Final Report Summary - STORM (Development and Application of Super-resolution Localization Microscopy)
Traditional fluorescence microscopes are not able to resolve structures finer than 250 nm due to the diffraction of the applied light. Consequently, in biological systems molecular-level biochemical processes could earlier be investigated only by using the SEM, TEM, AFM, etc. methods. Since these methods often require the special preparation of the samples, which can significantly alter their structure, they are not suitable for the investigation of the actual morphology of sensitive biological samples such as cells or tissues. In the last decade several so called optical super-resolution methods (STED, STORM, PALM, SIM etc.) have been developed that are able to provide a lateral (2D) spatial resolution below 50 nm. The goal of our project is to develop a dSTORM (direct stochastic optical reconstruction microscopy) super-resolution microscope operating on the basis of single molecule localisation (SML) principle. Development includes design, construction and application. In Hungary our custom-built system is a unique one, in which both the hardware and software elements can be modified to the needs of the end-users. The main directions of our development were (i) to improve the multimodality of the system, (ii) to understand and eliminate potential image artefacts and (iii) to apply the system for bio- and medical research.
Multimodal dSTORM microscopy aims to determine the axial position, orientation and local environment of single isolated fluorophores based on the measurement of the shape, the polarisation and the spectra of the modified point spread function (PSF). For the effective application of PSF engineering in SMLM techniques several requirements must be met. The technique must not reduce the field of view significantly, must be easily implemented, must use compact systems with few optical elements and must not limit the other functionalities of the microscope. And finally, the switch between the conventional and the PSF engineered modes must be simple and fast. An effective and feasible method was proposed and demonstrated for polarisation sensitive detection, which meets all of the above mentioned requirements. The polarisation degree (which, for example, provides direct information on the orientation of the dipole) of individual molecules was determined by measuring the two perpendicular polarisation states. Assuming rigid bonds between the target and the dye molecules, the high resolution polarisation degree images can reveal ordered, sub-diffraction sized structures. In the proposed method, a dual wedge plate made from a birefringent material was inserted into the detection path to slightly separate the polarisation components of the emitted beam. The plate was inserted into the Fourier plane of a 4f system where the beam is well collimated and therefore the aberrations are minimised. As a result, the separation of the two spots generated on the camera surface is approximately 10 pixels (~160 μm). This distance is determined by the refractive index of the birefringent material and the magnification of the microscope system. This method does not reduce the FOV, which is typically limited by the sensor size of the camera. An additional advantage of the proposed arrangement is the small deviation between the separated beams, which minimises optical aberrations and keeps the arrangement simple. It is worth noting here that the applied birefringent plate is a translation invariant optical component, and can be easily placed into and removed from the beam path by means of a filter wheel. Polarisation sensitive STORM imaging of the F-actins proved correlation between the orientation of fluorescent dipoles and the axis of the fibril.
Sample optimisation, imaging and data processing parameters are essential tasks in SMLM to understand and eliminate image artefacts. A test sample simulator software was developed for super-resolution localisation microscopy. The implemented advanced methods made the simulated data more realistic, providing a more useful tool to optimise the critical imaging parameters and understand the origin of different imaging artefacts. The axially symmetric Gaussian PSF was sensitive to the sigma value, but did not show any dependence on the residual value. In contrast, the asymmetric SD-PSF model showed direction dependent sensitivity to the sigma and residual values. Despite the significant difference between the two PSF- models, by applying appropriate thresholding processes the image quality of the final, high resolution images were found to be similar. Taking into consideration the direction of the dipoles, polarisation sensitive excitation and detection were also demonstrated using rings formed by actin filaments and labelled by rigid dye molecules. A significant difference between the captured images was realised under different dipole orientation and excitation conditions. Due to the rotation of the dipoles such characteristic features typically cannot be visualised, but using dyes rigidly bound to the target molecules anisotropy measurements at single molecule level are possible. It was also shown that optimised spectral filtering can reduce spectral crosstalk between the channels and hence improve the multi-coloured image quality. Separation of the spectral channels makes the software capable of providing rough data for testing the different multicolour imaging techniques. Image stacks with added structured background and/or mechanical drift of the sample can also be tested and used for the optimisation of either the sample preparation protocol or the applied algorithm. The above listed main features can be combined (just as they appear during a real measurement) and their effects can be evaluated and visualised by means of localisation software.
In cooperation with nearly ten research teams working in other units of the university and in the Biological Research Centre we study, among other things, degenerative diseases, actin cytoskeleton regulation, DNA break-repair mechanisms and injured motoneurons.
