Abstract
Multiphoton microscopy enables imaging deep into scattering tissues. The efficient generation of non-linear optical effects is related to both the pulse duration (typically on the order of femtoseconds) and the size of the focused spot. Aberrations introduced by refractive index inhomogeneity in the sample distort the wavefront and enlarge the focal spot, which reduces the multiphoton signal. Traditional approaches to adaptive optics wavefront correction are not effective in thick or multi-layered scattering media. In this report, we present sensorless adaptive optics (SAO) using low-coherence interferometric detection of the excitation light for depth-resolved aberration correction of two-photon excited fluorescence (TPEF) in biological tissue. We demonstrate coherence-gated SAO TPEF using a transmissive multi-actuator adaptive lens for in vivo imaging in a mouse retina. This configuration has significant potential for reducing the laser power required for adaptive optics multiphoton imaging, and for facilitating integration with existing systems.
Highlights
The adaptive optics (AO) techniques that have been developed for astronomical telescopes can be applied to microscopy and ocular imaging to correct for refractive errors and enable diffraction-limited focusing
Hartmann-Shack Wavefront Sensor (HS-WFS) AO has been successfully performed in living mouse retina[10,11,12,13,14,15], it was constrained by high-NA low depth-of-focus imaging to reduce the reflections from other surfaces, and aberration correction was only performed on the outer, most reflective, layer of the retina[11]
We have reported on an Sensorless Adaptive Optics (SAO) technique for high-resolution retinal imaging with Optical Coherence Tomography (OCT) in small animals and humans[9,21,22,23], as well as an SAO biomicroscope for imaging fluorescently labeled cells in the mouse retina[24]
Summary
The adaptive optics (AO) techniques that have been developed for astronomical telescopes can be applied to microscopy and ocular imaging to correct for refractive errors and enable diffraction-limited focusing. An SAO approach has been reported in the literature using TPEF images in mouse retina to guide aberration correction[26]; this system required 6–7 minutes to perform a single optimization using high power laser excitation. The OCT images constitute a coherence-gated, depth-resolved signal that can be used for image-guided SAO aberration correction of the excitation beam in the sample. The excitation laser intensity can be increased to perform the MPM imaging with a separate dedicated highly sensitive photodetector Both the MPM and the OCT-guided SAO share the same source and sample arm delivery optics to ensure exact co-registration of the images during acquisition. The image acquisition was performed with a home-built multiphoton microscope using a novel transmissive multi-actuator adaptive lens (MAL) as the deformable element[22]
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