New Haven, CT--Researchers at Yale University have taken the single-color superresolution technique known as stimulated emission depletion (STED) microscopy and gotten it to work with two colors on living cells. The technique will allow dynamic nanoscale processes to be studied by imaging differently labeled proteins at the same time.
In two-color STED microscopy, two sequential scans are taken using novel so-called SNAP-tags (which are mutants of a DNA-repair protein) or CLIP-tags that bind more quickly than commercially available versions, along with carefully chosen pairs of dyes. By incorporating fusion proteins, the researchers were able to improve the targeting between the protein and the dye, effectively bridging the gap. This allowed the researchers to achieve resolutions of 78 nm and 82 nm for 22 sequential two-color scans of two proteins—epidermal growth factor and epidermal growth factor receptor—in living cells.
Imaging of the two colors was accomplished by alternating line-by-line between different excitation beams to get the two images. The researchers believe that using two opposing microscope objectives or total internal reflection, the STED technique can be expanded to three or more colors and to 3D imaging.
The achievement was reported in the August issue of the Optical Society's (OSA's) open-access journal Biomedical Optics Express.1
1. Patrina A. Pellett et al., Biomedical Optics Express, Vol. 2, No. 8, p. 2364, 1 August 2011.