Stimulated Emission Depletion (STED) nanoscopy enables multi-color fluorescence imaging at the nanometer scale. Its typical single-point scanning implementation can lead to long acquisition times. In order to unleash the full spatiotemporal resolution potential of STED nanoscopy, parallelized scanning is mandatory. Here we present a dual-color STED nanoscope utilizing two orthogonally crossed standing light waves as a fluorescence switch-off pattern, and providing a resolving power down to 30 nm. We demonstrate the imaging capabilities in a biological context for immunostained vimentin fibers in a circular field of view of 20 µm diameter at 2000-fold parallelization (i.e. 2000 "intensity minima"). The technical feasibility of massively parallelizing STED without significant compromises in resolution heralds video-rate STED nanoscopy of large fields of view, pending the availability of suitable high-speed detectors.

Raman spectroscopy is a powerful tool for label-free, single cell characterization. In many reported studies, a Raman spectrum is acquired from a fraction of the cell volume and used as a representative signature of the whole cell to identify and discriminate between cell populations. It has remained an open question whether this is the most suitable approach since the spectra may not truly represent the cell as a whole and critical biochemical information could therefore be lost. To address this question, we developed a line-scan Raman microscope to acquire Raman images of single lymphocytes exposed to the chemotherapeutic drug doxorubicin for 24 to 96 hours. Principal component analysis was able to separate cells based on their drug-exposure times. Difference spectra on the mean data for the different time-points revealed that changes are related to a decrease in mean nucleic acid content and an increase in mean protein and lipid content. Vertex component analysis was used to extract the pure component spectra of lipids, nucleic acids, and proteins. Quantitative analysis of the data revealed that biochemical changes occurred at both local subcellular (i.e. molecular density) and global cellular (i.e. total observable molecular content) levels. However, significant differences between the trends in the local and global changes were observed. While local nucleic acid content decreased with increasing drug exposure time, the total cellular nucleic acid content remained relatively constant. For protein, local content remained relatively constant for all exposure times while the total protein content in the cell increased ∼3 fold. Lipid content in the entire cell increased ∼5 fold, compared to a smaller increase in lipid at the local level. These results show that valuable information about the biochemical changes throughout the entire cell can be missed if only Raman spectra of localized cell regions are used. These findings are expected to have a major impact on the future development of Raman spectroscopy for cytometry applications.

Background: Focusing with a 4π parabolic mirror allows for concentrating light from nearly the complete solid angle, whereas focusing with a single microscope objective limits the angle cone used for focusing to half solid angle at maximum. Increasing the solid angle by using deep parabolic mirrors comes at the cost of adding more complexity to the mirror's fabrication process and might introduce errors that reduce the focusing quality. Methods: To determine these errors, we experimentally examine the focusing properties of a 4π parabolic mirror that was produced by single-point diamond turning. The properties are characterized with a single 174 Yb + ion as a mobile point scatterer. The ion is trapped in a vacuum environment with a movable high optical access Paul trap. Results: We demonstrate an effective focal spot size of 209 nm in lateral and 551 nm in axial direction. Such tight focusing allows us to build an efficient light-matter interface. Conclusion: Our findings agree with numerical simulations incorporating a finite ion temperature and interferometrically measured wavefront aberrations induced by the parabolic mirror. We point at further technological improvements and discuss the general scope of applications of a 4π parabolic mirror.

Birthdays often evoke memories of the one who is celebrating. Sometimes it is a single question they have asked you that has stuck in your mind for a long time. Of the many times I met Ted Hänsch one comes to my mind in particular. It was when I saw him in a corridor at the Max Planck Institute of Quantum Optics, about thirty years ago-the building was quite new at the time. I vividly remember the question he asked me: 'Do you have a good explanation why the cross section of an atom for scattering light is as large as it is?' He was referring to the classical on-resonance cross section of an atom, σ sc = 3 2 /2π, being so much largeri.e. many orders of magnitude-than the cross section of the atomic charge distribution. Naturally, I knew the phenomenon and answered that in scattering processes the larger of the two following values dominates: the cross section of the atom as a massive object or the cross section of the particle you send in to probe the atom, namely a photon in the case under consideration. Obviously, the smallest cross section of an optical beam is limited by diffraction and this, I had thought, should define the cross section of the photon. I was surprised to see that Ted Hänsch did not seem satisfied as he slowly turned away. At the time, this made me think, and throughout the years since then I have returned to this thought every now and then.Ten years later, after I moved to Erlangen, this 'thinking' became more intense when within my group we started to first discuss spontaneous emission and the possibility of observing its time-reversed counterpart. In spontaneous emission, the energy is initially concentrated in a tiny volume, which is orders of magnitude smaller than the wavelength cubed-partially still stored in the atom-and begins to travel outwards. At first, the energy is both in the evanescent and propagating components of the field. Then, Abstract We discuss and measure the phase shift imposed onto a radially polarized light beam when focusing it onto an 174 Yb + ion. In the derivation of the expected phase shifts, we include the properties of the involved atomic levels. Furthermore, we emphasize the importance of the scattering cross section and its relation to the efficiency for coupling the focused light to an atom. The phase shifts found in the experiment are compatible with the expected ones when accounting for known deficiencies of the focusing optics and the motion of the trapped ion at the Doppler limit of laser cooling (Hänsch and Schawlow in Opt Commun 13: [68][69] 1975 This article is dedicated to Theodor W. Hänsch at the occasion of his 75th birthday. We are grateful to him for all he has done for our field and we wish him all the best for the next 25 years.