Heinz Eipel

Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range

We present a novel technique of far‐field localization nanoscopy combining spectral precision distance microscopy with widely used fluorochromes like the Green Fluorescent Protein (GFP) derivatives eGFP, EmGFP, Yellow Fluorescent Protein (YFP) and eYFP, synthetic dyes like Alexa 488 and Alexa 568, as well as fluoresceine derivates. Spectral precision distance microscopy allows the surpassing of conventional resolution limits in fluorescence far‐field microscopy by precise object localization after the optical isolation of single signals in time. Based on the principles of this technique, our novel nanoscopic method was realized for laser optical precision localization and image reconstruction with highly enhanced optical resolution in intact cells. This allows for spatial assignment of individual fluorescent molecules with nanometre precision. The technique is based on excitation intensity dependent reversible photobleaching of the molecules used combined with fast time sequential imaging under appropriate focusing conditions. A meaningful advantage of the technique is the simple applicability as a universal tool for imaging and investigations to the major part of already available preparations according to standard protocols. Using the above mentioned fluorophores, the positions of single molecules within cellular structures were determined by visible light with an estimated localization precision down to 3 nm; hence distances in the range of 10–30 nm were resolved between individual fluorescent molecules allowing to apply different quantitative structure analysis tools.

Automated microaxial tomography of cell nuclei after specific labelling by fluorescence in situ hybridisation

Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.

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

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.

Automated multi-view 3D image acquisition in human genome research

One of the main problems of 3D imaging in optical microscopy is nonisotropic optical resolution of acquired 3D data. For instance, in confocal laser scanning microscopy, axial resolution is 2-3 times worse than lateral resolution. A sophisticated solution to this problem is so-called micro-axial tomography that provides a good means for microscopic image acquisition of cells or subcellular components like cell nuclei with an improved resolution achieved by object tilting and acquisition of a series of mutually tilted 3D image data. Since the very early developments of micro-axial tomography a considerable drawback of this method has been the complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem, advanced microscopy and automated computer image acquisition and analysis were brought together. Special software that drives all hardware components required for automated micro-axial tomography was developed. It performs multi-view 3D image acquisition as well as related image processing.

Reconstruction of high-resolution fluorescence microscopy images based on axial tomography

For a reliable understanding of cellular processes, high resolution 3D images of the investigated cells are necessary. Unfortunately, the ability of fluorescence microscopes to image a cell in 3D is limited since the resolution along the optical axis is by a factor of two to three worse than the transversal resolution. Standard microscopy image deblurring algorithms like the Total Variation regularized Richardson Lucy algorithm are able to improve the resolution but the problem of a lower resolution in direction along the optical axis remains. However, it is possible to overcome this problem using Axial Tomography providing tilted views of the object by rotating it under the microscope. The rotated images contain additional information about the objects and an advanced method to reconstruct a 3D image with an isotropic resolution is presented here. First, bleaching has to be corrected in order to allow a valid registration correcting translational and rotational shifts. Hereby, a multi-resolution rigid registration method is used in our method. A single high-resolution image can be reconstructed on basis of all aligned images using an extended Richardson Lucy method. In addition, a Total Variation regularization is applied in order to guarantee a stable reconstruction result. The results for both simulated and real data show a considerable improvement of the resolution in direction of the optical axis.

Rapid image acquisition in multi-photon excitation fluorescence microscopy

Summary In conventional two-photon excitation fluorescence microscopy the excitation light is tightly focussed in the object plane in order to obtain the high field intensities needed for multi-photon excitation of fluorophores. However, a rough calculation suggested that focussing might not be necessary for many practical uses of multi-photon fluorescence microscopy. Here, we present experimental evidence that two-photon excitation widefield epifluorescence microscopy is indeed feasible using Ti-sapphire-laser excitation at λ = 760 nm. Finally, we discuss the possibilities of using a Free-Electron-Laser (FEL) as the excitation light source in two-photon epifluorescence microscopy.