Local monitoring of photosensitizer transient states provides feedback for enhanced efficiency and targeting selectivity in photodynamic therapy.

Photodynamic therapy (PDT) fundamentally relies on local generation of PDT precursor states in added photosensitizers (PS), particularly triplet and photo-radical states. Monitoring these states in situ can provide important feedback but is difficult in practice. The states are strongly influenced by local oxygenation, pH and redox conditions, often varying significantly at PDT treatment sites.

Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging.

Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7.

Fast, streamlined fluorescence nanoscopy resolves rearrangements of SNARE and cargo proteins in platelets co-incubated with cancer cells.

Background: Increasing evidence suggests that platelets play a central role in cancer progression, with altered storage and selective release from platelets of specific tumor-promoting proteins as a major mechanism. Fluorescence-based super-resolution microscopy (SRM) can resolve nanoscale spatial distribution patterns of such proteins, and how they are altered in platelets upon different activations. Analysing such alterations by SRM thus represents a promising, minimally invasive strategy for platelet-based diagnosis and monitoring of cancer progression.

Imaging Fluorescence Blinking of a Mitochondrial Localization Probe: Cellular Localization Probes Turned into Multifunctional Sensors.

Mitochondrial membranes and their microenvironments directly influence and reflect cellular metabolic states but are difficult to probe on site in live cells. Here, we demonstrate a strategy, showing how the widely used mitochondrial membrane localization fluorophore 10-nonyl acridine orange (NAO) can be transformed into a multifunctional probe of membrane microenvironments by monitoring its blinking kinetics. By transient state (TRAST) studies of NAO in small unilamellar vesicles (SUVs), together with computational simulations, we found that NAO exhibits prominent reversible singlet-triplet state transitions and can act as a light-induced Lewis acid forming a red-emissive doublet radical.

Deciphering Residual Emissions: Time-dependent Models for the Nonthermal Interstellar Radiation from the Milky Way.

Cosmic rays (CRs) in the Galaxy are an important dynamical component of the interstellar medium (ISM) that interact with the other major components (interstellar gas and magnetic and radiation fields) to produce broadband interstellar emissions that span the electromagnetic spectrum. The standard modeling of CR propagation and production of the associated emissions is based on a steady-state assumption, where the CR source spatial density is described using a smoothly varying function of position that does not evolve with time. While this is a convenient approximation, reality is otherwise, where primary CRs are produced in and about highly localized regions, e.

Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells.

Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine diphosphate and thromboxane A2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A).

Stimulated Emission Depletion Microscopy.

Despite its short history, diffraction-unlimited fluorescence microscopy techniques have already made a substantial imprint in the biological sciences. In this review, we describe how stimulated emission depletion (STED) imaging originally evolved, how it compares to other optical super-resolution imaging techniques, and what advantages it provides compared to previous golden-standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. We outline the prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics, and describe major evolving strategies for how to push the borders of STED imaging even further in life science.

Fluorescence-based characterization of non-fluorescent transient states of tryptophan - prospects for protein conformation and interaction studies.

Tryptophan fluorescence is extensively used for label-free protein characterization. Here, we show that by analyzing how the average tryptophan fluorescence intensity varies with excitation modulation, kinetics of tryptophan dark transient states can be determined in a simple, robust and reliable manner. Thereby, highly environment-, protein conformation- and interaction-sensitive information can be recorded, inaccessible via traditional protein fluorescence readouts.

STED microscopy - towards broadened use and scope of applications.

High resolution Stimulated Emission Depletion (STED) microscopy has been demonstrated for fundamental studies in cells, living tissue and organisms. Today, a major trend in the STED technique development is to make the instruments simpler and more user-friendly, without compromising performance. This has become possible by new low-cost, turn-key laser technology and by implementing specifically designed phase plates and polarization elements, extending and simplifying the shaping of the laser beam profiles.