3D IMAGING/FLUORESCENCE MICROSCOPY: Microscopy technique generates 3D image as quickly as 2D

Most microscopy approaches produce 3D imagery by scanning the depth of a sample, which is problematic for optically sensitive or fast-moving samples. But a simple, new technique correlates color and position information to enhance resolution in the third dimension, producing 3D images as quickly as 2D.

Researchers at the Institute of Molecular Pathology (IMP) Vienna and the Vienna University of Technology (Austria) developed the technique. Kareem Elsayad, a researcher working with Katrin Heinze at IMP, designed a thin, biocompatible nanostructure consisting of a quartz microscope slide with a thin silver film and a dielectric layer. He then labeled a specimen with a fluorescent dye and placed it above the coated slide. "The measured emission spectrum of a fluorescent dye above this substrate depends on its distance from the substrate," he explained. "In other words, the position information of a collection of fluorophores is translated into color information, and this is what we were measuring in the end."

A fluorophore's spectral light modified up to ~100 nm above a nanostructure-coated microscope slide
A fluorophore's spectral light modified up to ~100 nm above a nanostructure-coated microscope slide. (Image courtesy of IMP)

With their method, only one measurement is needed to determine the fluorophore distribution above the substrate, with a resolution down to 10 nm (in the direction away from the substrate). "Once the sample is placed on the substrate, which can be mass-produced, a confocal microscope with spectral detection is all that is needed," said Heinze.

Together with collaborators, Elsayad and Heinze used the method to study paxillin, a protein important for cell adhesion, in living cells. They also visualized the 3D dynamics of filopodia, small cell protrusions made of bundled actin-filaments that move very quickly and have a high turnover rate during cell migration.

Originally developed for a single fluorescent marker, the method could be adapted for more efficient DNA sequencing, among other applications.

1. K. Elsayad et al., Proc. Nat. Acad. Sci., doi:10.1073/pnas.1307222110 (2013).

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