Adaptive optics and active PSF shaping enable super resolution fluorescence microscopy in tissues
- Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA.
- Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
- Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio, USA.
- Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA.
- Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USA.
- Department of Neurosciences, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA.
- Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, Indiana, USA.
- Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, Indiana, USA.
- Current address: The Walter and Eliza Hall Institute, Melbourne, VIC, Australia.
Single molecule localization requires accurate and precise localization of the three-dimensional positions of single molecule point spread functions (PSFs) to reconstruct the 3D volume of a structure with high fidelity. Depth and sample induced optical aberrations make this task challenging when imaging more than just a few microns beyond the coverslip surface. These aberrations distort the PSFs of single molecules resulting in significant worsening of the localization precision, and therefore the resolution, while also introducing spatial localization biases. Optical aberrations can be compensated for using adaptive optics approaches, often with a deformable mirror, to restore high quality PSFs. We present here an efficient sensor-less adaptive optics approach using a deformable mirror for removal of aberrations for robust, 3D single molecule localization imaging. This method utilizes single molecule data as the base for the Nelder-Mead simplex algorithm to optimize the shape of the deformable mirror for removal of optical aberrations. We control the deformable mirror to include astigmatism for 3D localization information and adaptively control the magnitude of astigmatism to enforce a consistent, astigmatic PSF shape for a nearly uniform localization precision throughout the sample depth. We demonstrate this development by imaging through 30-μm thick brain tissue sections in order to visualize and reconstruct the 3D morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer’s disease.