GSBS Dissertations and Theses

Approval Date

1-23-2015

Document Type

Doctoral Dissertation

Academic Program

Biochemistry and Molecular Pharmacology

Department

Biochemistry and Molecular Pharmacology

First Thesis Advisor

Stephen C. Miller, PhD

Keywords

Near-Infrared Spectroscopy, Coloring Agents, Diagnostic Imaging, Esterases, Fluorescein, Fluorescence, Fluorescent Dyes, Indicators and Reagents, Light, Optical Imaging, Oxazines, Rhodamines

Subjects

Dissertations, UMMS; Spectroscopy, Near-Infrared; Coloring Agents; Diagnostic Imaging; Esterases; Fluorescein; Fluorescence; Fluorescent Dyes; Indicators and Reagents; Light; Optical Imaging; Oxazines; Rhodamines

Abstract

Fluorescence-based imaging techniques provide a simple, highly sensitive method of studying live cells and whole organisms in real time. Without question, fluorophores such as GFP, fluorescein, and rhodamines have contributed vastly to our understanding of both cell biology and biochemistry. However, most of the fluorescent molecules currently utilized suffer from one major drawback, the use of visible light. Due to cellular autofluorescence and the absorbance of incident light by cellular components, fluorescence imaging with visible wavelength fluorophores often results in high background noise and thus a low signal-to-noise ratio. Fortunately, this situation can be ameliorated by altering the wavelength of light used during imaging. Near-infrared (NIR) light (650-900 nm) is poorly absorbed by cells; therefore, fluorophores excited by this light provide a high signal-to-noise ratio and low background in cellular systems. While these properties make NIR fluorophores ideal for cellular imaging, most currently available NIR molecules cannot be used in live cells. The first half of this thesis addresses the synthetic difficulties associated with preparing NIR fluorophores that can be used within living systems. Small molecule NIR fluorophores are inherently hydrophobic which makes them unsuitable for use in the aqueous environment of the cell. Water-solubility is imparted to these dyes through highly polar sulfonates, which subsequently prevents the dyes from entering the cell. The novel work presented here details vii synthetic routes to aid in the development of sulfonated NIR fluorophores, which can be delivered into live cells through the inclusion of an esterase-labile sulfonate protecting group. Application of these synthetic techniques should allow for the development of novel NIR fluorophores with intracellular applications. The second half of this thesis addresses the need for novel NIR imaging reagents. Although several classes of NIR scaffolds do exist, most NIR probes are derivatives of a single class, heptamethine indocyanines. The work described here increases this palette by displaying the ability of NIR oxazines to function as an imaging reagent in live cells and in vivo and as a molecular sensor of biologically-relevant environmental conditions. Combined, the work contained herein has the capacity to not only advance the current NIR toolkit, but to expand it so that fluorescence imaging can move out of the dark and into the NIR light.

Comments

Date on signature page is incorrect.

This dissertation includes 8 videos that are available under "Additional Files."

DOI

10.13028/M2BG6B

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Movie 4.1_AcOTFMB-Ox1_DIC.AVI (27651 kB)
Real-time monitoring of AcOTFMB-Ox1 diffusion into U2-OS cells: DIC

Movie 4.2_AcOTFMB-Ox1_NIR.AVI (27651 kB)
Real-time monitoring of AcOTFMB-Ox1 diffusion into U2-OS cells: NIR

Movie 4.3_TFMB-Ox1_DIC.AVI (27651 kB)
Real-time monitoring of TFMB-Ox1 diffusion into U2-OS cells: DIC

Movie 4.4_TFMB-Ox1_NIR.AVI (27651 kB)
Real-time monitoring of TFMB-Ox1 diffusion into U2-OS cells: NIR

Movie 4.5_AcOTFMB-Ox2_DIC.AVI (27651 kB)
Real-time monitoring of AcOTFMB-Ox2 diffusion into U2-OS cells: DIC

Movie 4.6_AcOTFMB-Ox2_NIR.AVI (27651 kB)
Real-time monitoring of AcOTFMB-Ox2 diffusion into U2-OS cells: NIR

Movie 4.7_TFMB-Ox2_DIC.AVI (27651 kB)
Real-time monitoring of TFMB-Ox2 diffusion into U2-OS cells: DIC

Movie 4.8_TFMB-Ox2_NIR.AVI (27651 kB)
Real-time monitoring of TFMB-Ox2 diffusion into U2-OS cells: NIR

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.