Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence.

The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip.

Erratum: Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy: erratum.

[This corrects the article on p. 775 in vol. 11, PMID: 32206395.].

Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy.

Single molecule localization microscopy (SMLM) is one of the key techniques that break the classical resolution limit in optical imaging. It is based on taking multiple recordings of a sample, each showing only a sparse arrangement of spatially well separated fluorescent molecules which can be localized at nanometer precision. While localizing along the lateral directions is usually straightforward, estimating axial positions at a comparable precision is known to be much harder, which is due to the relatively large depth of focus provided by the microscope optics. Whenever a molecule is sufficiently close to the coverslip, it becomes feasible to draw additional information from near field coupling effects: super-critical angle fluorescence (SAF) appears and can be exploited to boost the axial localization precision. Here we propose defocused imaging as a SMLM strategy that is capable of leveraging the information contained in SAF. We show that, regarding axial localization precision, our approach is superior to established SAF-based approaches. At the same time it is simple and can be conducted on any research-grade microscope where controlled defocusing on the order of a few hundred nanometers is possible.

Metastatic tumors to the jaws: a report of eight new cases.

PURPOSE: The purpose of the article is to present 8 new cases of metastatic tumors occurring in the jawbones, their clinical features , diagnostic workup and management. PATIENTS AND METHODS: The records of 8 patients with metastatic jaw lesions were reviewed. Demographic data, presenting symptoms, primary tumor site, radiographic findings, bone scintigraphy , histopathology and clinical management were analyzed. RESULTS: The patients , ranged in age from 44 to 80 years, with a mean of 64.5 years. The primary malignant sites were: the lung , the breast , the rectum, the thyroid, the uterus and the parotid gland . The mandible was the site of oral involvement in seven cases and the maxilla in one. There was no gender difference with respect to the oral site affected. The clinical jaw presentations were: exophytic soft tissue mass, paresthesia of the lower lip and a periapical lesion The provided treatment protocols were: chemotherapy , radiotherapy and chemotherapy, surgery and chemotherapy and supportive care only. In one case the jaw lesion was the first indication of an unknown malignancy at a distant primary site. CONCLUSIONS: Metastatic jaw lesions are uncommon. Paresthesia of the lower lip and the chin is a sinister sign for patients with a metastatic jaw lesion. In view of these cases it can be said that meticulous work-up of of jaw lesions suspected of being metastatic, may be life saving or extend the patient s survival period.