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Spinning-disk microscope peers into the heart of a cell

BioPhotonics
Dec 2013
LONDON – A new microscopy technique with unprecedented focusing ability allows structures deep within cells – even inside viruses and bacteria – to be investigated for the first time.

Advances in optical physics, such as superresolution microscopy, have made it possible to use fluorescence to study complex structures smaller than 200 nm; however, only the structures at the bottom of the cell can be imaged clearly with these techniques. The nucleus and other vital information are in the middle of the cell, while bacterial and viral infections are scattered throughout, imposing considerable limitations for biologists.

The new technique, called spinning-disk statistical imaging (SDSI), images structures 80 nm or less anywhere in the cell. It was developed at Queen Mary University of London by Dr. Neveen Hosny, a bioengineer working with professor Martin Knight of the School of Engineering and Materials Science, and by Dr. Ann Wheeler, head of imaging at Queen Mary’s Blizard Institute.

“The spinning-disk microscope produces focused images at high speed because it has a disk with an array of tiny holes in it which remove the out-of-focus light,” Wheeler said. “We have combined this microscope with new fluorescent probes which switch between a bright and dark state rapidly. This system is now allowing us to see structures three times smaller than could usually be seen using standard light microscopes.”

The researchers have been able to visualize chromatin, the protein structure that controls DNA expression and the nuclear membrane, she said. “We have also used the method to get images of focal adhesions – subcellular macromolecules, which the cell uses to attach to its environment.”

Although it was possible to see these structures previously, the new method “provides a greater degree of detail. It also allows us to look at protein complexes which are smaller than 200 nm in the nucleus, which hasn’t been done before,” Wheeler said.

She has worked with colleagues across the university to make the technique cost-effective and easy to use for scientists who are not experts in optical physics. “We will be continuing to develop the technology to improve the fluorescent probes used for this technique and also applying it to cellular processes such as invasion in cancer,” she added.

The work was published in PLOS ONE (doi: 10.1371/journal.pone.0074604.g004).


GLOSSARY
fluorescence
The emission of light or other electromagnetic radiation of longer wavelengths by a substance as a result of the absorption of some other radiation of shorter wavelengths, provided the emission continues only as long as the stimulus producing it is maintained. In other words, fluorescence is the luminescence that persists for less than about 10-8 s after excitation.
probe
Acronym for profile resolution obtained by excitation. In its simplest form, probe involves the overlap of two counter-propagating laser pulses of appropriate wavelength, such that one pulse selectively populates a given excited state of the species of interest while the other measures the increase in absorption due to the increase in the degree of excitation.
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