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Live imaging in finer detail

01 Jun 2009

Cell biologists set to benefit from "super-resolution" video imaging.

US scientists have developed a microscope that is capable of live cell imaging at double the resolution of fluorescence microscopy. The team, from the Howard Hughes Medical Institute in Virginia, demonstrated the first use of structured illumination for live imaging to achieve 100 nm resolution at frame rates up to 11 Hz (Nature Methods DOI:10.1038/nmeth.1324).

"Many of life's processes involve size scales smaller than what can be seen using a conventional microscope," researcher Mats Gustafsson told optics.org. "Structured illumination microscopy (SIM) allows us to follow some of the fastest processes in a cell without the need for special fluorophores or extreme light intensities."

Until now, the resolution of fluorescence microscopy has been limited to around 200 nm and SIM has been too slow for dynamic live imaging. Now, Gustafsson and colleagues have boosted the speed of SIM by three orders of magnitude, making the technique suitable for studying live processes.

Structured illumination can be used to resolve features on a structured sample that are too fine to be imaged by a microscope. The technique involves projecting an illumination pattern onto the sample, which generates a set of resolvable fringes that contain information from the original sample. Multiple fringe sets are acquired with different illumination patterns to construct one high-resolution output image.

The secret to the team's success lies in how the structured illumination is produced. Gustafsson and colleagues used a ferroelectric-liquid-crystal spatial light modulator (SLM) to switch between patterns on a sub-ms timescale.

"We have increased the hardware speed by three orders of magnitude, which means that we can follow some of the fastest processes in a cell," explained Gustafsson. "SLM-based SIM offers a combination of increased resolution, multihertz live imaging, long time series and large field-of-view that other super-resolution techniques do not provide,"

In the set-up, a 512 × 512 pixel frame-transfer electron-multiplying charge-coupled-device camera is used to collect the images. The time required for one raw data image is limited by the readout time of the camera, which has a maximum full-frame rate of 35 Hz. The team believes that higher frame rates could be possible using faster cameras, though this usually entails taking a hit on either field-of-view or sensitivity.

The next step is to apply the technique in collaboration with cell biologists.

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