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Vol. LXIV, No. 4
February 17, 2012
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Made Ya Blink
Microscope Technique Turns Fluorescing Blobs into Well-defined Molecules
Dr. Bechara Kachar (l) of NIDCD and Dr. Dylan Burnette, an NIGMS fellow currently with NICHD, have developed a new microscope technique.
Dr. Bechara Kachar (l) of NIDCD and Dr. Dylan Burnette, an NIGMS fellow currently with NICHD, have developed a new microscope technique.

Seeing a lone molecule up close and personal just got faster and easier thanks to a new technique developed by scientists at NIDCD and NICHD.

“It’s very practical and very accessible,” said Dr. Bechara Kachar, head of the NIDCD Laboratory of Cell Structure and Dynamics and senior author on the study. “It doesn’t require further technical development and the software is freely available. We hope that more researchers will take advantage of it.”

Even the most powerful light microscopes have limitations, and so to discern very tiny structures, scientists label them with “probes.” The fluorophore, a probe that absorbs and gives off light, works like a pedestrian wearing a Day-Glo vest at night. If you see the vest, you can bet there’s a person underneath. If you see the fluorophore, you can safely assume a molecule is occupying the same spot.

Over time, as fluorophores emit light, they blink and bleach (lose color) like fireworks fading in the night sky. Kachar’s team wondered if measuring these changes could help track individual molecules.

“Fluorescence images often look like blobs, since molecules overlap one another,” said first author Dr. Dylan Burnette of NICHD. Seeing them more precisely would require more elaborate and costly equipment and expertise—at least that was the conventional opinion.

So they made a technical and conceptual leap. First, they videorecorded a sequence of images in real time. Then, using copyright-free software developed at NIH (called ImageJ), they digitally subtracted—from each image—the subsequent image overlapping it. This left the earlier image intact and let them see precisely where each molecule had been.

“If you take a photograph of the foliage of a tree in the summer, you cannot clearly visualize each leaf, although you know they are there,” said Kachar. “However, in autumn, when the leaves start falling individually, we can pinpoint the location of each leaf as it falls.”

Once they had detected each “fallen” or bleached molecule, they could calculate precisely its original location coordinates to make a comprehensive molecule-map within a cellular structure.

“Bleaching/blinking-assisted localization microscopy, or BaLM, is a relatively simple and accessible new technique,” said Kachar. “Molecular information can now be attained where before there were only fluorescing blobs.”

This may help find molecular hallmarks of diseases. The study appeared in the Dec. 13, 2011 online issue of the Proceedings of the National Academy of Sciences.

“We always knew that all the data was there,” said Kachar. “We just had to reveal it.” NIHRecord Icon


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