Title: Nonlinear optical microscopy for the invisible
Description: Nonlinear optical microscopy (NOM) has become increasingly popular in biomedical research in recent years with the developments of laser sources, contrast mechanisms, novel probes and etc. One of the advantages of NOM over the linear counterpart is the ability to image deep into scattering tissues or even on the whole animals. This is due to the adoption of near-infrared excitation that is of less scattering than visible excitation, and the intrinsic optical sectioning capability minimizing the excitation background beyond focal volume. Such an advantage is particularly prominent in two-photon fluorescence microscopy compared to one-photon fluorescence microscopy. In addition, NOM may provide extra molecular information (e.g. second harmonic generation and third harmonic generation) or stronger signal (e.g. stimulated Raman scattering and coherent anti-Stokes Raman scattering compared to spontaneous Raman scattering), because of the nonlinear interaction between strong optical fields and molecules.

However, the merits of NOM are not yet fully exploited to tackle important questions in biomedical research. This thesis contributes to the developments of NOM in two aspects that correspond to two fundamental problems in biomedical imaging: first, how to non invasively image small functional biomolecules in live biological systems (Chapters 1-4); second, how to extend the optical imaging depth inside scattering tissues (Chapters 5-6). The ability to non-perturbatively image vital small biomolecules is crucial for understanding the complex functions of biological systems. However, it has proven to be highly challenging with the prevailing method of fluorescence microscopy. Because it requires the utilization of large-size fluorophore tagging (e.g., the Green Fluorescent Protein tagging) that would severely perturb the natural functions of small bio-molecules. Hence, we devise and construct a nonlinear Raman imaging platform, with the coupling of the emerging stimulated Raman scattering (SRS) microscopy and tiny vibrational tags, which provides superb sensitivity, specificity and biocompatibility for imaging small biomolecules (Chapters 1-4).

Chapter 1 outlines the theoretical background for Raman scattering. Chapter 2 describes the instrumentation for SRS microscopy, followed with an overview of recent technical developments. Chapter 3 depicts the coupling of SRS microscopy with small alkyne tags (C≡C) to sensitively and specifically image a broad spectrum of small and functionally vital biomolecules (i.e. nucleic acids, amino acids, choline, fatty acids and small molecule drugs) in live cells, tissues and animals. Chapter 4 reports the combination of SRS microscopy with small carbon-deuterium (C-D) bonds to probe the complex and dynamic protein metabolism, including protein synthesis, degradation and trafficking, with subcellular resolution through metabolic labeling. It is to my belief that the coupling of SRS microscopy with alkyne or C-D tags will be readily applied in answering key biological questions in the near future. The remaining chapters of this thesis (Chapters 5-6) present the super-nonlinear fluorescence microscopy (SNFM) techniques for extending the optical imaging depth into scattering tissues.

Unlike SRS microscopy that is an emerging technique, multiphoton microscopy (mainly referred as two-photon fluorescence microscopy), has matured over 20 years with its setup scheme and biological applications. Although it offers the deepest penetration in the optical microscopy, it still poses a fundamental depth limit set by the signal-to-background ratio when imaging into scattering tissues. Three SNFM techniques are proposed to extend such a depth limit: unlike the conventional multiphoton microscopy whose nonlinearity stems from virtual-states mediated simultaneous interactions between the incident photons and the molecules, the high-order nonlinearity of the SNFM techniques that we have conceived is generated through real-state mediated population-transfer kinetics. In particular, Chapter 5 demonstrates the multiphoton activation and imaging (MPAI) microscopy, which adopts a new class of fluorophores, the photoactivatable fluorophores, to significantly extend the fundamental imaging depth limit.

Chapter 6 theoretically and analytically depicts two additional SNFM techniques of stimulated emission reduced fluorescence (SERF) microscopy and focal saturation microscopy. Both MPAI and focal saturation microscopies exhibit a fourth order power dependence, which is effectively a four-photon process. SERF presents a third order power dependence for a three-photon process.