| dc.description.abstracteng | The functionality of the human body and with it many of its diseases are based on single cells or even on single cellular components. It is therefore essential to gain insight into the intra- and intercellular processes in order to understand the overall physiological functions and the mechanisms of ailments. Among the many techniques which are available for investigation, taking microscopic images of regions of interest plays a major role.
Optical fluorescence microscopy is a powerful tool since it augments the advantages of optical microscopy, which are non-invasive imaging of the inside of sufficiently transparent samples, with the high specificity of molecular fluorescence labeling. This is in contrast to, for example, electron microscopy, which is limited to measuring ultra-thin slices, or atomic force microscopy which provides only information about the sample surface.
The microscope’s optical resolution determines the smallest structure size which can be distinguished in the image. In order to be able to visualize for example small cellular structures like single filaments or record molecular transport processes, a resolution in the range of typically several tens of nanometers or even better is needed. Unfortunately, light of wavelength λ, emitted by a point source and imaged by a lens, is always detected as a blurred spot. Adjacent objects which are closer than d = λ/2n sin α cannot be separated since their images are blurred by diffraction into a single pattern. At this, n is the refractive index of the medium and α is half the opening angle of the objective lens. Accordingly, it is not possible to focus visible light to a spot size smaller than 200 nm laterally and 400 to 700 nm axially.
This diffraction barrier was postulated by Émile Verdet, Ernst Abbe and Lord Rayleigh at the end of the 19th century and limited the resolution for all far-field light microscopes until the 1990s. 4Pi microscopy and I5M improved the resolution in axial direction up to a factor of 7 by using opposing objective lenses coherently. Still, this does not overcome the fundamental limitations due to the wave characteristics of light.
In the last decades, super resolution imaging techniques have been established which overcome this diffraction barrier. A review by Stefan W. Hell gives a comprehensive overview and is recommended for a deeper insight. The key element in order to distinguish fluorescent objects less than 200 nm apart is the on and off switching of their signal such that it can be separated in space and time. This typically requires specific fluorescent molecules which can be transferred resp. switched between a fluorescent on state and a dark off state.
The available switching variants are manifold. Most basic is the switching between a bright singlet S1 and a dark ground S0 electronic state. Alternatively, the molecule can be transferred between an excitable on state and a non-excitable off state, for example by a long lasting electronic triplet state or a state generated by chemical bonding. The mechanism used depends on the actual microscopy concept.
In order to increase the resolution well beyond the diffraction limit there are two complementary approaches. Either the region in which fluorescent molecules are in their on state is actively controlled by targeted switching, or single molecules at random positions are stochastically switched between on and off and their location is determined subsequently.
Targeted switching is used in methods such as stimulated emission depletion (STED), saturated pattern excitation microscopy (SPEM), saturated structured illumination microscopy (SSIM) and reversible saturable/switchable optically linear fluorescence transition (RESOLFT). For STED in particular the fluorescence excitation, induced by a diffraction-limited focused beam, is restricted in space by a second overlayed beam that features a central zero intensity area. This second beam de-excites the molecules to the electronic ground state, only at the zero intensity center fluorescence is still allowed. The extent of that defined area scales inversely with the square root of the applied STED intensity and is not limited by the diffraction barrier anymore. Within biological samples resolutions of about 20 nm full width at half maximum (FWHM) in the focal plane can be reached. Stochastic switching is used in the Single Marker Switching (SMS) schemes. Depending on the applied switching mechanism they are referred to as photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy with independently running acquisition (PALMIRA), ground state depletion microscopy followed by individual molecule return (GSDIM) and variants thereof. The probability for two fluorophores to be emitted at the same time within a diffraction limited volume has to be negligible. Therefore, the fraction of stochastically switched molecules in the on state needs to be restricted sufficiently. The burst of N photons, before the molecule subsequently transfers to the dark state, is detected as a diffraction limited pattern, spatially separated from the spots of other molecules. The centroid can be localized with a precision much better than the diffraction limit and scales with 1/√N. The hereby determined position is registered in a position histogram.
