A novel experimental approach for micro-absorption spectroscopy and high-pressure microscopy of single cells is developed and applied to the investigation of morphological, volume, and spectroscopic changes in healthy red blood cells (RBCs) and erythrocytes infected with the malaria parasite Plasmodium falciparum. Through real-time optical imaging of individual erythrocytes (size ~ 7[micrometer]) we determine the change in volume over the pressure range from 0.1 to 210 MPa. The lateral diameter of healthy RBCs decreases reversibly with pressure with an approximate slope of 0.015 [micrometer] / MPa. In infected cells, clear differences in the deformability and between the compression and decompression curves are observed. The results are discussed with respect to the elasticity of the phospholipid membrane and the spectrin molecular network. Employing micro-absorption spectroscopy with spatial resolution of 1.4 [micrometer] in the lateral and 3.6 [micrometer] in the axial direction the visible absorption spectrum of hemoglobin in a single red blood cell is measured under physiological conditions. The spectra of cells infected with the malaria parasite show changes in peak positions and relative intensities in the Soret and [alpha]- and [beta]- bands. These indicate hemoglobin degradation that can be correlated with the stages of the parasite multiplication cycle and can be used as a potential diagnostic marker. The research is further extended towards the understanding of pressure effects on the ligand binding kinetics to heme proteins. For a well characterized reaction at ambient pressure, CO binding to myoglobin in solution, we investigate the transient absorption following laser flash photolysis over eight decades in time at variable pressure and temperature. The data demonstrate that pressure significantly affects the amplitudes (not just the rates) of the component processes. The amplitude of the geminate process increases with pressure corresponding to a smaller escape fraction of ligands into the solvent and a smaller inner barrier.

Medical devices are used widely at every stage of disease diagnosis and treatment. To eradicate certain infectious diseases, the development of highly sensitive diagnostic tools and techniques is essential. The work reported in this thesis presents a novel approach, which can be used for the diagnosis of various diseases in the field of clinical cytology. The central theme of this approach was to develop a simple, holistic and completely automated system for point-of-care (POC) diagnostics. This is realized through the Development of an Absorption Flow-Cytometer with Synergistic Integration of Microfluidic, Optics and simple Electronics. Quantitative diagnosis of malaria has been taken as test case for the characterization and validation of the developed technology. Malaria is a life-threatening disease widely prevalent in developing countries. Approximately half the world population undergoes a test of malaria and it kills close to half a million people every year. Early detection and treatment will reduce the number of fatalities and also decrease its transmission rate. In the recent past, several diagnostic tools have been developed to detect malaria but there are varied demands on diagnostic instruments in healthcare settings and endemic contexts. The objective of this thesis is to develop an instrument capable of identifying malaria-infected red blood cells (i-RBCs) from a given few micro-liters of whole blood. The optical absorption properties of blood cells were measured at a single-cell level to diagnose malaria. The proof-of-concept for the instrument was established in four stages, after which a prototype was also developed and validated. In the first stage, a system capable of simultaneously imaging cells and also measuring their optical absorbance properties was developed. The developed system was employed to characterize absorption properties of red blood cells (malaria-infected and healthy ones) on blood-smear. A custom-made bright-field transmission microscope in combination with a pair of laser diode and photo-detector was used to simultaneously image and measure transmittance of infected and uninfected RBCs. In the second stage, the technique was extended to enable high-throughput measurements with the use of microfluidic sample handling and synchronous data acquisition. Using this technique, the optical absorbance and morphology of infected and healthy RBCs have been characterized in statistically significant numbers. The correlation between cell morphology (from images) and single-cell optical absorbance level helped to establish the thresholds for differentiating healthy and infected cells. In the third stage, a portable prototype capable of assessing optical absorbance levels of single cells was fabricated. The developed prototype is capable of assessing cells at throughputs of about 1800 cells/ second. It was initially validated with sample suspensions containing infected and healthy RBCs obtained from malaria cultures. For the device to be usable at the field-level, it has to function in the presence of all other cellular components of whole blood. The optical absorbance of other cellular components of blood like white blood cells and platelets, were characterized. The device was finally tested with blood samples spiked with malaria-infected RBCs validating the overall proof-of-concept and the developed prototype. The deployment of such cost-effective, automated POC system would enable malaria diagnosis at remote locations and play a crucial role in the ongoing efforts to eradicate malaria. In future, the presented technology can be extended to develop POC diagnostic tool for other diseases as well. As it enables quantitative estimation of malaria, the present optical absorption flow analyzer would also find application in disease prognosis monitoring, anti-malarial drug development and other studies requiring measurements on a single-cell basis. The hyper-imaging system can be used to characterize and validate the threshold information, and can be incorporated in the prototype. Thus, it is a continuous process to characterization and implementation in the prototype. The optofluidic absorption flow analyzer will help enable affordable clinical diagnostic testing in resource limited settings. This approach will be extended to diagnose other diseases, using differences in optical absorption as criteria for differentiating healthy and infected cells.

Introduction Nigeria has the highest malaria burden worldwide. The 2010 and 2015 Nigeria Malaria Indicator Surveys (NMIS) suggest an improvement in malaria indicators, with the North West zone lagging behind. This study aimed to identify the individual and household-level malaria determinants in north-west and southern Nigeria, using Rapid Diagnostic Testing (RDT) and microscopy for malaria diagnosis. Methods Data on 3,358 children aged 6-59 months from north-west and southern Nigeria from the 2015 NMIS was used. The two populations were compared using chi-square tests, and logistic regression analysis was done for determinants of malaria infection, based on RDT and microscopic malaria test results. Results Malaria prevalence by RDT in the north-west and south was 55.8% and 29.2%, respectively (37.0% and 14.9%, respectively by microscopy). In both populations, a higher age, positive RDT in an additional household member and rural residence increased the odds of malaria infection; while higher education of the head of household and greater household wealth lowered the odds of malaria infection. Household clustering of RDT-positive cases appeared to be stronger in the south compared to the north-west. There were no statistically significant differences between the results using RDT or microscopy. Conclusion Irrespective of the diagnostic tool used, malaria determinants were similar in north-west and southern Nigeria. However, poorer social circumstances were observed in the north-west, and may account for the delayed progress in malaria control in the region. There may be a need to intensify malaria control efforts, particularly in the north-west, while awaiting socio-economic development.