Biological Applications of Fluorescent Imaging

Abstract

The use of fluorescent microscopy as a tool for studying biological systems has exploded with the development of the laser scanning confocal microscope. In conjunction with traditional biochemical techniques, it can plan an indespensible role in answering questions of subcellular processing and interactions. Here we have applied this powerful tool to three independent systems. The first of these is investigating the role of the malaria pigment hemozoin in immunomodulation. This project focused on looking at the biomineral core and the lipid peroxidation product coating which together make up native hemozoin. By exploring each of these elements the origin of the immunomodulatory effects seen by hemozoin could be elluciated. The second application of fluorescent imaging explored the ability of oxidative stress marker 4-hydroxy-2-nonenal (HNE) to form adducts on Protein Kinase C (PKC) and the resulting functional effects of these adducts. The use of fluorescent imaging allowed the functional effects of HNE adduction to PKC to be visualized and conclusions about the adducts to be drawn. The final application of fluorescent imaging focused on the development of a new probe to image specific strands of RNA. Named nanoblossoms, this probe would allow for the real time imaging of specific sequences of RNA relaying spatial information about RNA trafficking in live cells. Cast in the context of the detection and monitoring of Respiratory Syncytial Virus (RSV), this probe utilizes a RSV-specific DNA hairpin conjugated to a gold nanoparticle. Nanoblossoms represent a new method for studying RNA trafficking in live cell applications of fluorescent imaging. Combined, the chapters within this document provide specific examples of the utility of fluorescent imaging to solve a specific problem and the conclusions that were drawn as well as demonstrate the potential for improvement in fluorescent imaging techniques.