Sensorless adaptive optics optical coherence tomography for two photon excited fluorescence mouse retinal imaging

Two-Photon Excited Fluorescence (TPEF) is a common modality for volumetric imaging within a biological sample. The molecule-specific contrast of TPEF imaging of the retina enables novel in vivo studies of disease and retina physiology. Furthermore, retinal studies can be aided by imaging the relevant cells, which can be fluorescently labelled with fluorophores such as GFP. The multiphoton absorption suppresses the out-of-focus background signal and improves with the axial sectioning without a confocal aperture in the optical detection path. Imaging the retina with near infrared (NIR) light is ideal since the retina contain visual pigments that are sensitive to visible wavelengths and NIR light has less scattering within biological tissue than shorter wavelengths. However, high pulse energy is required to generate the TPEF, yet minimizing the incident exposure energy is required for non-invasive imaging. TPEF signal intensity increases quartically with the spot size, which leads to high sensitivity to aberrations that distribute the energy deposition of the focused light¹. For retinal imaging, decreasing the spot size requires the imaging beam to fill a larger area of the eye, which corresponds to an increase in the aberration amplitudes and often results in low TPEF. Recent research has demonstrated improvements to the TPEF signal for retinal imaging using AO for aberrations correction^2,3,4. Theoretically, the numerical aperture through the pupil of the mouse eye permits sub-micrometer imaging of the retina. However, optical aberrations introduced by the tear film, cornea and intraocular lens reduce the actual resolution. In order to approach diffraction limited imaging, these aberrations can be corrected with Adaptive Optics (AO) using a wavefront corrector such as Deformable Mirror (DM). The traditional approach to AO is to use a Wavefront Sensor (WFS) to measure the ocular aberrations directly. Performing accurate wavefront measurements for WFS AO imaging in a small animal eye requires a high system complexity due to the short length of the eye creating an optically thick sample with multiple scattering surfaces. Alternatively, Sensorless AO (SAO) has the potential to overcome the limitations of the SH-WFS. SAO does not require direct measurement of the optical wavefront but instead uses an image-based approach. SAO methods have the ability to provide depth resolved aberration correction by performing the aberration correction at different layers within the retina. Our imaging system provides volumetric TPEF imaging in the retina using SAO-OCT for depth-specific aberration correction, using same light source to generate the OCT and TPEF. Here, we present our progress since our previous report4 with improvements to the light delivery, aberration correction, and TPEF detection.