Applications of Ultrafast Pulse Shaping to Two-Photon Fluorescence Microscopy.

[摘要] Two-photon fluorescence microscopy is a popular method for imaging biological systems, offering benefits over one-photon fluorescence microscopy including increased penetration depth in tissues and reduced photobleaching. Experiments requiring selective excitation of multiple fluorophores are challenging to perform with existing two-photon fluorescence methods. Conventional two-photon microscopes employ tunable laser systems with limited temporal resolution. Other approaches include multiple-beam setups which may have complicated alignments, and ultrabroadband lasers, which simultaneously excite all fluorophores. The use of ultrafast pulse shaping methods in two-photon fluorescence microscopy offers the ability to tailor an ultrabroadband pulse to excite specific fluorophores for multicolor two-photon microscopy applications.This thesis demonstrates three applications of ultrafast pulse shaping to multicolor two-photon fluorescence microscopy. The first is the use of pulse shaping with linear unmixing of fluorescence signals to perform three-color two-photon imaging of live cells expressing fluorescent proteins. Resulting images show that the method can identify cells expressing single fluorescent proteins as well as cells expressing three fluorescent proteins simultaneously. This ultrafast pulse shaping approach has a simpler setup compared to other three-color imaging techniques, and can be easily expanded to more fluorophores.Two applications of ultrafast pulse shaping to Fluorescence Resonance Energy Transfer (FRET) microscopy are also presented. FRET microscopy is a powerful tool for observing interacting molecular species. While significant progress has been made in obtaining quantitative one-photon FRET measurements, similar progress in two-photon FRET measurements has been lacking. The techniques presented here use the selective excitation provided by pulse shaping to pave the way towards quantification of two-photon FRET. First, the use of pulse shaping to distinguish between FRET and no FRET conditions in live cells expressing fluorescent proteins is demonstrated. Differing intensity contrast results between cells imaged with different shaped pulses indicate that selective excitation can be applied to donor and acceptor fluorophores in FRET constructs and FRET can be detected. The second application discussed is the generalization of FRET stoichiometry to two-photon FRET measurements. A new theory for two-photon FRET stoichiometry is derived, and initial experimental measurements on live cells expressing fluorescent proteins are presented, yielding results that are largely consistent with expectations.

[发布机构] University of Michigan

[效力级别] Pulse Shaping

[关键词] Ultrafast Optics;Pulse Shaping;Two-Photon Microscopy;Fluorescence Microscopy;Fluorescence Resonance Energy Transfer;Physics;Science;Applied Physics