An adaptive scanning optical microscope with extended depth of field (ASOM-EDOF) is described. The system is based on the ASOM developed previously*, and uses a custom-built Thorlabs 0.21NA objective with a 75mm pupil diameter that allows scanned imaging over a circular region of 40mm diameter using fast steering mirror. The microscope is configured for fluorescence imaging with epi-illumination. A 140 actuator, 5.5µm stroke DM is conjugated to the pupil of the objective, and is used in conjunction with a Shack Hartmann wavefront sensor in an adaptive optics loop to measure and compensate errors of the objective as a function of nominal scan angle. At a given scan angle, the microscope camera forms an image of a 200µm x 200µm region with resolution of about 1.4µm. Images recorded at different scan angles can be stitched together to form a larger image mosaic. At each scan angle, the DM has been calibrated not only to compensate astatic aberrations, but also to perform an axial focal sweep: changing shape from concave to convex at high speed during a single camera exposure. This type of extended depth of field (EDOF) imaging (without aberration compensation) has been reported previously**. By combining these two techniques (ASOM and EDOF), a single recorded camera frame includes in-focus light from objects at depths from the nominal objective focus to depths +/-250µm from that focus, corresponding to an extension of the depth of focus by a factor of 100x for this microscope. The image also includes out-of-focus light from all depths. After a simple deconvolution, one can recover the in-focus light from all swept layers, condensed into a 2D image. Calibration details and performance metrics are described, along with example images from large volumetric samples.
*Potsaid B, Wen JTY, “Adaptive scanning optical microscope: large field of view and high-resolution imaging using a MEMS deformable mirror,” Journal of Micro-Nanolithography Mems and Moems, , 10, (2008).
**Shain WJ, Vickers NA, Goldberg BB, Bifano T, Mertz J, “Extended depth-of-field microscopy with a high-speed deformable mirror,” Optics Letters, , 995-998, (2017).