Abstract
We report the use of adaptive optics with coherent anti-Stokes Raman scattering (CARS) microscopy for label-free deep tissue imaging based on molecular vibrational spectroscopy. The setup employs a deformable membrane mirror and a random search optimization algorithm to improve signal intensity and image quality at large sample depths. We demonstrate the ability to correct for both system and sample-induced aberrations in test samples as well as in muscle tissue in order to enhance the CARS signal. The combined system and sample-induced aberration correction increased the signal by an average factor of approximately 3x for the test samples at a depth of 700 microm and approximately 6x for muscle tissue at a depth of 260 microm. The enhanced signal and higher penetration depth offered by adaptive optics will augment CARS microscopy as an in vivo and in situ biomedical imaging modality.
Highlights
Optical microscopy has proved to be an immensely powerful tool for biomedical applications, allowing for subcellular-level image resolution in living systems [1, 2]
We have demonstrated aberration correction in a coherent anti-Stokes Raman scattering (CARS) microscope using an adaptive optic element
Maximum enhancement factors of approximately six times are of the same order as observed in comparable two-photon microscopy experiments [20]
Summary
Optical microscopy has proved to be an immensely powerful tool for biomedical applications, allowing for subcellular-level image resolution in living systems [1, 2]. It is possible to improve penetration depth by utilizing longer excitation wavelengths in the near-IR region [5]. In this regime single-photon absorption and scattering cross-sections decrease while absorption, dominated by water, is still weak. A successful technique for deep tissue imaging using a near-IR excitation wavelength is two-photon fluorescence microscopy, which routinely images at depths of 500 μm and is capable of probing tissues at depths in excess of 1 mm [6]. Two-photon microscopy relies on the presence of fluorescent species that are either intrinsic to the tissue [7] or introduced as exogenous labels. Small molecules often cannot be labeled without perturbing their functions and exogenous labeling is, in many cases, not possible for in situ imaging of patients
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