&lt;title&gt;Correlation of chromatic shifts and focal depth in spectral precision distance microscopy measured by microaxial tomography&lt;/title&gt;

Edelmann, Peter; Esa, Arif; Bornfleth, Harald; Heintzmann, Rainer; Hausmann, Michael; Cremer, Christoph

doi:10.1117/12.336821

Cited by 7 publications

(1 citation statement)

References 13 publications

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“…Nevertheless, it was also used to precisely measure focal depth dependent chromatic shifts. 13 The aim of the design described and applied here was an improvement and a miniaturization of the micro axial tomography setup in such a way that it can be easily mounted on any given type of light microscopes with a stage suitable for standard glass slides (76 mm × 26 mm). The precision mechanics of a fully adjustable glass fiber carrier was constructed which allows for improved isotropic precision in 3D localization and distance measurements.…”

Section: Introductionmentioning

confidence: 99%

Micro axial tomography: A miniaturized, versatile stage device to overcome resolution anisotropy in fluorescence light microscopy

Staier

Eipel

Matula

et al. 2011

Review of Scientific Instruments

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With the development of novel fluorescence techniques, high resolution light microscopy has become a challenging technique for investigations of the three-dimensional (3D) micro-cosmos in cells and sub-cellular components. So far, all fluorescence microscopes applied for 3D imaging in biosciences show a spatially anisotropic point spread function resulting in an anisotropic optical resolution or point localization precision. To overcome this shortcoming, micro axial tomography was suggested which allows object tilting on the microscopic stage and leads to an improvement in localization precision and spatial resolution. Here, we present a miniaturized device which can be implemented in a motor driven microscope stage. The footprint of this device corresponds to a standard microscope slide. A special glass fiber can manually be adjusted in the object space of the microscope lens. A stepwise fiber rotation can be controlled by a miniaturized stepping motor incorporated into the device. By means of a special mounting device, test particles were fixed onto glass fibers, optically localized with high precision, and automatically rotated to obtain views from different perspective angles under which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopic equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives. Only very few images registered under different rotation angles are sufficient for full 3D reconstruction. The results indicate the principal advantage of the micro axial tomography approach for many microscopic setups therein and also those of improved resolutions as obtained by high precision localization determination.

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Section: Introductionmentioning

confidence: 99%