Optimization of imaging depth and spectral content in multi-photon microscopy

Multi-photon microscopy is a biological imaging technique that relies on nonlinear light-matter interactions to provide high contrast and optical sectioning capabilities. Due to the near-infrared excitation wavelengths used in multi-photon microscopy, scattering is significantly reduced. Also, photodamage and photobleaching are practically eliminated since they are confined to a sub-femptoliter excitation volume. These features of multi-photon microscopy make it ideal for in vivo imaging of highly scattering biological tissues.; A major concern when imaging turbid tissues is the maximum probing depth where useful information can be obtained. Theoretical studies have shown that the maximum imaging depth in multi-photon microscopy depends on the optical properties of tissues and on instrumentation parameters, such as the numerical aperture of the microscope objective. One of the goals of this thesis is to validate these theoretical findings and use experiments and models to quantitatively characterize tissues. In keeping with this goal, experiments were conducted for the determination of the optimal numerical aperture for imaging tissues at depth. The exponential decay of the detected signal as a function of depth was used to determine tissue optical properties, which provide unique and clinically relevant functional and structural information about tissues.; The nonlinear signals responsible for forming images in multi-photon microscopy are of two primary types: second-harmonic generation and two-photon excited fluorescence. Although both types of nonlinear interactions have long been known to occur in biological tissues at the absence of any contrast agents, there is discrepancy as to their precise origin. Understanding light-tissue interactions and the type(s) of signals generated from the individual tissue constituents is essential in order to fully capitalize on the useful features of multi-photon microscopy. This dissertation presents novel findings on the characterization of structural and functional origin of signals in multi-photon microscopy of biological tissues by means of spectral measurements. Determination of the image-forming signals provides excellent contrast enhancing opportunities and prompts the development of models for the quantitative characterization of tissues. This, in turn, will help establish multi-photon microscopy as a powerful technique for the qualitative and quantitative characterization of tissue pathophysiology.