The succession of switching on, emitting/detecting photons and switching off of random fluorescent molecules needs to be repeated a sufficient number of times in order to achieve a detailed image of the sample. Typical resolutions are in the range of several 10 nm FWHM in the focal plane.
Both the targeted and stochastic approach need adaptations in the optics and/or light sources of the microscope setups. An alternative strategy to overcome the diffraction barrier is to analyze the independent stochastic intensity fluctuations of fluorescent emitters in the super-resolution optical fluctuation imaging (SOFI) concept. For this, just a short video of the sample with labels switching repeatedly and independently between a fluorescent and a non-fluorescent state is required. The cumulant of the original pixel time series, related to the correlation function, gives the pixel value of the final SOFI image, calculable up to different orders. Non-correlated fluctuations cancel each other out whereby only highly correlated fluctuations remain. The resolution improvement depends on the order of cumulant reached which again requires i.a. high signal intensities. A 5-fold improvement in spatial resolution beyond the diffraction barrier can typically be achieved. Since cells in their natural environment have a distinct spatial extension, resolution increases not only in two, but in all three spatial dimensions is indispensable to super-resolve their three-dimensional (3D) structure. The SOFI concept is intrinsically three-dimensional by taking a video z-stack and the STED technique of depletion can equally be extended to the axial direction. In contrast the expansion of the SMS-based techniques to the third dimension is typically realized by breaking the axial symmetry of the detection point spread function (PSF). By using two opposing objective lenses in a 4Pi like geometry the detection efficiency can be increased twofold, improving the resolution by a factor of √2. Interference between the signals detected through both lenses increases the axial resolution even further, resulting in an overall resolution of about 6 nm FWHM in the axial and 8–22 nm in the lateral direction. However, such methods are restricted to thin layers which are in the range of about 0.25 µm and 1 µm for the interferometric PALM (iPALM) and the 4Pi-SMS implementation respectively. The restriction is caused by the limited focal length of the required high numerical objective lenses. Recently with the whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN) setup the 4Pi-SMS scheme is optimized i.a. by deformable mirrors such that whole cells along a 10 µm axial range can be imaged with isotropic resolution. Since the focal depth of the high numerical aperture (NA) objective lens is still limited to about 1.2 µm the concatenation of optical slices is necessary.
A new concept for super resolution imaging is needed which provides a much greater axial range, preferably fully isotropic. In this dissertation a stereo three-dimensional Single Marker Switching (Stereo 3D-SMS) microscope is presented which is capable to image large sample volumes.
Multiple objective lenses image the same emitter from different perspectives which are not on the same optical axis. Similar to the concept of stereo view the spatial position of the emitter can be calculated from the respective two-dimensional (2D) detection patterns. In order to optimize the detection efficiency and to achieve an isotropic resolution over a great volume four objective lenses are used simultaneously, arranged in a tetrahedron like manner. This stereo SMS concept applies the basic principle of localizing the detection patterns even for the expansion to the third dimension and has no need for any PSF modifications.
The dissertation covers the whole development process from the plain idea towards the first applications with the following main points:
• Conceiving an implementation of the stereo view procedure.
• Simulation of the expected capabilities in terms of resolution and spatial volume.
• Computer aided design of the setup.
• Programming the control of the electronic devices.
• Development of the concept as well as the related algorithms to generate a 3D image from the measured raw data.
The thesis in hand starts with the theoretical background of fluorescent imaging and super resolution. Then, the implementation of the experimental setup is presented. Beside the technical components and the light paths, this also contains the electronic control and the sample as well as the buffer preparation. Afterwards, the concept for the image analysis and the simulation of artificial data for determining the capabilities of the setup are explained in detail. Next, the theoretical and experimental results are presented. Finally, the discussion of the results and the whole setup is adjoined along with an outlook to possible advancements. | de |