Vignetting and photo-bleaching correction in automated fluorescence microscopy from an array of overlapping images

We propose a novel acquisition scheme and non-parametric multi-image based method for correcting illumination in fluorescence images. Our approach measures changes in intensity of the subject by moving the microscope stage at regularly spaced intervals, and exploits this information to learn the correction function. The acquisition process and learning are performed prior to imaging, and take only a few minutes. Afterwards, images can be corrected for vignetting and photo-bleaching effects on the fly. Our approach can be implemented in any microscope with a motorized stage, and does not require a reference calibration slide. Experiments demonstrate that our method outperforms standard approaches to illumination correction

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

<p>Abstract</p> <p>Background</p> <p>Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture.</p> <p>Methods</p> <p>Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale <it>in silico </it>image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM).</p> <p>Results</p> <p>We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates.</p> <p>Conclusion</p> <p>The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes.</p

Automatic Tumor-Stroma Separation in Fluorescence TMAs Enables the Quantitative High-Throughput Analysis of Multiple Cancer Biomarkers

The upcoming quantification and automation in biomarker based histological tumor evaluation will require computational methods capable of automatically identifying tumor areas and differentiating them from the stroma. As no single generally applicable tumor biomarker is available, pathology routinely uses morphological criteria as a spatial reference system. We here present and evaluate a method capable of performing the classification in immunofluorescence histological slides solely using a DAPI background stain. Due to the restriction to a single color channel this is inherently challenging. We formed cell graphs based on the topological distribution of the tissue cell nuclei and extracted the corresponding graph features. By using topological, morphological and intensity based features we could systematically quantify and compare the discrimination capability individual features contribute to the overall algorithm. We here show that when classifying fluorescence tissue slides in the DAPI channel, morphological and intensity based features clearly outpace topological ones which have been used exclusively in related previous approaches. We assembled the 15 best features to train a support vector machine based on Keratin stained tumor areas. On a test set of TMAs with 210 cores of triple negative breast cancers our classifier was able to distinguish between tumor and stroma tissue with a total overall accuracy of 88%. Our method yields first results on the discrimination capability of features groups which is essential for an automated tumor diagnostics. Also, it provides an objective spatial reference system for the multiplex analysis of biomarkers in fluorescence immunohistochemistry

Super-resolution Microscopy: Novel Developments and Optimisations

This thesis describes the design, development and optimisation of a multifunctional localisation based super-resolution microscope at Cambridge Advanced Imaging Centre. The microscope is optimised to perform single and dual-colour imaging with high localisation precision and accuracy. Moreover, three-dimensional imaging capability is included in the microscope using the double-helix point spread function and the light field imaging modality. The thesis also describes the application of the microscope to image challenging biological samples, in collaboration with the research groups at the Department of Physiology, Development and Neuroscience. This includes, studying dynamics of a DNA-binding transcription factor for the Notch signalling pathway, deep within the whole salivary glands of Drosophila. Characterisation and optimisation of the microscope and the subsequent image analysis pipeline, to extract dynamics of single molecules at such depths is also discussed. Another application of the microscope, discussed in the thesis, is the study of chromatin architecture in primary spermatocytes of Drosophila. This includes optimisation of imaging conditions and data analysis software to reconstruct features with different densities of labelling dye in the imaged nuclei. Calibration and application of dual-colour localisation microscopy, to visualise the arrangement of active transcription sites in chromatin fibres is also discussed. Finally, the thesis also presents the application of light field imaging technique to extend the depth of field of localisation microscopy to over 20 μm. Modification of the microscope for light field imaging and a method to localise point emitters with high precision in all three spatial dimensions is discussed. The effectiveness of the technique for single molecule imaging is shown by detecting emissions from single fluorophores in labelled cells

The development of a time-resolved confocal microscope for single molecule detection of luminescent polymers

