Superresolution imaging in sharper focus: An optical microscope cannot distinguish objects separated by less than half the wavelength of light. Superresolution techniques have broken this “diffraction limit” and provided exciting new insights into cell biology. Still, such techniques hit a limit at a resolution of about 10 nm. Balzarotti et al. describe another way of localizing single molecules called MINFLUX (see the Perspective by Xiao and Ha). As in photoactivated localization microscopy and stochastic optical reconstruction microscopy, fluorophores are stochastically switched on and off, but the emitter is located using an excitation beam that is doughnut-shaped, as in stimulated emission depletion. Finding the point where emission is minimal reduces the number of photons needed to localize an emitter. MINFLUX attained ∼1-nanometer precision, and, in single-particle tracking, achieved a 100-fold enhancement in temporal resolution. Science , this issue p. 606 ; see also p. 582

Significance: Popular localization of single molecules through calculating the centroid of the diffraction pattern produced by molecular fluorescence on a camera is typically limited to spatiotemporal resolutions of >10 nm per >10 milliseconds. By requiring at least 10–100 times fewer detected photons and being free of bias due to molecular orientation, the localization concept called MINFLUX propels molecular tracking to the hitherto-unachievable regime of single-digit nanometer precision within substantially less than a millisecond. Our experiments herald the feasibility to detect molecular interactions and conformational changes at microsecond timescales.

Significance: The emission of light generated in a process referred to as bioluminescence can be used for imaging of living cells over long timespans without phototoxicity or bleaching. The amounts of light produced in the bioluminescence process are very low, and exogenous substrate molecules are often required. We improved the brightness of bacterial bioluminescence, a system that features the advantage that all of the required molecular components are genetically encoded within a single operon. Consequently, we have engineered an improved operon ilux , which enables long-term visualization of single bacterial cells while simultaneously providing information about cellular viability.

Radiotherapy with narrow scanned carbon ion beams enables a highly accurate treatment of tumours while sparing the surrounding healthy tissue. Changes in the patient's geometry can alter the actual ion range in tissue and result in unfavourable changes in the dose distribution. Consequently, it is desired to verify the actual beam delivery within the patient. Real-time and non-invasive measurement methods are preferable. Currently, the only technically feasible method to monitor the delivered dose distribution within the patient is based on tissue activation measurements by means of positron emission tomography (PET). An alternative monitoring method based on tracking of prompt secondary ions leaving a patient irradiated with carbon ion beams has been previously suggested. It is expected to help in overcoming the limitations of the PET-based technique like physiological washout of the beam induced activity, low signal and to allow for real-time measurements. In this paper, measurements of secondary charged particle tracks around a head-sized homogeneous PMMA phantom irradiated with pencil-like carbon ion beams are presented. The investigated energies and beam widths are within the therapeutically used range. The aim of the study is to deduce properties of the primary beam from the distribution of the secondary charged particles. Experiments were performed at the Heidelberg Ion Beam Therapy Center, Germany. The directions of secondary charged particles emerging from the PMMA phantom were measured using an arrangement of two parallel pixelated silicon detectors (Timepix). The distribution of the registered particle tracks was analysed to deduce its dependence on clinically important beam parameters: beam range, width and position. Distinct dependencies of the secondary particle tracks on the properties of the primary carbon ion beam were observed. In the particular experimental set-up used, beam range differences of 1.3 mm were detectable. In addition, variations in the beam width could be measured with a precision of 0.9 mm. Furthermore, shifts of the lateral beam position could be monitored with a sub-millimetre precision. The presented investigations demonstrate experimentally that the non-invasive measurement and analysis of secondary ion distributions around head-sized homogeneous objects provide information on the actual beam delivery. Beam range, width and position could be monitored with a precision attractive for therapeutic situations.

Bioluminescence-based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has, so far, not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells. Significance Bioluminescence is generated by luciferases that oxidize a specific luciferin. The enzymes involved in the synthesis of the luciferin from widespread cellular metabolites have so far been identified for only 2 bioluminescence systems, those of bacteria and fungi. In these cases, the complete reaction cascade is genetically encodable, meaning that heterologous expression of the corresponding genes can potentially produce autonomous bioluminescence in cell types other than the bacterial or fungal host cells. However, the light levels achieved in mammalian cells so far are not sufficient for single-cell applications. Here we present autonomous bioluminescence images of single mammalian cells by coexpression of the genes encoding the 6 enzymes from the bacterial bioluminescence system.