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<p> Analyzing single molecule emission patterns plays a critical role in retrieving the structural and physiological information of their tagged targets, and further, understanding their interactions and cellular context. These emission patterns of tiny light sources (i.e. point spread functions, PSFs) simultaneously encode information such as the molecule’s location, orientation, the environment within the specimen, and the paths the emitted photons took before being captured by the camera. However, retrieving multiple classes of information beyond the 3D position from complex or high-dimensional single molecule data remains challenging, due to the difficulties in perceiving and summarizing a comprehensive yet succinct model. We developed smNet, a deep neural network that can extract multiplexed information near the theoretical limit from both complex and high-dimensional point spread functions. Through simulated and experimental data, we demonstrated that smNet can be trained to efficiently extract both molecular and specimen information, such as molecule location, dipole orientation, and wavefront distortions from complex and subtle features of the PSFs, which otherwise are considered too complex for established algorithms. </p>
<p> Single molecule localization microscopy (SMLM) forms super-resolution images with a resolution of several to tens of nanometers, relying on accurate localization of molecules’ 3D positions from isolated single molecule emission patterns. However, the inhomogeneous refractive indices distort and blur single molecule emission patterns, reduce the information content carried by each detected photon, increase localization uncertainty, and thus cause significant resolution loss, which is irreversible by post-processing. To compensate tissue induced aberrations, conventional sensorless adaptive optics methods rely on iterative mirror-changes and image-quality metrics to compensate aberrations. But these metrics result in inconsistent, and sometimes opposite, metric responses which fundamentally limited the efficacy of these approaches for aberration correction in tissues. Bypassing the previous iterative trial-then-evaluate processes, we developed deep learning driven adaptive optics (DL-AO), for single molecule localization microscopy (SMLM) to directly infer wavefront distortion and compensate distortion near real-time during data acquisition. our trained deep neural network monitors the individual emission patterns from single molecule experiments, infers their shared wavefront distortion, feeds the estimates through a dynamic filter (Kalman), and drives a deformable mirror to compensate sample induced aberrations. We demonstrated that DL-AO restores single molecule emission patterns approaching the conditions untouched by specimen and improves the resolution and fidelity of 3D SMLM through brain tissues over 130 µm, with as few as 3-20 mirror changes.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/22704316 |
| Date | 27 April 2023 |
| Creators | Peiyi Zhang (15361429) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY-NC-SA 4.0 |
| Relation | https://figshare.com/articles/thesis/SINGLE_MOLECULE_ANALYSIS_AND_WAVEFRONT_CONTROL_WITH_DEEP_LEARNING/22704316 |
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