Intrinsic Nonlinear Microscopy: From Neuronal Firing to Historical Artwork

<p>Imaging based on nonlinear processes takes advantage of the localized excitation to achieve high spatial resolution, optical sectioning, and deeper penetration in highly scattering media. However, the use of nonlinear contrast for imaging has conventionally been limited to processes that create light of wavelengths that are different from the wavelengths used for excitation. Intrinsic nonlinear contrasts that do not generate light at distinct wavelengths are generally difficult to measure because of the overwhelming background from the excitation light. This dissertation focuses on extension of nonlinear microscopy to these new intrinsic processes by using femtosecond pulse shaping to encode the nonlinear information as new frequency components in the spectrum. We will present a pump-probe microscopy technique based on pulse train shaping technology to sensitively access nonlinear transient absorption or gain processes. This technique has recently been used to uniquely identify a variety of biological pigments with high spatial resolution. Here, we extend this technique to image and characterize several inorganic and organic pigments used in historical artwork. We also present a spectral reshaping technique based on individual femtosecond pulse shaping to sensitively access nonlinear refractive contrasts in scattering media. We will describe an extension of this technique to utilize two distinct wavelengths and discuss its application in biological imaging. This two-color implementation would allow the extension of widely employed phase contrast to the nonlinear regime.</p>Dissertatio

Rapid pulse shaping with homodyne detection for measuring nonlinear optical signals

We have designed a common-mode interferometric acousto-optic pulse shaper that is capable of shaping individual pulses differently from a mode-locked laser. The design enables the measurement of weak nonlinear optical signals such as two-photon absorption and self-phase modulation at megahertz rates. The experimental apparatus incorporates homodyne detection as a means of resolving the phase of the detected signals. The fast data acquisition rate and the ability to perform measurements in scattering media make this experimental apparatus amenable to imaging applications analogous to measurements of two-photon fluorescence using a mode-locked laser

Label-Free Pump–Probe Nanoscopy

In the last few decades fluorescence microscopy has been the most widely used microscopy technique and much effort has been put into the development of advanced super-resolution fluorescence microscopy techniques to circumvent the diffraction limit. Despite their well-established benefits, these techniques have to rely on the photo-physical properties of fluorescent molecules to obtain the desired contrast and spatial resolution. The labeling procedure may cause unwanted alterations in the sample. With the advent of ultrashort-pulsed laser sources, it became possible to better explore novel non-fluorescent-based contrast mechanisms that rely solely on intrinsic properties of the molecules of interest and which led to the development of label-free microscopy approaches. In this chapter, the imaging capabilities of absorption-based pump\u2013probe microscopy are presented. This technique explores the ultrafast dynamic properties of the sample with high spatial and temporal resolution, as well as high sensitivity and chemical specificity. Two pulses, a pump and a probe, with a proper spatial and temporal overlap are used. The pump is absorbed, inducing a measurable change in the sample carrier population, which is then monitored by a delayed probe pulse. The development of new label-free approaches also represents a key challenge for the exploration of super-resolution approaches in non-fluorescence-based methods