The microscope system is housed in an optical laboratory set up specifically for this purpose in the basement of the Bolyai Building of the University of Szeged. The Advanced Optical Imaging (http://titan.physx.u-szeged.hu/~adoptim/) research team currently consists of 5 undergraduate students, 3 PhD students and 1 post doc fellow. Since the start of the Marie Curie grant the scientific results have been published and presented in 12 refereed journals (cumulative impact factor >60) and at more than 15 national and international conferences or workshops.
Multimodal dSTORM microscopy aims to determine the axial position, orientation and local environment of single isolated fluorophores based on the measurement of the shape, the polarisation and the spectra of the modified point spread function (PSF). For the effective application of PSF engineering in SMLM techniques several requirements must be met. The technique must not reduce the field of view significantly, must be easily implemented, must use compact systems with few optical elements and must not limit the other functionalities of the microscope. And finally, the switch between the conventional and the PSF engineered modes must be simple and fast. An effective and feasible method was proposed and demonstrated for polarisation sensitive detection, which meets all of the above mentioned requirements. The polarisation degree (which, for example, provides direct information on the orientation of the dipole) of individual molecules was determined by measuring the two perpendicular polarisation states. Assuming rigid bonds between the target and the dye molecules, the high resolution polarisation degree images can reveal ordered, sub-diffraction sized structures. In the proposed method, a dual wedge plate made from a birefringent material was inserted into the detection path to slightly separate the polarisation components of the emitted beam. The plate was inserted into the Fourier plane of a 4f system where the beam is well collimated and therefore the aberrations are minimised. As a result, the separation of the two spots generated on the camera surface is approximately 10 pixels (~160 μm). This distance is determined by the refractive index of the birefringent material and the magnification of the microscope system. This method does not reduce the FOV, which is typically limited by the sensor size of the camera. An additional advantage of the proposed arrangement is the small deviation between the separated beams, which minimises optical aberrations and keeps the arrangement simple. It is worth noting here that the applied birefringent plate is a translation invariant optical component, and can be easily placed into and removed from the beam path by means of a filter wheel. Polarisation sensitive STORM imaging of the F-actins proved correlation between the orientation of fluorescent dipoles and the axis of the fibril.
Sample optimisation, imaging and data processing parameters are essential tasks in SMLM to understand and eliminate image artefacts. A test sample simulator software was developed for super-resolution localisation microscopy. The implemented advanced methods made the simulated data more realistic, providing a more useful tool to optimise the critical imaging parameters and understand the origin of different imaging artefacts. The axially symmetric Gaussian PSF was sensitive to the sigma value, but did not show any dependence on the residual value. In contrast, the asymmetric SD-PSF model showed direction dependent sensitivity to the sigma and residual values. Despite the significant difference between the two PSF- models, by applying appropriate thresholding processes the image quality of the final, high resolution images were found to be similar. Taking into consideration the direction of the dipoles, polarisation sensitive excitation and detection were also demonstrated using rings formed by actin filaments and labelled by rigid dye molecules. A significant difference between the captured images was realised under different dipole orientation and excitation conditions. Due to the rotation of the dipoles such characteristic features typically cannot be visualised, but using dyes rigidly bound to the target molecules anisotropy measurements at single molecule level are possible. It was also shown that optimised spectral filtering can reduce spectral crosstalk between the channels and hence improve the multi-coloured image quality. Separation of the spectral channels makes the software capable of providing rough data for testing the different multicolour imaging techniques. Image stacks with added structured background and/or mechanical drift of the sample can also be tested and used for the optimisation of either the sample preparation protocol or the applied algorithm. The above listed main features can be combined (just as they appear during a real measurement) and their effects can be evaluated and visualised by means of localisation software.
In cooperation with nearly ten research teams working in other units of the university and in the Biological Research Centre we study, among other things, degenerative diseases, actin cytoskeleton regulation, DNA break-repair mechanisms and injured motoneurons.
The microscope system is housed in an optical laboratory set up specifically for this purpose in the basement of the Bolyai Building of the University of Szeged. The Advanced Optical Imaging (http://titan.physx.u-szeged.hu/~adoptim/) research team currently consists of 5 undergraduate students, 3 PhD students and 1 post doc fellow. Since the start of the Marie Curie grant the scientific results have been published and presented in 12 refereed journals (cumulative impact factor >60) and at more than 15 national and international conferences or workshops.