double helix point spread function dhpsf  (Double Helix)

Journal: bioRxiv

Article Title: Organization of Myosin H in the Apical Complex of Toxoplasma Gondii Revealed by 3D Single-Molecule Super-Resolution Microscopy

doi: 10.64898/2026.04.23.720434

Figure Lengend Snippet: A) Toxoplasma Gondii utilizes an apical complex to drive invasion of host cells. The conoid is a prominent feature of the apical complex. The precise organization of MyoH within the apical complex is unknown. B) Cryo-electron tomography has provided a density map of the conoid fibrils in situ (modified from Ref. ). C) The conoid fibrils are comprised of a comma-shaped arrangement of tubulin monomers with several conoid-associated proteins coating the tubulin. D) Two lines of parasite are created to label either terminus of MyoH, as indicated by the relative location of the red star on the miniaturized cartoon of a myosin monomer. MyoH-ALFA possesses a C-terminal fusion of the ALFA tag, while ALFA-MyoH possesses an N-terminal fusion. The ALFA tag is targeted by a nanobody conjugated to Alexa Fluor 647. E) The Double-Helix Point Spread Function (DHPSF) allows for scanning-free imaging of single-molecule fluorescence over a ∼2.5-μm axial range. Fluorophores (red stars 1, 2, and 3) have their z position encoded in the angle (θ) formed between the line bisecting the two lobes of the PSF (cyan) and the horizontal (dashed white). F) Gel expansion has been shown to increase protein accessibility, which is useful for the dense protein environment of the apical complex. Scale Bar: (E) 1 μm.

Article Snippet: We used the double-helix point spread function (DHPSF) and (d)STORM-style blinking ( , ) to localize single MyoH molecules in 3D with high-precision (AF647: 12 nm lateral, 20 nm axial; CF583R: 27 nm lateral, 40 nm axial; Fig. S4) across a 2.5-μm axial range ( , ).

Techniques: Tomography, In Situ, Modification, Imaging, Fluorescence