Quantitative Two-Photon Laser Scanning of Living Cells

Abstract

Two-photon laser scanning identifies the location and oligomeric state of fluorescently tagged proteins within a living cell. Knowledge of these properties is important because the cell function critically depends on the assembly state of protein complexes, as well as their subcellular localization. This thesis focuses on advancing the capabilities of two-photon laser scanning microscopy by improving the sensitivity and accuracy of the Number and Brightness technique in identifying the oligomeric state of proteins and by developing a novel dual color (DC) z-scan technique that can identify protein locations at cellular membranes with nanometer level resolution. The DC z-scan technique was applied to the nuclear envelope (NE) in living cells to identify its thickness and study the distribution of NE proteins across its inner and outer nuclear membrane. Finally, DC z-scan experiments were utilized to directly observe the translocation of the NE protein SUN2 from the outer to the inner nuclear membrane in real time.