74 MICROSCOPY & IMAGING
PERFECTPARTNERS
Jan Barghaan explains why live-cell imaging and silicone objectives are the perfect match
L
ive-cell imaging applications require stable, long-term, high-resolution imaging that is unaffected by changes in temperature and optical aberrations. However, the use of standard immersion objectives in live-cell imaging can introduce issues with spherical aberration caused by refractive index mismatch. Te refractive index of living cells is 1.38, whereas the refractive index of water immersion objectives is 1.33 and 1.52 for oil objectives. Tis difference in comparison to living cells introduces spherical aberrations and means that light rays are unable to come to a single focal point in the Z plane, causing blurring and spatial distortions in 3D reconstructions. Silicone immersion objectives on the other hand have a refractive index of 1.4 – much closer to that of living cells – reducing spherical aberration to a minimum. Tis results in brighter and higher-resolution 3D images of living cells and tissues without spatial distortions, particularly at deeper sample depths. In addition, silicone immersion objectives
are resistant to temperature and humidity changes, with virtually no evaporation at 37°C – the temperature of incubation chambers. Tese properties are aiding researchers
in live-cell imaging experiments across a wide range of applications – and even helping to drive technological advances.
A host of applications Tanks to the advantages discussed, silicone immersion objectives are proving vital across a wealth of live-cell imaging applications. Tese include the macro and micro observation of embryos, zebrafish and other model organisms in developmental biology; and in regenerative biology for the development and differentiation of embryonic stem and induced pluripotent stem (iPS) cells. Te ability to obtain high-resolution imaging of brain slices and neural cultures makes them well suited to neurobiology. Silicone immersion objectives are also ideal for certain superresolution
applications due to their ability to produce an excellent point spread function at greater depths, where they easily outperform oil immersion objectives. Tey are also more effective than water immersion objectives as they have a larger NA for comparable magnifications, reduced refractive index mismatch (Fig.1) and less evaporation. In addition, certain silicone objectives
are ideal for multiphoton imaging. Tese objectives typically have special coatings with high transmission up to 1,600nm wavelengths. Silicone objectives also have a similar refractive index to some clearing agents such as Olympus Scaleview-A2 (1.38), meaning they can be used in advanced clearing techniques to produce less light scatter for better resolution and deeper imaging.
Comparison case study Highlighting the advantages of silicone immersion objectives over traditional oil, researchers at the Department of Anatomy at Japan’s Hokkaido University Graduate School of Medicine compared images of
Fig. 1. Matching the refractive index of a sample and immersion media is important, particularly in 3D imaging. Only with silicone immersion objectives is it is possible to reconstruct the real shape of the sample without distortion in the Z plane
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