Fig. 1. The experimental setup. (a) Schematic representation of a full-field epifluorescence inverted microscope with the conjugate-AO extension. The microscope, represented by the objective, DM, and the tube lens, images the sample on the intermediate image plane (IIP), where the image sensor is normally located. To be able to position the DPP at a plane conjugate to the aberrating layer, the extension features 4f optics that re-images the IIP on the image sensor with minimal aberrations. The inset depicts the structure of the synthetic samples manufactured to test the performance of the AO extension. (b) Photograph of the conjugate-AO extension attached to a commercial microscope.

Fig. 2. Impact of the conjugate-AO extension on the native imaging performance of the microscope. (a) Approximate PSF of the microscope at five different points on the FoV in the absence of the DPP within the AO extension in the imaging path. The insets on the left in each figure depict the spot images, plotted in negative color for clarity. The respective location of each PSF within the FoV is indicated by the column labels. The FWHM spot size of each PSF along the x− and y−axes are indicated on the plots as well. (b) The same plots with the AO attachment and the DPP at its initial state. The initial flatness error of the DPP leads to a slight increase in the PSF widths. (c) The DPP is actively brought to the best-flat state.

Fig. 3. Estimation of the aberration mode amplitudes for the modal decomposition algorithm, demonstrated for the Zernike mode Z31. (a) The image quality metric as a function of the mode amplitude coefficient, which is a convex function whose maximum corresponds to the contribution of this specific mode. (b) Calculation of the image quality metric based on the integration of the PSD within a spatial frequency range of k1 and k2. To minimize the measurement time, this metric is calculated at different bias points (c31=−0.4,−1.5, 0, 1.5), and the results are fitted to a Gaussian curve. The mode coefficient corresponding to the maximum of this curve is chosen as the amplitude of that mode.

Fig. 4. Correction performance of the conjugate-AO extension with APP1 as the sample. (a) Uncorrected (left) and corrected (right) images of the beads. The cross-sectional intensity profiles along the eight lines are indicated on the uncorrected image. (b) Close-up view of the six square areas within the FoV indicated on the corrected image. (c) The cross-sectional intensity profiles along the eight lines are indicated on the uncorrected image. (d) Measured APP1 profile (orange), and the DPP correction profile calculated using the open-loop model and the drive signals (purple). (e) Zernike decomposition of both profiles indicating a significant qualitative difference between the ground truth and the estimated profiles.

Fig. 5. Correction performance of the conjugate-AO extension with APP2 as the sample. (a) Uncorrected (left) and corrected (right) images of the beads. The cross-sectional intensity profiles along the eight lines indicated on the uncorrected image. (b) Close-up view of the six square areas within the FoV indicated on the corrected image. (c) The cross-sectional intensity profiles along the eight lines indicated on the uncorrected image. (d) Measured APP2 profile (orange), and the DPP correction profile calculated using the open-loop model and the drive signals (purple). (e) Zernike decomposition of both profiles indicating a significant qualitative difference between the ground truth and the estimated profiles.

Fig. 6. Maximum image quality metric as a function of correction plane location with a new correction routine run at every data point. The DPP position on the x axis refers to the distance from the intermediate image plane.