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NASHVILLE, Tenn. – Cells aren’t flat, but normal microscopy images are. Researchers work around this by sequentially collecting images focused at different depths and combining them into a three-dimensional representation. However, this approach requires relatively expensive equipment.
Now a group from Vanderbilt University has demonstrated an inexpensive technique based on mirrored pyramidal wells to image cells from multiple vantage points simultaneously. The scheme could prove useful in a number of ways, said team member and assistant professor of biological sciences Christopher J. Janetopoulos.
Centromere movement in a dividing cell is viewed simultaneously from the left to right with a mirrored pyramidal well.
For example, Janetopoulos noted that single-molecule detection sometimes is difficult because it is hard to distinguish the signal from noise. However, a source would appear in the same spot when viewed from another location, while noise most likely would not. He added that this would be especially clear for a moving source viewed from different perspectives. “You can track it moving around, and you know for sure that it’s a real signal.”
In building the wells, the group used long-established silicon-processing methods to create what looks like inverted pyramids with sides that slope inward at 54.7°. When an object is placed inside a well, it can be viewed simultaneously from five directions: the top and four others at nearly right angles. Not only does this provide various perspectives, but it also theoretically should be five times as efficient in collecting light, which could be important in bioluminescence and other low-light-imaging situations.
This arrangement also means that it is possible to illuminate cells from one side while viewing them from the top or from another side. Team member and fabrication facility manager Ronald S. Reiserer said that side illumination can improve contrast. Birefringent coatings, which are easy to implement, also can be used, making it possible to investigate fluorescence, reflectance and absorption at variable wavelengths.
Fabricated on a piece of silicon smaller than a dime, an array of mirrored pyramidal wells, each only microns across, allows researchers to see an object from different vantage points simultaneously. Images courtesy of Vanderbilt University.
The size of the wells at the surface can range from a few to several hundred microns and, thus, can be custom-sized for a particular specimen. They also can be produced in customized layouts. The wells could be inexpensive tools for 3-D visualization, Janetopoulos noted, along with team members Kevin T. Seale and John P. Wikswo, in a recent ACS Nano paper.
Finally, it also is possible to build wells with no bottom by etching through the silicon, although such structures have not yet been fabricated. These open wells could image cells as they flowed through. This capability could be useful in areas of the world without nearby hospitals or reliable electricity, said Seale, an assistant professor of the practice of biomedical engineering.
“The devices could be used for very inexpensive diagnostics in those locations, where you could begin with a drop of blood, and you could make some important measurements,” he added.