This report details the construction and testing of a confocal microscope for use in single molecule fluorescence spectroscopy. With the prospect of making time-resolved lifetime and spectral measurements of π-conjugated polymers in the condensed phase, the system employs a pulsed 390nm, 65ps diode laser along with compact electronics for time-correlated single photon counting. Following optimisation the microscope records continuous photon arrival times with l00ps resolution, and fluorescence lifetime decays with 40ps resolution. Isolation of polymer chains has been investigated as a function of dilution by the dispersion of poly[9,9di(ethylhexyl)fluorene] (PF2/6) in a variety of host matrices. Films have been prepared by spin coating from toluene and the emission spectra, confocal lifetime and anisotropy have been investigated. Whilst the choice of host does not show any considerable influence on the spectral characteristics of the luminescent polymer, concentration and spin speed were observed to significantly influence the photophysical properties of the film. Spectral blue shifts in the emission band have been observed as the dilution of the luminescent polymer increases, reaching a maximum shift of 20nm in the most dilute samples. In line with the spectral shift, lifetime measurements show that the fluorescence decay becomes increasingly mono-exponential with dilution. In films exhibiting the maximum spectral blue shift, mono-exponential fluorescence decay was observed, for the oligomer oligo [9,9di(ethylhexyl)fluorene] (_N=20) (OF2/6) in zeonex the lifetime is found to be τ = 0.92ns. The dilution at which this occurs varies from 1x10(^-4) % w/w to 1x10(^-6) % w/w depending on spin speed and host matrix. Significantly longer that the lifetime of OF2/6 in solution, this is thought to be very close to the natural radiative lifetime of the polymer and is taken as a clear indication of chain isolation within the host matrix. Spectral blue shift and progression to mono-exponential fluorescence decay are explained in terms of the decline of low energy aggregate states and a reduction in non-radiative interchain energy transfer due to increased interchain separation. Confocal imaging using the specimen scanning technique has been developed using an electronically controlled x y piezo-stage. By optimising the optical set up the confocal image resolution is currently 0.369nm. The maximum theoretical confocal resolution at 390nm excitation is 0.268nm; the cause of this loss in resolution is thought to originate from slight non-uniformity in the excitation source

Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy

Multiphoton microscopy is a powerful optical imaging modality renowned for its non-invasive nature and relatively affordable characteristics. In particular, it has found its niche in neuroimaging due to its ability to probe in vivo biological processes in scattering brain tissue approaching millimeter depths with cellular resolution. However, the brain is a large and complex organ, and in order to fully understand its heterogeneous architecture and associated functional roles, several distal regions must be imaged simultaneously. Moreover, due to the critical implications of organelle features in various macroscale processes, whole-brain imaging at subcellular resolution scales presents itself as one of the outstanding challenges faced by the neuroscientific community today. Primarily, this research aims to expand the depth, field-of-view, and temporal throughput of multiphoton microscopy to enable large volume imaging of microvasculature at greater acquisition speeds. To accomplish this, we combine multi-faceted efforts focused on the engineering and development of advanced multiphoton microscopy techniques and technologies. This includes the characterization of novel contrast agents, the optimization of scan system optics, and the integration of high-repetition rate lasers with a resonant galvanometer. In addition, we develop a two-color imaging system capable of enhancing excitation efficiency, improving signal-to- background ratio, and further extending imaging depth. Finally, we present a novel application for two-color non-degenerate mode mixing to effectively circumvent the diffraction-limited nature of optical resolution and enable subcellular imaging. Collectively, these efforts advance the state-of-the art of multiphoton microscopy for routine cerebrovascular and neuroimaging.Biomedical Engineerin

