Photobleaching is a general hurdle of fluorescence-based techniques especially in high-resolution microscopy that relies on prolonged and complex illumination. Strategies to reduce photobleaching require chemical modifications of the cell medium, which often compromise physiological cellular conditions. Here, we outline an all-optical strategy to minimize photobleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we develop imaging schemes to increase the number of on-off photoswitching cycles, either modulating the on-switching light or co-irradiating the RSFPs with light at longer wavelengths with respect to fluorescence excitation. We apply the optimized imaging scheme to achieve imaging multiplexing at high-spatiotemporal resolutions and to record longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

Fluorescence-based single-molecule and fluctuation spectroscopy in the near-IR can open avenues for biomolecular dynamic studies in biological media with suppressed autofluorescence and scattering background. However, further implementation is limited by the lower brightness of NIR fluorophores and available single-photon detector technologies that are still to be explored and adapted. Superconducting nanowire single-photon detectors (snSPDs) have found increasing use in quantum optics and optical communication applications thanks to high sensitivity in the near-infraed (NIR), low dark-counts, no after-pulsing, and high time resolution. Here, we present characterization of fluorescence intensity fluctuations from single vesicles and NIR fluorophores based on fluorescence correlation spectroscopy (FCS), specifically taking advantage of these snSPD properties. We present a concept allowing multiplexed readouts based on only one snSPD, in which the emitted photons are separated by their emission wavelength into different optical paths, thereby translating the emission wavelengths into different arrival times onto the snSPD. This concept allows one-laser-one-detector, dual-color fluorescence cross-correlation spectroscopy (FCCS) measurements, with fluorescence intensity fluctuations of two fluorophore species separately analyzed and cross-correlated. It is shown how two fluorophore species in a sample can be distinguished by their different blinking kinetics, fluorescence lifetimes, and/or diffusion properties. Apart from differences in emission spectra, the presented concept for multiplexing using a single detector can also be applied to distinguish emitters by properties such as polarization, coherence lengths, and fluorescence bunching and antibunching signatures. It can also be generalized to other modalities than FCS, including single-molecule detection, confocal microscopy, and imaging.

Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans-cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.

Lanthanide-doped upconversion nanoparticles (UCNPs) have attractive emission properties but suffer from weak light-absorbing capacities and thereby relatively low brightnesses. This motivates using strongly absorbing dye molecules as antennas and sensitizers. However, despite much effort, understanding of this dye-UCNP interplay is still limited. Major sensitization mechanisms are still under discussion, largely because there is a lack of effective means to observe key factors such as dark state transitions within the dyes. Here, a combined spectroscopic procedure is established to systematically investigate the photophysics behind the dye-UCNP interaction, embracing fluorescence-based transient-state excitation-modulation, lifetime and correlation spectroscopy, and spectrofluorometry/spectrophotometry. With this procedure the heptamethine cyanine dye IR806, a typical UCNP sensitizer is studied, its photophysical model is established, its photophysics in UCL-sensitization-related environments is deciphered, and the energy transfer from the IR806 singlet excited state to Yb³⁺ (UCNP sensitizer ion) can be identified as the dominant sensitization mechanism. These studies suggest that IR806 can form non-emissive H-aggregates at the nanoparticle surfaces, which can be dissociated after certain light excitation duration (typically>100 µs). Moreover, buildup of a non-fluorescent, photo-redox state of IR806 after longer irradiation times (10–100 ms) can deleteriously affect its UCL sensitization effect, inferring an optimal excitation duration for dye-sensitized UCNPs, relevant for, e.g., optical imaging applications.

We have developed an Ir(PPy)₃ photoredox-catalyzed cross-coupling reaction that allows installation of quinoxalinones at the C7 position of thiazolino ring-fused 2-pyridones (TRPs) under mild conditions. The methodology tolerates various substituted quinoxalinones and biologically relevant substituents on the C8 position of the TRP. The TRP scaffold has large potential in the development of lead compounds, and while the coupled products are interesting from a drug-development perspective, the methodology will be useful for developing more potent and drug-like TRP-based candidates.