Eva Rittweger

STED microscopy with a supercontinuum laser source.

We report on a straightforward yet powerful implementation of stimulated emission depletion (STED) fluorescence microscopy providing subdiffraction resolution in the far-field. Utilizing the same super-continuum pulsed laser source both for excitation and STED, this implementation of STED microscopy avoids elaborate preparations of laser pulses and conveniently provides multicolor imaging. Operating at pulse repetition rates around 1 MHz, it also affords reduced photobleaching rates by allowing the fluorophore to relax from excitable metastable dark states involved in photodegradation. The imaging of dense nanoparticles and of the microtubular network of mammalian cells evidences a spatial resolution of 30-50 nm in the focal plane, i.e. by a factor of 8-9 beyond the diffraction barrier.

Three-Dimensional Stimulated Emission Depletion Microscopy of Nitrogen-Vacancy Centers in Diamond Using Continuous-Wave Light

Charged nitrogen-vacancy (NV) color centers in diamond are excellent luminescence sources for far-field fluorescence nanoscopy by stimulated emission depletion (STED). Here we show that these photostable color centers can be visualized by STED using simple continuous-wave or high repetition pulsed lasers (76 MHz) at wavelengths >700 nm for STED. Furthermore, we show that NV centers can be imaged in three dimensions (3D) inside the diamond crystal and present single-photon signatures of single color centers recorded in high density samples, demonstrating a new recording scheme for STED and related far-field nanoscopy approaches. Finally, we exemplify the potential of using nanodiamonds containing NV centers as luminescence tags in STED microscopy. Our results offer new experimental avenues in nanooptics, nanotechnology, and the life sciences.

Far-field fluorescence nanoscopy of diamond color centers by ground state depletion

We report on two modalities of lens-based fluorescence microscopy with diffraction-unlimited resolution relying on the depletion of the fluorophore ground state. The first version utilizes a beam with a deep intensity minimum, such as a doughnut, for intense excitation followed by mathematical deconvolution, whereas in the second version, a regularly focused beam is added for generating the image directly. In agreement with theory, the subdiffraction resolution scales with the square root of the intensity depleting the ground state. Applied to the imaging of color centers in diamond our measurements evidence a resolving power down to ≈7.6 nm, corresponding to 1/70 of the wavelength of light employed. Our study underscores the key role of exploiting (molecular) states for overcoming the diffraction barrier in far-field optical microscopy.

Direct Light‐Driven Modulation of Luminescence from Mn‐Doped ZnSe Quantum Dots

Quantum dot (QD) nanocrystals remain at the forefront of fluorescence microscopy as they have the advantages of enhanced photostability, high quantum yield, and macromolecular size. Furthermore, the ability to tune the QD fluorescence, either by changing their size or by doping, allows for multiplexed imaging. The range of applications extends well beyond the realm of microscopy: QDs may also play a major role in developing novel photonic devices including lasers, light-emitting diodes, and displays. Despite significant advancements in nanocrystal research, the inability to directly modulate the fluorescence from QDs has precluded their implementation in several areas. In particular, emerging far-field diffraction-unlimited microscopy techniques uniquely benefit from the capability to reversibly modulate/switch fluorescent ensembles from a bright “on” state to a dark “off” state. This activation must occur as a response to optical stimuli which do not contain spectral components within the excitation kernel of the fluorescent markers. With the need for optical control over QD fluorescence, indirect methods have been conceived by using hybrid QD structures that incorporate a photochromic activator/quencher. Although the concept has been clearly established, hybrid QD structures suffer from inherent drawbacks, such as inadequate photostability, limited fluorescence quenching, and sensitivity to local environment/ solvent. Herein we report on the direct light-driven modulation of QD fluorescence. The mechanism for the fluorescence modulation relies only on internal electronic transitions within Mn-doped ZnSe quantum dots (Mn-QDs). It is demonstrated that the fluorescence of the QD can be reversibly depleted with efficiencies of over 90% by using continuous-wave optical intensities of approximately 1.9 MWcm . Time-domain measurements during the modulation indicate that the number of fluorescent on–off cycles exceeds 10 before a significant reduction in the fluorescence quantum efficiency occurs. Such robust nanometric probes having remotely controllable optical transitions are useful in many areas of research, particularly in far-field nanoscopy based on reversible saturable or switchable optical fluorescence transitions (RESOLFT). Consequently, we show that implementation of Mn-QDs for imaging leads to an increase in the resolution by a factor of 4.4 over that of confocal microscopy. A schematic diagram of the electronic transitions involved in light-modulated fluorescence from Mn-QDs is shown in Figure 1a. Initially, electrons are photoexcited from the

