6 research outputs found
A divide and conquer strategy for the maximum likelihood localization of low intensity objects
In cell biology and other fields the automatic accurate localization of sub-resolution objects in images is an important tool. The signal is often corrupted by multiple forms of noise, including excess noise resulting from the amplification by an electron multiplying charge-coupled device (EMCCD). Here we present our novel Nested Maximum Likelihood Algorithm (NMLA), which solves the problem of localizing multiple overlapping emitters in a setting affected by excess noise, by repeatedly solving the task of independent localization for single emitters in an excess noise-free system. NMLA dramatically improves scalability and robustness, when compared to a general purpose optimization technique. Our method was successfully applied for in vivo localization of fluorescent proteins
Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices
The high quantum efficiency of the charge-coupled device (CCD) has rendered it the imaging technology of choice in diverse applications.However, under extremely lowlight conditions where few photons are detected from the imaged object, the CCD becomes unsuitable as its readout noise can easily overwhelm the weak signal. An intended solution to this problem is the electron-multiplying charge-coupled device (EMCCD), which stochastically amplifies the acquired signal to drown out the readout noise. Here, we develop the theory for calculating the Fisher information content of the amplified signal, which is modeled as the output of a branching process. Specifically, Fisher information expressions are obtained for a general and a geometric model of amplification, as well as for two approximations of the amplified signal. All expressions pertain to the important scenario of a Poisson-distributed initial signal, which is characteristic of physical processes such as photon detection. To facilitate the investigation of different data models, a "noise coefficient" is introduced which allows the analysis and comparison of Fisher information via a scalar quantity.We apply our results to the problem of estimating the location of a point source from its image, as observed through an optical microscope and detected by an EMCCD.</p
Improving FRAP and SPT for mobility and interaction measurements of molecules and nanoparticles in biomaterials
An increasing amount of pharmaceutical technologies are being developed in which nanoparticles play a crucial role. The rational development of these technologies requires detailed knowledge of the mobility and interaction of the nanoparticles inside complex biomaterials. The aim of this PhD thesis is to improve fluorescence microscopy based methods that allow to extract this information from time sequences of images. In particular, the fluorescence microscopy techniques Fluorescence Recovery After Photobleaching (FRAP) and Single Particle Tracking (SPT) are considered. FRAP modelling is revisited in order to incorporate the effect of the microscope's scanning laser beam on the shape of the photobleached region. The new model should lead to more straightforward an accurate FRAP measurements. SPT is the main focus of the PhD thesis, starting with an investigation of how motion during image acquisition affects the experimental uncertainty with which the nanoparticle positions are determined. This knowledge is used to develop a method that is able to identify interactions between nanoparticles in high detail, by scanning their trajectories for correlated positions. The method is proven to be useful in the context of drug delivery, where it was used to study the intracellular trafficking of polymeric gene complexes. Besides SPT data analysis, it is also explored how light sheet illumination, which allows to strongly reduce the out of focus fluorescence that degrades the contrast in SPT experiments, can be generated by a planar waveguide that is incorporated on a disposable chip. The potential as platform for diagnostic measurements was demonstrated by using the chip to perform SPT size and concentration measurements of cell-derived membrane vesicles. The results of this PhD thesis are expected to contribute to the effort of making accurate SPT and FRAP measurements of nanoparticle properties in biomaterials more accessible to the pharmaceutical research community
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High-resolution detectors for soft X-ray spectroscopy
Resonant Inelastic X-ray Scattering (RIXS) is a modern soft X-ray spectroscopy technique used to investigate the structure of and excitations in materials. It requires high resolution spectrometers and a brilliant, tunable, X-ray source and therefore is carried out at spectrometers such as SAXES at the Swiss Light Source Light, a synchrotron at the Paul Scherrer Institut.
SAXES uses a grating to disperse X-rays scattered from a sample across a position sensitive detector, a Charge-Coupled Device (CCD). It has been recognised that the spatial resolution of the CCD currently limits the spectrometer resolution and therefore the investigations described in this thesis focus on developing and testing methods of improving the detector resolution. Whilst this thesis addresses improving the resolution of the detector at SAXES specifically, the methods and results are applicable to other applications requiring high spatial resolution soft X-ray detection.
After an introduction, Chapters 2 and 3 describe the importance of RIXS, operation of SAXES and background of soft X-ray detection in CCDs. A comparison of models that describe charge spreading in a CCD is in Chapter 4, and the best model is implemented in a simulation package that generates populations of soft X-ray events. Chapter 5 explores the resolution improvements possible through applying centroid algorithms to simulated X-ray events, and Chapter 6 begins by describing experimental work undertaken to verify simulation results. Due to the limitations of applying centroiding algorithms to the current SAXES camera, a small-area Electron Multiplying CCD (EM-CCD) is experimentally tested (Chapter 7). Results with the EM-CCD proved positive, therefore in Chapter 8 the spatial resolution achievable with a large area EM-CCD is verified for a future SAXES camera upgrade.
Due to the successful results presented in this thesis, negotiations to develop a new camera system for SAXES are underway, and interest from other RIXS beamlines in the community may lead to the work also being applied elsewhere. The detection of soft X-rays with high spatial resolution is applicable to other future CCD and EM-CCD instruments, such as astronomical X-ray observatories