Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.

Angle analysis of Haglund syndrome and its relationship with osseous variations and Achilles tendon calcification.

BACKGROUND: Haglund syndrome is a cause of posterior heel pain. The prominent posterosuperior projection into the retrocalcaneal bursa is thought to be a major etiology. Many methods have been proposed to measure the posterosuperior projection of the tuberosity into this bursa. The Fowler angle and the parallel pitch lines are the most frequently used. However, the relation between symptomatic Haglund syndrome and the measuring methods, especially the Fowler angle and parallel pitch lines, is not clear. The purposes of this paper were to study the predictive value of the most frequently used measurement methods to evaluate bursal impingement and to determine if other osseous variations and Achilles tendon calcification are associated with the development of Haglund syndrome. METHODS: From October, 1996, to March, 2003, we evaluated 37 heels in 31 patients with symptomatic Haglund syndrome, and 40 heels in 27 individuals without posterior heel pain. On a lateral view radiograph, the Fowler angle, and the parallel pitch lines were measured, in addition to Achilles tendon calcification and the osseous variations, such as a posterior calcaneal step spur or plantar osseous projection. RESULTS: The average Fowler angles in the control group and study group were 62.31 +/- 7.79 degrees and 60.14 +/- 7.01 degrees, respectively. There was no statistically significant difference (p = 0.490). The positive parallel pitch lines in the symptomatic group were 56.8% and in the control group 42.5%. There was no statistically significant difference (p = 0.474) between the groups. CONCLUSIONS: No statistically significant differences were noted between the groups concerning the Fowler angle and parallel pitch lines. The posterior calcaneal step spur and Achilles tendon calcification were statistically significant between these two groups. The Fowler angle and parallel pitch lines were of little predictive value for the Haglund syndrome.