STED with wavelengths closer to the emission maximum

In stimulated emission depletion (STED) nanoscopy the wavelength of the STED beam is usually tuned towards the red tail of the emission maximum of the fluorophore. Shifting the STED wavelength closer to the emission peak, i.e. towards the blue region, favorably increases the stimulated emission cross-section. However, this blue-shifting also increases the probability to excite fluorophores that have remained in their ground state, compromising the image contrast. Here we present a method to exploit the higher STED efficiency of blue-shifted STED beams while maintaining the contrast in the image. The method is exemplified by imaging immunolabeled features in mammalian cells with an up to 3-fold increased STED efficiency compared to that encountered in standard STED nanoscopy implementations.

Far-field optical nanoscopy with reduced number of state transition cycles.

We report on a method to reduce the number of state transition cycles that a molecule undergoes in far-field optical nanoscopy of the RESOLFT type, i.e. concepts relying on saturable (fluorescence) state transitions induced by a spatially modulated light pattern. The method is exemplified for stimulated emission depletion (STED) microscopy which uses stimulated emission to transiently switch off the capability of fluorophores to fluoresce. By switching fluorophores off only if there is an adjacent fluorescent feature to be recorded, the method reduces the number of state transitions as well as the average time a dye is forced to reside in an off-state. Thus, the photobleaching of the sample is reduced, while resolution and recording speed are preserved. The power of the method is exemplified by imaging immunolabeled glial cells with up to 8-fold reduced photobleaching.

Microscopy: Light from the dark.

Fluorescence microscopy is the most popular way to image biomolecules, but it leaves many of them in the dark. Non-fluorescent, light-absorbing molecules can now be viewed by a method that turns them into mini-lasers.

Dark state photophysics of nitrogen-vacancy centres in diamond

Nitrogen-vacancy (NV) colour centres in diamond are attractive fluorescence emitters owing to their unprecedented photostability and superior applicability to spin manipulation and sub-diffraction far-field optical microscopy. However, some applications are limited by the co-occurrence of dark state population and optical excitation. In this paper, we use fluorescence microscopy and correlation spectroscopy on single negatively charged NV centres in type IIa bulk diamond to unravel the population kinetics of a >100s long-lived dark state. The bright-dark state interconversion rates show a quadratic dependence on the applied laser intensity, which implies that higher excited states are involved. Depopulation of the dark state becomes less effective at wavelengths above 532nm, resulting in a complete fluorescence switch-off at wavelengths >600nm. This switch is reversible by the addition of shorter wavelengths. This behaviour can be explained by a model consisting of three dark and three bright states of different excitation levels, with the most efficient interconversion via the respective higher excited states. This model accounts for

STED and related concepts for far-field optical nanoscopy

Far-field fluorescence microscopy is the most frequently applied microscopy technique in life sciences. Its strength is the unique combination of highly attractive features such as molecular specificity, simple sample preparation, possibility of 3D imaging and operation under ambient, live cell compatible, conditions. The main shortcoming in comparison to methods, such as electron microscopy, is its resolution which is limited by diffraction to Deltar ~ lambda /(2NA) where lambda denotes the wavelength and NA refers to the numerical aperture. This leads typically to a resolution of approx 200nm laterally and 500nm axially. In 1994 STED (stimulated emission depletion) was invented providing the possibility to combine the advantages of far-field fluorescence microscopy with virtually unlimited resolution.

Light from the dark: Microscopy

Fluorescence microscopy is the most popular way to image biomolecules, but it leaves many of them in the dark. Non-fluorescent, light-absorbing molecules can now be viewed by a method that turns them into mini-lasers.