as well as scanning electron microscopy, quantifying both the number of cells to penetrate a chemoattractant-filled droplet, and the distance they travel.
The assay does have one significant drawback, Rappoport says: It isn't easy to quantify the chemical gradient at any location in the culture dish. Still, he hopes to use it to understand vesicle trafficking in migrating cells. Preliminary data suggest that blocking trafficking pathways can inhibit chemotaxis, an observation with potential therapeutic implications.
Indeed, studies of cell migration can often yield unexpected insights into fundamental biological processes. In February, Lawrence Berkeley National Laboratory investigator Mina Bissell, an expert in tumor microenvironments, used time-lapse confocal microscopy to demonstrate that mammary gland epithelial cells organize themselves into acini by spinning, a “unique cellular movement” they called “coherent angular motion,” which appears to be disrupted in cancerous cells.
That observation could never have been made without the proper tools, but someone also had to be watching. And with more and more researchers doing exactly that, expect other exciting – and unexpected – findings to follow.