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Precisely measuring the three-dimensional position and orientation of individual fluorophores is challenging due to the substantial photon shot noise in single-molecule experiments. Facing this limited photon budget, numerous techniques have been developed to encode 2D and 3D position and 2D and 3D orientation information into fluorescence images. In this work, we adapt classical and quantum estimation theory and propose a mathematical framework to derive the best possible precision for measuring the position and orientation of dipole-like emitters for any fixed imaging system. We find that it is impossible to design an instrument that achieves the maximum sensitivity limit for measuring all possible rotational motions. Further, our vectorial dipole imaging model shows that the best quantum-limited localization precision is 4%–8% worse than that suggested by a scalar monopole model. Overall, we conclude that no single instrument can be optimized for maximum precision across all possible 2D and 3D localization and orientation measurement tasks.
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Single-molecule orientation localization microscopy II: a performance comparison
Various techniques have been developed to measure the 2D and 3D positions and 2D and 3D orientations of fluorescent molecules with improved precision over standard epifluorescence microscopes. Due to the challenging signal-to-background ratio in typical single-molecule experiments, it is essential to choose an imaging system optimized for the specific target sample. In this work, we compare the performance of multiple state-of-the-art and commonly used methods for orientation localization microscopy against the fundamental limits of measurement precision. Our analysis reveals optimal imaging methods for various experiment conditions and sample geometries. Interestingly, simple modifications to the standard fluorescence microscope exhibit superior performance in many imaging scenarios.
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- Award ID(s):
- 1653777
- PAR ID:
- 10211937
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Journal of the Optical Society of America A
- Volume:
- 38
- Issue:
- 2
- ISSN:
- 1084-7529; JOAOD6
- Format(s):
- Medium: X Size: Article No. 288
- Size(s):
- Article No. 288
- Sponsoring Org:
- National Science Foundation
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