Nano Bugle

A window into applied science supported by INL

Observing biological complexes at the single molecule level with optical microscopy at room temperature

The contribution of optical microscopy to life sciences can hardly be underestimated. Ever since the popularization of the optical microscope among biologists in the 17th century, mainly fueled by advances made by Anton van Leeuwenhook leading to the observation of single cell organisms, a long journey of technological development started to improve optical resolution over the complete frequency spectrum of light. Nowadays optical microscopes have achieved a lateral and longitudinal resolution around 200 and 600nm respectively. Unfortunately, further improvements are hampered by the diffraction of light waves.

Since the beginning of the 90s several new techniques have been introduced to circumvent the diffraction limitation for far-field optical image formation by fluorescence, light emitted from samples after absorption of excitation light. One of these techniques is based on the detection of single nano-sized fluorescence sources. Recently, a team of researchers under supervision of Steven Chu, Nobel laureate and current United States Secretary of Energy, have improved distance measurements between two molecular fluorescence centers from the 5-20nm range down to 0.77nm thereby bringing optical localization with visible light in the angstrom regime.

This major breakthrough was realized by a thorough analysis of the noise sources that influence the optical localization measurements. Two closed feedback loops were implemented to perform dual color image registration in order to reduce systematic effects such as drift and vibration of the instrumentation. Further, it was postulated that previous attempts to improve resolution were hampered by lack of characterization of the interpixel photoresponse non-uniformity of charge-coupled device (CCD) cameras used in these studies.

As a demonstration of future applications for this new measurement concept, a structural analysis of epithelial cadherin dimers, complexes that mediate cell adhesion but whose molecular functionality remains elusive, was demonstrated. Hereby, it was demonstrated that biological structures can be analyzed with 1-2nm resolution at room temperatures and in physiological buffer conditions. This new technique is likely to provide new insights in biological complexes that cannot be studied with nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) under these conditions.

Pieter De Beule

18/10/10

Pieter de Beule with confocal microscope add-on he is building with his group.

INL is interested in new high resolution imaging instrumentation and started research collaboration in 2009 with the Max Planck Institute for Biophysical Chemistry. In this project, Pieter De Beule, an INL postdoctoral researcher, works on the design and implementation of a new microscope for optical sectioning of live cells under the supervision of Thomas Jovin. This microscope will help scientists to further develop the understanding of basic cell biology, especially by capturing events occurring on fast timescales such as signal transduction.

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October 21, 2010 - Posted by | INL Community

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