Browsing by Subject "Pulse shaping"

Two-photon excited fluorescence microscopy (TPM) has become a standard biological imaging tool due to its simplicity and versatility. The fundamental contrast mechanism is derived from fluorescence of intrinsic or extrinsic markers via simultaneous two-photon absorption which provides inherent optical sectioning capabilities. The NIR-II wavelength window (1000–1350 nm), a new biological imaging window, is promising for TPM because tissue components scatter and absorb less at longer wavelengths, resulting in deeper imaging depths and better contrasts, compared to the conventional NIR-I imaging window (700–1000 nm). However, the further enhancement of TPM has been hindered by a lack of good two-photon fluorescent imaging markers in the NIR-II.

In this dissertation, we design and characterize novel two-photon imaging markers, optimized for NIR-II excitation. More specifically, the work in this dissertation includes the investigation of two-photon excited fluorescence of various highly conjugated porphyrin arrays in the NIR-II excitation window and the utilization of nanoscale polymersomes that disperse these highly conjugated porphyrin arrays in their hydrophobic layer in aqueous environment. The NIR-emissive polymersomes, highly conjugated porphyrins-dispersed polymersomes, possess superb two-photon excited brightness. The synthetic nature of polymersomes enables us to formulate fully biodegradable, non-toxic and surface-functionalized polymersomes of varying diameters, making them a promising and fully customizable multimodal diagnostic nano-structured soft-material for deep tissue imaging at high resolutions. We demonstrated key proof-of-principle experiments using NIR-emissive polymersomes for in vivo two-photon excited fluorescence imaging in mice, allowing visualization of blood vessel structure and identification of localized tumor tissue. In addition to spectroscopic characterization of the two-photon imaging agents and their imaging capabilities/applications, the effect of the laser setup (e.g., repetition rate of the laser, peak intensity, system geometry) on two-photon excited fluorescence measurements is explored to accurately measure two-photon absorption (TPA) cross-sections. A simple pulse train shaping technique is demonstrated to separate pure nonlinear processes from linear background signals, which hinders accurate quantification of TPA cross-sections.

Nonlinear optical microscopy serves as a great tool for biomedical imaging due to its high resolution, deep penetration, inherent three dimensional optical sectioning capabilities and superior performance in scattering media. Conventional nonlinear optical microscopy techniques, e.g. two photon fluorescence and second harmonic generation, are based on detecting a small light signal emitted at a new wavelength that is well separated from the excitation light. However, there are also many other nonlinear processes, such as two-photon absorption and self-phase modulation, that do not generate light at new wavelengths and that have not been extensively explored for imaging. This dissertation extends the accessible mechanisms for contrast to the later nonlinear optical processes by combining femtosecond laser pulse shaping and homodyne detection. We developed a rapid pulse shaper with a relatively simple and compact instrument design that modifies the spectrum of individual laser pulses from an 80 MHz mode-locked laser. The pulse shaper enables simultaneous two-photon absorption and self-phase modulation imaging of various nanoparticles in-vitro with high sensitivity. We also applied this imaging technique to study the nonlinear optical response in graphene. Because our technology detects the nonlinear signature encoded within the laser pulse itself, we achieve intrinsic contrast of biological and non-biological samples in highly scattering media. These capabilities have significant implications in biomedical imaging and nanophotonics.