Nonlinear microscopies (most commonly, two-photon fluorescence, second harmonic generation, and coherent
anti-Stokes Raman scattering (CARS)) have had notable successes in imaging a variety of endogenous and exogenous targets
in recent years. These methods generate light at a color different from any of the exciting laser pulses, which makes the
signal relatively easy to detect. Our work has focused on developing microscopy techniques using a wider range of
nonlinear signatures (two-photon absorption of nonfluorescent species, self phase modulation) which have some specific
advantages - for example, in recent papers we have shown that we can differentiate between different types of melanin
in pigmented lesions, image hemoglobin and its oxygenation, and measure neuronal activity. In general, these signatures
do not generate light at a different color and we rely on the advantages of femtosecond laser pulse shaping methods to
amplify the signals and make them visible (for example, using heterodyne detection of the induced signal with one of the
co-propagating laser pulses). Here we extend this work to stimulated Raman and CARS geometries. In the simplest
experiments, both colors arise from filtering a single fs laser pulse, then modulating afterwards; in other cases, we
demonstrate that spectral reshaping can retain high frequency resolution in Raman and CARS geometries with
femtosecond laser pulses.
The ability to perform high-resolution imaging of microvasculature and its oxygenation is very important in studying early tumor development. Toward this goal, we improved upon our excited state absorption (ESA)–based imaging technique to allow us to not only image hemoglobin directly but also differentiate between oxy- and deoxyhemoglobin in tissue. We demonstrate the separation of arterioles from venules in a live nude mouse ear using our imaging technique.
Even though multi-photon fluorescence microscopy offers higher resolution and better penetration depth than traditional
fluorescence microscopy, its use is restricted to the detection of molecules that fluoresce. Two-photon absorption (TPA)
imaging can provide contrast in non-fluorescent molecules while retaining the high resolution and sectioning capabilities
of nonlinear imaging modalities. In the long-wavelength water window, tissue TPA is dominated by the endogenous
molecules melanin and hemoglobin with an almost complete absence of endogenous two-photon fluorescence. A
complementary nonlinear contrast mechanism is self-phase modulation (SPM), which can provide intrinsic signatures
that can depend on local tissue anisotropy, chemical environment, or other structural properties. We have developed a
spectral hole refilling measurement technique for TPA and SPM measurements using shaped ultrafast laser pulses. Here
we report on a microscopy setup to simultaneously acquire 3D, high-resolution TPA and SPM images. We have
acquired data in mounted B16 melanoma cells with very modest laser power levels. We will also discuss the possible
application of this measurement technique to neuronal imaging. Since SPM is sensitive to material structure we can
expect SPM properties of neurons to change during neuronal firing. Using our hole-refilling technique we have now
demonstrated strong novel intrinsic nonlinear signatures of neuronal activation in a hippocampal brain slice. The
observed changes in nonlinear signal upon collective activation were up to factors of two, unlike other intrinsic optical
signal changes on the percent level. These results show that TPA and SPM imaging can provide important novel
functional contrast in tissue using very modest power levels suitable for in vivo applications.
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