Bringing life into focus

May 17, 2013
Figure 1: Imaging using conventional spinning-disk confocal microscopy (left) and the new configuration (right) developed by Yuko Mimori-Kiyosue and colleagues at RIKEN.

Spinning-disk confocal microscopy is an optical imaging technique that can be used to generate detailed three-dimensional fluorescence images of living cells and their contents. Although a powerful tool for observing dynamic processes in living organisms, it has proved difficult to use for all but the thinnest biological specimens. Motivated by a need to see more deeply into living cells, Yuko Mimori-Kiyosue at the RIKEN Center for Developmental Biology and colleagues have now made major technical improvements to the technique that deliver greatly improved resolution and clarity.

Mimori-Kiyosue's team conducts research on cellular structures called microtubules. Visualizing their dynamic behavior and arrangement in live mice proved frustrating. "We didn't have a microscope that enabled imaging of fast-moving, submicrometer-sized structures in cells within thick specimens," she says.

In confocal microscopy, a laser scans a fluorescently labeled sample and the emitted fluorescence is observed through a pinhole that blocks most of the out-of-focus light. Although the technique is able to generate sharp three-dimensional reconstructions, the scanning process is too slow for use on . The spinning-disk method accelerates the process through the rotation of a disk containing many pinholes, which splits the laser into multiple beams. Unfortunately, this approach is limited by 'pinhole cross-talk'—the unwanted intrusion of background fluorescence through adjacent pinholes. Pinhole cross-talk becomes particularly severe in thick tissue samples due to high background levels.

The researchers overcame this problem with a strategy known as 'two-photon illumination', which generates fluorescence only within the focal plane of the image. While background signal is eliminated, the strategy does require the use of more powerful lasers. In response, they developed new spinning-disk units with special microlenses that efficiently condensed , with pinholes spaced further apart to further minimize cross-talk.

When combined with a more sensitive high-resolution digital camera, these modifications yielded remarkably improved image quality (Fig. 1), allowing the research team to clearly image the of microtubules in fruit fly and worm embryos as well as mouse oocytes at depths of up to 100 micrometers. The researchers now intend to use the technique to investigate genetically modified mice expressing fluorescently tagged microtubule-associated proteins.

The maximum tissue depth and sample area that can be imaged by this technique are currently limited by the power of the lasers used, but such limitations are anticipated to be short-lived. "Following the development of the next generation of high-powered lasers, we expect this system to have a large impact in the field of life sciences," Mimori-Kiyosue says.

Explore further: Scientists use X-ray diffraction to image whole, hydrated cells in their natural state for the first time

More information: Shimozawa, T. et al. Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging. Proceedings of the National Academy of Sciences USA 110, 3399–3404 (2013). dx.doi.org/10.1073/pnas.1216696110

Related Stories

Three-photon microscopy improves biological imaging

Jan 22, 2013

(Phys.org)—Scientists may be a step closer to cracking one of the world's most compelling mysteries: the impossible complexity of the brain and its billions of neurons. Cornell researchers have demonstrated ...

Recommended for you

Using antineutrinos to monitor nuclear reactors

14 hours ago

When monitoring nuclear reactors, the International Atomic Energy Agency has to rely on input given by the operators. In the future, antineutrino detectors may provide an additional option for monitoring. ...

Imaging turns a corner

18 hours ago

(Phys.org) —Scientists have developed a new microscope which enables a dramatically improved view of biological cells.

Mapping the road to quantum gravity

Apr 23, 2014

The road uniting quantum field theory and general relativity – the two great theories of modern physics – has been impassable for 80 years. Could a tool from condensed matter physics finally help map ...

User comments : 0

More news stories

Phase transiting to a new quantum universe

(Phys.org) —Recent insight and discovery of a new class of quantum transition opens the way for a whole new subfield of materials physics and quantum technologies.

When things get glassy, molecules go fractal

Colorful church windows, beads on a necklace and many of our favorite plastics share something in common—they all belong to a state of matter known as glasses. School children learn the difference between ...

A 'quantum leap' in encryption technology

Toshiba Research Europe, BT, ADVA Optical Networking and the National Physical Laboratory (NPL), the UK's National Measurement Institute, today announced the first successful trial of Quantum Key Distribution ...

Genetic code of the deadly tsetse fly unraveled

Mining the genome of the disease-transmitting tsetse fly, researchers have revealed the genetic adaptions that allow it to have such unique biology and transmit disease to both humans and animals.