Chip-Based Nanoscopy in HD Quality
Waveguide Chips Enable Super-Resolution Microscopy on Conventional Microscopes
- Fig. 1: A laser is coupled from the left into the waveguide chip. The evanescent field of the light propagating inside the planar waveguide can excite fluorescence along its entire stretch. As the signal is detected by a standard upright microscope, it is easy to retrofit this technique to many existing instruments.
- Fig. 2: Different imaging modalities are provided by the developed chip-based approach, exemplary shown for the cytoskeleton of a liver cell. While no algorithmic reconstruction is necessary for the generation of a diffraction limited image (left), ESI analysis based on only a few hundred frames already allows to increase the resolution (middle). Accordingly, both approaches are suitable for a quick detection of cells of interest, that can subsequently be imaged by dSTORM in multiple thousands of frames for uncompromised resolution (right).
- Fig. 3: Waveguides of almost arbitrary extend can be designed on chip. Using a 0.5 mm wide waveguide enables super-resolution imaging of the actin network in more than 50 cells at once.
A waveguide chip that enables optical super-resolution microscopy (“nanoscopy”) on conventional microscopes with a resolution better than 50 nm has been developed by physicists in Germany and Norway . The new technique is easy to use and does not rely on complex and expensive microscopes. Furthermore, the mass-producible waveguide chip offers a much larger field-of-view as compared to current techniques. This has the great potential to open up optical super-resolution microscopy to many more people with even new applications.
The optical microscope, beginning with the first apparatus built by Galilei, has been a core instrument in science for centuries. For some time, it was thought that the resolution achievable by microscopes would only be limited by the skills of builders. However, in 1873, Ernst K. Abbe discovered that the resolution of the optical microscope is limited by the diffraction barrier of visible light to roughly 200 nm. More than a century later, various approaches to circumvent this fundamental limit and to achieve higher optical resolution (“super-resolution”) have been demonstrated . These include Structured Illumination Microscopy (SIM)  and STimulated Emission Depletion (STED)  microscopy as well as temporal signal fluctuation-based techniques such as Super-resolution Optical Fluctuation Imaging (SOFI)  and Entropy-based Super-resolution Imaging (ESI) . However, single molecule localization microscopy (SMLM)  approaches are possibly the most widespread implementations currently used in applied super-resolution imaging.
In 2006, three research groups independently developed similar fluorescence based super-resolution methods that exploit the very precise localization of single molecules [8-10]. In 2014, the Nobel Prize in Chemistry was awarded, among others, to Eric Betzig who already in 1995 proposed the general principle of single molecule isolation and localization . All other methods of localization microscopy are therefore considered specific embodiments. SMLM techniques overcome the problem of so far optically unresolvable structures by the use of photoactivatable or photoswitchable fluorescent proteins or fluorophores.
The molecules are optically switched between a fluorescent bright (“on”) and a non-fluorescent dark (“off”) state. directSTORM (dSTORM) utilizes the photoswitching properties that can be observed from a large set of commercially available fluorophores [7,12,13]. In single-molecule localization based super-resolution microscopy a pretty small subset of all fluorophores attached to the structure of interest is activated stochastically at any time, effectively confining the fluorescence emission of the activated fluorophores. A sequence of several hundred to thousand fluorescence images (“image stack”) is recorded using either wide-field or total internal reflection fluorescence (TIRF) microscopy whereupon localization with nanometer precision is performed for each fluorescence emission detected in the image. All localized fluorophores are afterwards combined into one super-resolved image.
Other approaches to super-resolution microscopy are also based on fluorescence signal dynamics. However, in contrast to localization-based methods, fluctuation-based techniques do not require strictly separated emitters but use temporal fluctuations of the fluorescence intensity in several to hundreds of imaging frames. The super-resolved image is generated by time-dependent statistical analysis on a pixel basis in the fluorescence image stack, e.g. the calculation of correlations or cumulants in time (SOFI)  or entropy values (ESI) . This results in typically worse spatial resolution in comparison to localization microscopy but images can be acquired at shorter timescales, thus, enhancing the temporal resolution.
Super-Resolved Images with Low-Cost Microscope
Generally, present optical nanoscopy techniques use rather complex microscopes for imaging and just a simple glass slide to hold the sample. In collaboration with scientists from the Arctic University of Norway in Tromsø, we have developed the inverse: the use of a complex and mass-producible optical chip (fig. 1) that provides a waveguide for the illumination-source and hosts the sample, and a standard low-cost microscope to acquire super-resolved images. Optical waveguides are based on the propagation of light inside a core with higher refractive index than the surrounding material. In the optical chip the guided light is confined inside by total internal reflection. Physical continuity forces the field to leak out of the planar waveguide, generating an evanescent field, where it decays exponentially within a few hundred nanometers but excites fluorescence in samples positioned directly on the waveguide.
Using the waveguide chips, we are able to simultaneously image up to 0.5 mm × 0.5 mm in evanescent excitation. This is made possible because the evanescent field is generated by the waveguide, independent of the microscope detection optics. Consequently, the illumination and detection light paths are decoupled and arbitrary objective lenses can be utilized instead of specific TIRF lenses. The strong evanescent field of the waveguide is used for single molecule switching and fluorescence excitation and, thus, enables chip based single molecule localization microscopy. But we also applied another technique: multi-mode interference patterns induce spatial fluorescence intensity variations that allow for fluctuation-based super-resolution imaging by ESI analysis of the raw data. Imaging of the tubulin cytoskeleton in a liver cell reveals the specific strengths of the different approaches: While no algorithmic reconstruction is necessary for TIRF-like diffraction limited imaging (fig. 2, left), ESI analysis based on only about 200 frames already results in increased resolution (fig. 2, middle). The highest resolution is obtained by reconstructing the dSTORM image from 40.000 frames (fig. 2, right). These results nicely demonstrate the trade-off between temporal resolution and spatial resolution. Switching to a low magnification objective lens, more than 50 cells can be imaged simultaneously with dSTORM (fig. 3).
Waveguide chips dramatically reduce the complexity and potentially costs of the microscopes and, thus, have the potential to make nanoscopy accessible for a wide range of users. By guiding the illumination light through optical fibers and waveguides, many standard optical microscopes can be retrofitted for super-resolution imaging. Future chip developments towards on-chip laser generation and steering of entire illumination systems will potentially further extend the capabilities. Additionally, the integrated platform makes it straightforward to implement combinations with different lab-on-a-chip methods, such as microfluidics, optical trapping or other detection techniques.
We thank our collaborators Øystein I. Helle, Cristina I. Øie, Peter McCourt, Thomas R. Huser and Balpreet S. Ahluwalia. Further thanks to Matthias Simonis for the photograph of figure 1 and Rajwinder Singh and Deanna L. Wolfson for cell preparations shown in figure 3. This work was supported by the German Academic Exchange Service (DAAD).
R. Diekmann1, M. Schüttpelz1
1Department of Physics, Bielefeld University, Bielefeld, Germany
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