Oral Presentation 26th ACMM “2020 Visions in Microscopy”

Adaptive optics and active PSF shaping enable super resolution fluorescence microscopy in tissues (#8)

Michael J Mlodzianoski 1 , Paul J Cheng-Hathaway 2 3 , Shane M Bemiller 4 , Tyler J McCray 4 , Sheng Liu 5 , David A Miller 5 , Bruce T Lamb 4 6 7 , Gary E Landreth 2 3 4 , Fang Huang 5
  1. The Walter and Eliza Hall Institute, Parkville, VICTORIA, Australia
  2. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA
  3. Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, OH, USA
  4. Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA
  5. Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA
  6. Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis , IN, USA
  7. Department of Neurosciences , Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA

Single molecule localization microscopy requires precise and accurate localization of the three-dimensional positions of single molecule point spread functions (PSFs) so that the 3D volume of a structure can be reconstructed with high fidelity. Sample and depth induced optical aberrations make this task challenging when imaging beyond the coverslip by more than just a few microns. These aberrations distort the PSFs of single molecules resulting in significant deterioration of the localization precision, and therefore the resolution, while also introducing spatial localization biases. Adaptive optics approaches, often using a deformable mirror, compensate for optical aberrations to restore high quality PSFs. We present an efficient sensor-less adaptive optics approach using a deformable mirror for the removal of aberrations for robust, three-dimensional single molecule localization imaging. This method utilizes raw single molecule data during imaging as the inputs for the Nelder-Mead simplex algorithm for optimization of the shape of the deformable mirror for removal of optical aberrations. We control the deformable mirror to include astigmatism for three-dimensional localization information and, further, adaptively adjust the magnitude of astigmatism to enforce a consistent, astigmatic PSF shape for a nearly uniform localization precision throughout the sample depth. We demonstrate this approach by imaging through 30-μm thick brain tissue sections to visualize and reconstruct the 3D morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer’s disease.