METABOLIC HETEROGENEITY IN ISCHEMIA REPERFUSION INJURY: THE INSIDE STORY

It is well established that ischemia reperfusion (IR) injury can lead to life-threatening arrhythmias. Our group introduced the theory of metabolic sinks as a novel cause of arrhythmogenesis in a heart exposed to IR injury. Metabolic sinks are clusters of myocytes in which ischemia-induced reduction of ATP:ADP ratio increases open probability of sarcolemmal ATP-sensitive K+ channels. This in turn leads to hyperpolarization of membrane potential that reduces or ablates electrical excitability within the affected region of myocardium. To date, studies of metabolic sinks in intact hearts have been largely limited to use of fluorescent indicators to image mitochondrial membrane potential on the epicardial surface. This has revealed the existence of spatio-temporally evolving regions of myocardium within which mitochondrial membrane potential is depolarized, and whose presence influences electrical conduction and action potential duration. In order to explore the three-dimensional structure of metabolic sinks within the myocardium, we have developed a protocol for labeling an intact guinea pig heart exposed to IR injury and imaging any portion of the labeled heart using a custom designed automated volume imaging microtome (AVIM) to overcome the limited imaging field of laser scanning microscopy (LSM). Our AVIM is composed of low-cost components that can be easily installed and removed from a shared microscope. We have developed an open-source software signal processing pipeline to correct for imaging artifacts inherent to LSM and effectively reconstruct the acquired image volumes. Using this approach, we show that hearts undergoing reperfusion arrhythmias have an endocardium containing mostly depolarized mitochondria, with an abrupt transition to repolarized mitochondria in mid-myocardial to epicardial regions. Hearts not exhibiting reperfusion arrhythmias show a much more uniform distribution of depolarized mitochondria as a function of transmural location. These results show different stereotypical patterns of mitochondrial depolarization that are correlated with the presence and absence of reperfusion arrhythmias

Design and implementation of the SBX1: a smart environment chamber for biological research and discovery

2021 Summer.Includes bibliographical references.Modern biomedical laboratories make significant use of environmentally controlled chambers for incubation and examination of live cell samples. They require precise control over temperature, humidity, and gas concentration to mimic natural conditions for cell survival and growth. Many incubators and live cell imaging systems exist as commercial products; however, they are prohibitively expensive, costing tens or hundreds of thousands of dollars depending on capabilities of the system. This thesis presents the electrical, optical, mechanical, and software design of the SBX1Smart Environment Chamber. This device aims to fulfill the needs of most users at a lower cost than current commercial offerings, providing an opportunity for less funded labs to pursue biomedical research and development. The chamber provides temperature, humidity, and gas concentration controls, an internal microscope with an automated stage, and an integrated ARM microcomputer to with a graphical user interface for control and monitoring of the system. A patent has been filed for the SBX1; application no. US 2020/0324289 A1

Content-aware approach for improving biomedical image analysis: an interdisciplinary study series

Biomedicine is a highly interdisciplinary research area at the interface of sciences, anatomy, physiology, and medicine. In the last decade, biomedical studies have been greatly enhanced by the introduction of new technologies and techniques for automated quantitative imaging, thus considerably advancing the possibility to investigate biological phenomena through image analysis. However, the effectiveness of this interdisciplinary approach is bounded by the limited knowledge that a biologist and a computer scientist, by professional training, have of each other’s fields. The possible solution to make up for both these lacks lies in training biologists to make them interdisciplinary researchers able to develop dedicated image processing and analysis tools by exploiting a content-aware approach. The aim of this Thesis is to show the effectiveness of a content-aware approach to automated quantitative imaging, by its application to different biomedical studies, with the secondary desirable purpose of motivating researchers to invest in interdisciplinarity. Such content-aware approach has been applied firstly to the phenomization of tumour cell response to stress by confocal fluorescent imaging, and secondly, to the texture analysis of trabecular bone microarchitecture in micro-CT scans. Third, this approach served the characterization of new 3-D multicellular spheroids of human stem cells, and the investigation of the role of the Nogo-A protein in tooth innervation. Finally, the content-aware approach also prompted to the development of two novel methods for local image analysis and colocalization quantification. In conclusion, the content-aware approach has proved its benefit through building new approaches that have improved the quality of image analysis, strengthening the statistical significance to allow unveiling biological phenomena. Hopefully, this Thesis will contribute to inspire researchers to striving hard for pursuing interdisciplinarity