Extending the reach and contrast of pump-probe microscopy

Femtosecond pump-probe microscopy is a specific implementation of nonlinear microscopy that can measure transient absorption processes like two-photon absorption, excited state absorption, stimulated emission, ground state depletion, and stimulated Raman scattering for structural and functional imaging. Transient absorption can provide molecular specificity without the need for exogenous dyes and labels, and measure the electronic and vibrational dynamics of materials with high specificity. Because of its good sensitivity, pump-probe microscopy has found applications in the fields of material science, biomedicine, and art conservation. For example, in biomedicine, our group (Warren lab at Duke University) have shown that the characteristic pump-probe signature of melanin changes with increased metastatic potential, which can directly impact patient treatment and prognosis by assisting pathologists in the diagnostic process.

However, current pump-probe microscopy suffers from several limitations. Firstly, the system is bulky and not user-friendly, which makes clinical translation difficult. Secondly, the pump-probe signal is in general overlaid with parasitic signals stemming from fluorescence and thermal lensing. The spurious fluorescence and thermal background not only limit the detection of molecules with weak pump-probe response, but also lead to inaccurate interpretation of signal magnitude in quantitative data analysis. Finally, the field-of-view, working-distance and penetration depth of pump-probe imaging needs to be improved for broader ranges of applications.

In this work, I address these issues by exploiting a broadband laser source, as well as new temporal and spatial configurations of pump and probe beams. In particular, in Chapter 2, I discuss a compact and user-friendly pump-probe system with high temporal resolution, based on a single, ultra-broadband, femtosecond laser source. In Chapter 3, I propose a novel detection method, featuring a MHz time-delay, polarization, and pulse-width modulation scheme to suppress unwanted signals and enhance the nonlinear imaging contrast. Finally, in Chapters 4 and 5, I propose a crossed-beam method for both Gaussian and Airy beams and demonstrate their promise for depth-resolved, long working-distance, large field-of-view imaging in scattering media. My work thus overcomes many of the issues in current instrumentation and paves the way for the next-generation femtosecond pump-probe microscopy.