Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level. / <p>QC 20121017</p> / The Human Protein Atlas

Optical microscopy is the simplest and the gold standard method adopted for the screening and subsequent diagnosis of various hematological and infectious diseases like malaria, sickle cell disease, tuberculosis etc. In addition to infectious disease diagnosis, its applications range from routine blood tests to the more sophisticated cancer biopsy sample analysis. Microscopy Tests (MTs) follow a common procedural workflow: (1) A technician prepares a smear of the given sample on a glass slide in a specific manner depending on the sample and the disease to be diagnosed; (2) The smeared slide is subsequently exposed to fixative agents and different histochemical stains specific to the diagnosis to be performed and (3) the prepared slide is then observed under a high quality bright- field bench-top microscope. An expert pathologist/cytologist is required to manually examine multiple fields-of-views of the prepared slide under appropriate magnification. Multiple re-adjustments in the focus and magnification makes the process of microscopic examination time consuming and tedious. Further, the manual intervention required in all the aforementioned steps involved in a typical MT, makes it inaccessible to rural/resource limited conditions and restricts the diagnostics to be performed by trained personnel in laboratory settings. To overcome these limitations, there has been considerable research interest in developing cost-effective systems that help in automating MTs. The work done in this thesis addresses these issues and proposes a two-step solution to the problem of affordable automation of MTs for cellular imaging and subsequent diagnostic assessment. The first step deals with the development of a low cost portable system that employs custom-built microscopy setup using o -the-shelf optical components, low cost motorized stage and camera modules to facilitate slide scanning and digital image acquisition. It incorporates a novel computational approach to generate good quality in-focus images, without the need for employing high-end precision translational stages, thereby reducing the overall system cost. The process of slide analysis for result generation is further automated by using image analysis and classification algorithms. The application of the developed platform in automating slide based quantitative detection of malaria is reported in this thesis. The second aspect of the thesis addresses the automation of slide preparation. A major factor that could influence the analysis results is the quality of the prepared smears. The feasibility of automating and standardizing the process of slide preparation using Microfluidics with appropriate surface fictionalization is explored and is demonstrated in the context of automated semen analysis. As an alternative to the mechanism of fixing the spermatozoa to the glass slide by smearing and chemical treatment with fixative, microfluidic chips pre-coated with adhesive protein are employed to capture and immobilize the cells. The subsequent histochemical staining is achieved by pumping the stains through the microfluidic device. The proof-of-principle experiments performed in this thesis demonstrate the feasibility of the developed system to provide an end-to-end cost-effective alternative solution to conventional MTs. This can further serve as an assistive tool for the pathologist or in some cases completely eliminate the manual intervention required in MTs enabling repeatability and reliability in diagnosis for clinical decision making