טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRevital Halevy
SubjectMapping of Oxygen Concentration in Biological Samples by
Electron Spin Resonance Microscopy
DepartmentDepartment of Chemistry
Supervisor Full Professor Blank Aharon
Full Thesis textFull thesis text - English Version


Abstract

Electron Spin Resonance Microcopy (ESRM) is an imaging method aiming at the observation of stable free radicals in samples with a spatial resolution of ~1?m. One of the important potential applications of ESRM is the accurate measurement of oxygen concentration levels.          Such measurements take advantage of the fact that the ESR linewidth (and the inverse of the relaxation time, T2) is linearly proportional to the oxygen concentration at all levels from 0 to 1 atm.

This study demonstrates for the first time the application of ESR micro-imaging to oxygen concentration mapping in biological samples. It employed pulsed ESRM experiments using a unique "home-made" high resolution system that was recently developed at the Technion. The first part of the work focuses on the development of ESRM methodology and biological sample preparation procedures. Following this stage, measurements were made on references samples. These have shown the experimental capability of ESRM to achieve a resolution of a few ?m and demonstrated our ability for quick and easy sample preparation procedure.  In the next stage, measurements were carried out on cyanobacteria Synechocystis sp. PCC 6803 cells (a model live sample with photosynthetic activity).  The cells were placed in special glass sample holders with stable paramagnetic species (trityl) water solution.  The spin-spin relaxation time, T2, was imaged at high spatial resolution of ~30?30?100 ?m in the volume around the cells under conditions of dark and light. The results of T2 distribution and their corresponding oxygen concentrations were in agreement with the expected ones based on the photosynthetic activity of the sample. The measurement was found to have ~?M oxygen concentration sensitivity and sub-fmol absolute oxygen sensitivity per voxel. The next challenge that was only partly addressed is to image the oxygen concentration inside cells. Since the trityl is not cell permeable we had to inject it to the C. elegans nematode, which up to now provided only initial results.

The ESR micro-imaging techniques for oxygen mapping near and inside cells complements the currently available techniques based on Clark oxygen microelectrodes and fluorescence. Our hope is that further system and spin probe development would enable to address many problems of interest in the field of intercellular oxygen micro-imaging of cells. One example for such probes are the newly developed esterified trityl radicals, which can cross the cell membranes and may be used for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.