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

Exploring B cell responses in Plasmodium falciparum malaria

Plasmodium falciparum malaria remains one of the most devastating infectious diseases today. Small children in sub-Saharan Africa carry the heaviest burden of morbidity and mortality. Immunity to malaria is not well understood, although humoral immunity has proven an integral part of protection from disease. Antibodies are produced by B cells and have been directly linked to clinical immunity to malaria. There is a need to further characterize the development of B cell responses during and following a malaria infection, in order to understand the basis of clinical immunity. In Study I we developed a method for the identification of P. falciparum-specific B cells using Quantum dots in flow cytometry. We found almost a third of B cells from individuals living in a malaria-endemic area to be specific for P. falciparum. In Study II we followed a cohort of mothers and infants in Uganda with prospective blood sampling from birth up to nine months. Levels of the cytokine, B cell activating factor, were measured and in infants found to be highest in cord blood with a subsequent decrease, while the levels in mothers remained stable. Furthermore, B cell activating factor was inversely correlated with IgG+ memory B cells and CD27- memory B cells at different time points in infants and mothers. Study III was a prospective study in Stockholm enrolling individuals with acute malaria with subsequent sampling over a year. We found that B cells responding to infection with P. falciparum expressed CD11c with a dynamic shift within B cell compartments. Differences between individuals with a primary malaria infection and those previously infected, revealed differential expansion with a higher frequency of atypical memory B cells in previously infected individuals. In Study IV we established a novel co-culture method for human B cells and P. falciparum-infected red blood cells to mimic in vivo conditions. Parasitemia increased more rapidly when parasites were cultured with B cells than when cultured alone, and B cells exhibited phenotypic changes after ten days in co-culture with P. falciparum. Within the scope of this thesis we provide new methodology for the study of B cell responses to malaria, and present longitudinal data on B cell remodeling after acute malaria, as well as B cell activating factor in an endemic area. These novel methods and findings contribute valuable knowledge and can be used to inform the design of future studies to increase our understanding of the immune system in malaria