Limits of 3D optical microscopy extended

05 Dec 2013

Novel approach to biological imaging developed by Vienna-based consortium; suited to sensitive and dynamic samples.

A new microscopy technique that greatly enhances resolution in the third dimension has been developed by a group of researchers from the IMP Vienna and Vienna University of Technology, Austria. In a relatively simple set-up, the scientists use the translation of position information of fluorescent markers into color information.

Overcoming the need for scanning the depth of a sample, they were able to generate the precise 3D information about a sample at the same speed as it would take to acquire a 2D image. The general principle of this innovative approach can be used for broader applications and is published this week online in the PNAS Early Edition (Proceedings of the National Academy of Sciences of the USA).

For many research activities in the natural sciences it is necessary to achieve highly enlarged, precise pictures of specimens such as cells. Depending on the purpose of an experiment and the preparation of the sample, different microscopy techniques are used to analyze small structures or objects.

But a drawback of most current approaches is the need to scan the full depth of a sample in order to develop a 3D picture. In particlar for optically sensitive or highly dynamic (moving) samples this can represent a serious problem. Katrin Heinze and Kareem Elsayad, lead authors of the PNAS article, managed to circumvent this difficulty during their work at Vienna’s IMP.

Delicate samples

Elsayad employed fluorescence microscopy for his experimental set-up. In fluorescence microscopy fluorescent dyes, known as fluorophores, are switched on by light of certain wavelengths and, as a consequence, “spontaneously” emit light of a different characteristic wavelength.

He designed a thin biocompatible nanostructure consisting of a quartz microscope slide with a thin silver film and a dielectric layer. The IMP scientist then labeled the sample – fixed or live cells – with a fluorescent dye and placed it above the coated slide. Elsayad commented, “The measured emission spectrum of a fluorescent dye above this substrate depends on its distance from the substrate. In other words, the position information of a collection of fluorophores is translated into color information, and this is what we were measuring in the end.”

With this elaborate method, only one measurement is required to determine the fluorophore distribution above the substrate, with a resolution – in the direction away from the substrate – down to 10nm. Heinze added, “The beauty of our method is its simplicity. No elaborate set-up or machines are required to achieve this high resolution. Once the sample is placed on the substrate, which can be mass produced, a confocal microscope with spectral detection is all that is needed.”

Simple method - great potential

The novel technique was already successfully tested by Elsayad and Heinze. Together with collaborators at the Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences, they used it to study paxillin, a protein important for cell adhesion, in living cells. The scientists also visualized the 3D dynamics of filopodia, small cell protrusions made of bundled actin-filaments that move very quickly and have a high turnover-rate during cell migration.

Originally developed for a single fluorescent marker, the new method can be adapted for others as well.“There are numerous possibilities for further development and additional applications of the technique”, Elsayad points out. “From optical readout on chips to make faster computers, to more efficient DNA sequencing methods.” The novel technique patented by the IMP has already attracted a lot of interest from several big optical companies.

The Research Institute of Molecular Pathology (IMP) in Vienna is a basic biomedical research institute largely sponsored by Boehringer Ingelheim. With over 200 scientists from 30 nations, the IMP is committed to scientific discovery of fundamental molecular and cellular mechanisms underlying complex biological phenomena. Research areas include cell and molecular biology, neurobiology, disease mechanisms and computational biology.

About the Author

Matthew Peach is a contributing editor to optics.org.