Everlasting rhodamine dyes and true deciding factors in their STED microscopy performance.

The authors took an independent and closer look at the family of red-emitting rhodamine dyes known for a decade due to their excellent performance in STED microscopy. After the family was further extended, the true grounds of this performance became clear. Small-molecule protective agents and/or auxiliary groups were attached at two different sites of the dye's scaffold. Thus, a rhodamine core, which is already quite photostable as it is, and an intramolecular stabilizer - a 4-nitrobenzyl or a 4-nitrobenzylthio group were combined to give potentially "everlasting dyes". The fluorescence quantum yields (Φf) and the fluorescence lifetimes (τ) of the modified dyes were thoroughly measured with comparison to those of the parent dyes. The correlation of their STED performance with photostability and fluorescence color stability under illumination in water were explored. Unexpectedly, the anaerobic GSDIM (GOC) buffer proved unhelpful with respect to STED performance. It was demonstrated that, even dyes with a Φf of only 14-17% allow STED imaging with a sufficient photon budget and good signal-to-noise ratio. For the dyes with photostabilizing groups (PSG) the Φf values are 4-5 times lower than in the reference dyes, and lifetimes τ are also strongly reduced. Noteworthy are very high fluorescence color stability and constant or even increasing fluorescence signal under photobleaching in bulk aqueous solutions, which suggests a sacrificing role of the 4-nitrobenzyl-containing moieties. Straightforward and improved recipes for "last-minute" modifications and preparations of "self-healing" red-emitting fluorescent tags are described.

Three dimensional live-cell STED microscopy at increased depth using a water immersion objective.

Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 µm.

Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection.

The extension of fluorescence nanoscopy to larger numbers of molecular species concurrently visualized by distinct markers is of great importance for advanced biological applications. To date, up to four markers had been distinguished in STED experiments featuring comparatively elaborate imaging schemes and optical setups, and exploiting various properties of the fluorophores. Here we present a simple yet versatile STED design for multicolour imaging below the diffraction limit. A hyperspectral detection arrangement (hyperSTED) collects the fluorescence in four spectral channels, allowing the separation of four markers with only one excitation wavelength and a single STED beam. Unmixing of the different marker signals based on the simultaneous readout of all channels is performed with a non-negative matrix factorization algorithm. We illustrate the approach showing four-colour nanoscopy of fixed and living cellular samples.

SRpHi ratiometric pH biosensors for super-resolution microscopy.

Fluorescence-based biosensors have become essential tools for modern biology, allowing real-time monitoring of biological processes within living cells. Intracellular fluorescent pH probes comprise one of the most widely used families of biosensors in microscopy. One key application of pH probes has been to monitor the acidification of vesicles during endocytosis, an essential function that aids in cargo sorting and degradation. Prior to the development of super-resolution fluorescence microscopy (nanoscopy), investigation of endosomal dynamics in live cells remained difficult as these structures lie at or below the ~250 nm diffraction limit of light microscopy. Therefore, to aid in investigations of pH dynamics during endocytosis at the nanoscale, we have specifically designed a family of ratiometric endosomal pH probes for use in live-cell STED nanoscopy.Ratiometric fluorescent pH probes are useful tools to monitor acidification of vesicles during endocytosis, but the size of vesicles is below the diffraction limit. Here the authors develop a family of ratiometric pH sensors for use in STED super-resolution microscopy, and optimize their delivery to endosomes.