Keywords

Erythrocytes, Hemoproteins, Microscopy, Plasmodium falciparum, Spectrum analysis, malaria diagnostic

Abstract

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.

Notes

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Graduation Date

2011

Semester

Summer

Advisor

Schulte, Alfons

Degree

Doctor of Philosophy (Ph.D.)

College

College of Sciences

Department

Physics

Degree Program

Physics

Format

application/pdf

Identifier

CFE0004039

URL

http://purl.fcla.edu/fcla/etd/CFE0004039

Language

English

Release Date

August 2011

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

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