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

Abstract

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.

Resolution enhancement techniques in microscopy

“…So far, micro-axial tomography has been applied to 3D studies of fluorescence labeled cellular structures ] using a setup with an external stepping motor and a flexible shaft, and also to perform highly precise measurements of focal depth dependent chromatic shifts . Recently, a miniaturized device has been presented which can be implemented in a motor driven microscope stage [Staier et al 2011].…”

Three-dimensional quantitative phase imaging via tomographic deconvolution phase microscopy

“…The configuration utilized in the present work is designed to implement tomography on optical fibers using an upright microscope (Olympus BX60) and has been described elsewhere [52,53]. In addition to this configuration, TDPM can immediately benefit from the groundwork laid for object rotation in similar fluorescent [30,31,34] and phase [24][25][26] methods. For the measurements presented here, the illuminating source was a mercury-arc lamp with a λ 546 nm green interference filter with a full width at halfmaximum bandwidth of Δλ 10 nm.…”

“…Techniques associated with 3D cell fixation and culture using glass capillaries and other cylindrical housings are still a matter of research [24][25][26]34]. However, it is anticipated that such techniques will be enabling in the application of TDPM to both fixed and living cells.…”

“…Assuming the addition of an experimental configuration for object rotation, which in practice will likely be a glass fiber or capillary coupled to a rotation stage/device [30,31,34], we are ready to devise a strategy for implementing TDPM. In order to sample the object spectrum in an isotropic fashion near the origin, the object must be rotated at least N times, where N ≥ ⌈π∕2θ c ⌉, θ c sin −1 N A c ∕n 0 is the marginal illumination angle in radians, and N A c is the NA of the illuminating condenser lens adjustable via an aperture diaphragm in the front focal plane.…”

“…TDPM is analogous to methods used in 3D fluorescence microscopy [30][31][32][33][34] which are capable of 3D spatial resolution better than confocal microscopy. The extension to QPI was originally suggested by Cogswell et al [32], although, to the authors' knowledge, this concept was never realized.…”