Single Cell Protein Analysis PDF Download

SADRI, BEHROKH BAGHERIFAR. Protein Analysis at the Single Cell Level by Nonlinear Laser Wave-Mixing Spectroscopy for High Throughput Capillary Electrophoresis Applications. (Under the direction of William G. Tong and Morteza G. Khaledi) Nonlinear degenerate four-wave mixing is presented as an ultrasensitive optical absorption-based method for detection and measurement of biological samples. Wave-mixing imaging detection technique can localize and quantify biomolecules in single cells and tissue sections with excellent spatial distribution of light absorbed by a target sample. Cellular components can be label-free or labeled with a chromophore or a fluorophore and imaged by wave mixing using a CCD camera. In a 2-D forward-scattering wave-mixing geometry, two overlapping laser beams form interference gratings and transfer their energy to an absorbing medium, creating thermal gratings followed by changes in the refractive index. The probe beam diffracts off these laser- induced gratings to produce the signal beam, which is detected by a CCD camera or a photodiode. A single bio cell can be placed in a glass slide and as the laser beams probe the labeled cellular component, the CCD camera captures wave-mixing signals corresponding to the absorbing cellular components. This nonlinear imaging technique can be used for both live and fixed cells in real time to obtain information on sequential changes in the number, morphology and distribution of cellular components in a single cell. Nonlinear laser wave-mixing spectroscopy coupled with capillary electrophoresis provides a novel ultrasensitive method for single-cell protein analysis. This method is used to detect proteins separated within a single cell. Nonlinear wave mixing has many advantages including quadratic dependency on analyte concentration, high spatial resolution and small sample requirements. Furthermore, wave mixing offers excellent detection sensitivity levels even when using very short optical path lengths, an.