University of Twente Student Theses


Detection and analysis of neutrophilic granulocytes on opsonized gold islets with Raman spectroscopy

Boerboom, G.P.M. (2013) Detection and analysis of neutrophilic granulocytes on opsonized gold islets with Raman spectroscopy.

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Abstract:Neutrophil granulocytes are important leukocytes in the innate immune system. They are the most abundant white blood cells and are capable of phagocytosis. They can engulf pathogens like bacteria, and digest them via an respiratory burst. This respiratory burst is activated by fusion of the phagosome with lysosomes and includes the enzyme myeloperoxidase and reactive oxygen species from the NADPH oxidase complex. These neutrophils can be imaged with Raman spectroscopy. This technique is based on molecular vibrations. A laser beam radiates on a focus point on the cell. The molecules in this focus point can scatter the photons with the same or a different frequency than the incident beam. Changes in the frequency are caused by the molecular vibrations of the molecules. Each kind of molecule causes a very specific shift in wavelength. By measuring the full spectra in a raster of focus points it is possible to visualize composition of a cell. When a Raman spectrum is measured in the near vicinity of a metal with free electrons, called surface plasmons, the Raman signal is immensely enhanced. The neutrophils are capable of phagocytosis, and an investigation is done to see if it is possible to use this mechanism to adhere the cells to a surface coated with gold islets. Glass and CaF2 substrates are vapor deposited with a 3 nm thick layer of gold. The absorption spectra of both substrates showed different absorption peaks, which indicate differences in the morphology of the gold. These differences are measured with SEM and AFM and showed that on CaF2 the gold formed islets of 40 nm in size. The glass substrates showed no differences in grain size between empty glass and vapor deposited gold on glass. NGs are isolated from donor blood using a dextran solution, which promotes erythrocytes rouleaux formation, a Ficoll-Paque centrifugation, which separates cells based on their density, and a hypotonic lysis of remaining red blood cells. The cells are added to substrates, which are opsonized in FBS, immersed in a phagocytosis buffer. After fixation with formaldehyde the cells should be ready for measuring with Raman spectroscopy. Unfortunately the cells did not adhere to the surface via the phagocytosis mechanism. Further investigation of the possibility to adhere NGs to a gold coated surface might include different distributions of the gold islets. Also a opsonization with human serum instead of FBS might be considered. Detection
Item Type:Essay (Bachelor)
Faculty:TNW: Science and Technology
Subject:33 physics
Programme:Applied Physics BSc (56962)
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