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Ulrich Nienhaus
Project Leader
Prof. Dr. Ulrich Nienhaus


Karlsruher Institut für Technologie (KIT)
Institut für Angewandte Physik
Wolfgang-Gaede-Strasse 1

D-76133 Karlsruhe

Phone:
+49 (0)721 608 - 43410

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E4: Super-Resolution Optical Microscopy

Optical Microscopy

Since its invention more than 400 years ago, far-field optical microscopy has provided a wealth of scientific knowledge, especially in the life sciences because is a minimally invasive technique that is perfectly suited for the study of live specimens over extended periods of time. Toward the end of the 19th century, it was realized that the wave nature of light poses a fundamental limitation on the resolving power of a far-field microscope. Consequently, two point sources will only be resolved in an image if they are spatially separated by more than half the wavelength of light. Typical biological macromolecules have dimensions of a few nanometers and, consequently, their interactions are not directly accessible to observation by standard optical microscopy.

Fluorescence Nanoscopy

In recent years, various fluorescence-based microscopy techniques have been introduced that circumvent the resolution limitation (‘Abbe barrier’) and permit optical microscopy images to be acquired with, at least in principle, unlimited resolution, including localization microscopy (PALM, FPALM, STORM), saturable optical fluorescent transition (RESOLFT, STED) microscopy, saturated structured illumination microscopy (SSIM) and super-resolution optical fluctuation imaging (SOFI). Featuring spatial resolutions in the range 10 – 50 nm in cellular imaging, these nanoscopy methods narrow the resolution gap between light and electron microscopy significantly and permit subcellular imaging at or close to the molecular scale.

Super-Resolution Imaging Goes Live

Super-resolution optical imaging is technically demanding and, as yet, has mainly been applied to fixed specimens. It is obvious, however, that a deep understanding of biomolecular interactions and the resulting functional behavior requires observations while life’s processes are ongoing in space and time. Therefore, the challenge is to increase the speed of data acquisition so as to keep up with the intrinsic dynamics of the processes investigated (see subproject E4.1: Super-resolution Optical Microscopy). This goal can be approached by technical improvements including optimization of the fluorescence excitation, scanning speed, detection and data acquisition and image reconstruction algorithms. However, all nanoscopy techniques rely on the photo-induced control of the fluorescence emission from marker dyes. Therefore, future progress will crucially hinge on the availability of advanced fluorescence markers, especially in regard to their photostability.  

Beyond Imaging

While there are many biological processes accessible to live-cell microscopy, others are too fast for plain imaging. For fast dynamics, one has to resort to methods such as particle tracking or fluctuation correlation techniques, which are not based on acquiring entire images.