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Interactions between biomolecules are characterized by where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial and angular information are mixed within the image of a fluorescent molecule–the microscope’s dipole-spread function (DSF). We demonstrate the pixOL algorithm to simultaneously optimize all pixels within a phase mask to produce an engineered Green’s tensor–the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision to simultaneously measure the 3D orientation and 3D location of a single molecule, i.e., 4.1° orientation, 0.44 sr wobble angle, 23.2 nm lateral localization, and 19.5 nm axial localization precisions in simulations over a 700 nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration in six dimensions.
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Three-dimensional nanoscale localization of point-like objects using self-interference digital holography
We propose localizing point-like fluorescent emitters in three dimensions with nanometer precision throughout large volumes using self-interference digital holography (SIDH). SIDH enables imaging of incoherently emitting objects over large axial ranges without refocusing, and single molecule localization techniques allow sub-50 nm resolution in the lateral and axial dimensions. We demonstrate three-dimensional localization with SIDH by imaging 100 and 40 nm fluorescent nanospheres. With 49,000 photons detected, SIDH achieves a localization precision of 5 nm laterally and 40 nm axially. We are able to detect the nanospheres from as few as 13,000 detected photons.
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- Award ID(s):
- 1555576
- PAR ID:
- 10130684
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Optics Letters
- Volume:
- 45
- Issue:
- 2
- ISSN:
- 0146-9592; OPLEDP
- Format(s):
- Medium: X Size: Article No. 591
- Size(s):
- Article No. 591
- Sponsoring Org:
- National Science Foundation
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