By combining experiments with calculations and simulations, researchers in Germany have gained new insights into why placing transparent microspheres on a sample improves the resolution of an interferometry-based microscopy technique. By examining how light interacts with the microspheres, Lucie Hüser and colleagues at the University of Kassel have opened the door to understanding the mysterious enhancement.
A Linnik interferometer microscope is designed to take high-resolution images of the surface topography of a sample. The device works by splitting a beam of illuminating light in two, with one beam sent to the sample and the other to a mirror. The reflected beams are recombined at a detector, creating an image of the interfering light. By scanning the height of the sample, an accurate representation of the 3D topography of the sample is obtained.
However, like all microscopy techniques, this method faces a fundamental limit in the size of features it can resolve. This is a result of the diffraction limit, which means that the technique cannot resolve features that are smaller than half the wavelength of the imaging light.
Mysterious effect
However, microscopists have known for some time that the diffraction limit can be overcome by simply placing micron-sized transparent spheres on the surface of a sample. This has proven to be a very useful technique, but despite its efficacy, researchers do not fully understand the physics behind the enhancement. Explanations include the creation of highly focused photonic nanojets as light passes between the microspheres and the sample; an increase in the numerical aperture of the microscope that is caused by the microspheres; near-field (evanescent) effects; and the excitation of whispering-gallery modes of light within the microspheres.
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To gain a better understanding of why microsphere enhancement works for interference microscopy, Hüser’s team combined rigorous experimental measurements with new computer simulations. These included ray tracing calculations that use simple mathematics to track changes in the paths of light beams travelling through the spheres.
The study suggests that evanescent and whispering-gallery effects are negligible when it comes to resolution enhancement. Instead, the researchers found that the microspheres increase the effective size of the numerical aperture of the microscope – which improves the resolution of the instrument. The research also suggests that photonic nanojets may be involved in the improvement of the resolution.
This result brings a robust theoretical basis for microsphere-enhanced optical interference microscopy a step closer. Hüser and colleagues hope that their work may soon lead to better methods for the rapid and non-invasive imaging of the surfaces of microscopic structures. This could be especially useful for probing delicate samples, such as biological systems, that cannot be studied with high-resolution techniques such as electron microscopy and atomic force microscopy.
The research is described in the Journal of Optical Microsystems.
