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Luminescence thermometry represents a technique of choice for measurements in small objects and imaging of temperature distribution. However, most state‐of‐the‐art luminescent probes are limited in spectral characteristics, brightness, photostability, and sensitivity. Molecular thermometers of the new generation utilizing air and moisture‐stable zirconium(IV) pyridinedipyrrolide complexes can address all these limitations. The dyes emit pure thermally activated delayed fluorescence without any prompt fluorescence and show a unique combination of attractive features: a) visible light excitation and emission in the orange/red region, b) high luminescence brightness (quantum yields ≈0.5 in toluene and 0.8–1.0 in polystyrene matrix), c) excellent photostability, d) suitability for two‐photon excitation and e) mono‐exponential decay on the order of tens to hundreds of microseconds with strongly temperature‐dependent lifetimes (between −2.5 and −2.9% K−1 in polystyrene at 25 °C). Immobilization in gas‐blocking polymers yields sensing materials for self‐referenced decay time read‐out that are manufactured in two common formats: planar optodes and water‐dispersible nanoparticles. Positively charged nanoparticles are demonstrated to be suitable for nanothermometry in live cells and multicellular spheroids. Negatively charged nanoparticles represent advanced analytical tools for imaging temperature gradients in samples of small volumes such as microfluidic devices.

The importance of extracellular gradients of biomolecules becomes increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, dynamics of extracellular calcium concentration is rarely studied. Here, we present low affinity Ca 2+ biosensor based on Twitch-2B fluorescent protein fused with the celluloseand collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable for scaffold-based biosensing of Ca 2+ in proximity of live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one-and twophoton excitation modes. Furthermore, the donor fluorescence lifetime of ColBD-Twitch displays response to [Ca 2+ ] over a range of ~2-2.5 ns, making it attractive biosensor for multiplexed FLIM microscopy assays. To evaluate performance of this biosensor in physiological measurements, we applied ColBD-Twitch to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca 2+ upon chelation with EGTA. When we combined it with spectrally resolved FLIM of lipid droplets using Nile Red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the

To achieve safer patient treatments, serum-free cell culture conditions have to be established for cell therapies. In previous studies, we demonstrated that serum-free culture favored the proliferation of MSCA-1+ osteoprogenitors derived from the jaw periosteum. In this study, the in vitro formation of bone-specific matrix by MSCA-1+ jaw periosteal cells (JPCs, 3 donors) was assessed and compared under serum-free and serum-containing media conditions using the marker-free Raman spectroscopy. Based on a standard fluorescence assay, JPCs from one patient were not able to mineralize under serum-containing culture conditions, whereas the other cells showed similar mineralization levels under both conditions. Raman spectra from mineralizing MSCA-1+ JPCs revealed higher levels of hydroxyapatite formation and higher mineral to matrix ratios under serum-free culture conditions. Higher carbonate to phosphate ratios and higher crystallinity in JPCs cultured under serum-containing conditions indicated immature bone formation. Due to reduced collagen production under serum-free conditions, we obtained significant differences in collagen maturity and proline to hydroxyproline ratios compared to serum-free conditions. We conclude that Raman spectroscopy is a useful tool for the assessment and noninvasive monitoring of in vitro mineralization of osteoprogenitor cells. Further studies should extend this knowledge and improve JPC mineralization by optimizing culture conditions.

In a variety of diseases, from benign to life-threatening ones, inflammation plays a major role. Monitoring the intensity and extent of a multifaceted inflammatory process has become a cornerstone in diagnostics and therapy monitoring. However, the current tools lack the ability to provide insight into one of its most crucial aspects, namely, the alteration of the extracellular matrix (ECM). Using a radiolabeled platelet glycoprotein VI-based ECM-targeting fusion protein (GPVI-Fc), we investigated how binding of GPVI-Fc on fibrous tissue could uncover the progression of several inflammatory disease models at different stages (rheumatoid arthritis, cutaneous delayed-type hypersensitivity, lung inflammation and experimental autoimmune encephalomyelitis). Methods: The fusion protein GPVI-Fc was covalently linked to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and subsequently labeled with 64 Cu. We analyzed noninvasively in vivo 64 Cu-GPVI-Fc accumulation in murine cutaneous delayed-type hypersensitivity, anti-glucose-6-phosphate isomerase serum-induced rheumatoid arthritis, lipopolysaccharide-induced lung inflammation and an experimental autoimmune encephalomyelitis model. Static and dynamic Positron Emission Tomography (PET) of the radiotracer distribution was performed in vivo , with ex vivo autoradiography confirmation, yielding quantitative accumulation and a distribution map of 64 Cu-GPVI-Fc. Ex vivo tissue histological staining was performed on harvested samples to highlight the fusion protein binding to collagen I, II and III, fibronectin and fibrinogen as well as the morphology of excised tissue. Results: 64 Cu-GPVI-Fc showed a several-fold increased uptake in inflamed tissue compared to control tissue, particularly in the RA model, with a peak 24 h after radiotracer injection of up to half the injected dose. Blocking and isotype control experiments indicated a target-driven accumulation of the radiotracer in the case of chronic inflammation. Histological analysis confirmed a prolonged accumulation at the inflammation site, with a pronounced colocalization with the different components of the ECM (collagen III and fibronectin notably). Binding of the fusion protein appeared to be specific to the ECM but unspecific to particular components. Conclusion: Imaging of 64 Cu-GPVI-Fc accumulation in the ECM matrix appears to be a promising candidate for monitoring chronic inflammation. By binding to exposed fibrous tissue (collagen, fibronectin, etc.) after extravasation, a new insight is provided into the fibrotic events resulting from a prolonged inflammatory state.

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The importance of extracellular gradients of biomolecules becomes increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, dynamics of extracellular calcium concentration is rarely studied. Here, we present low affinity Ca 2+ biosensor based on Twitch-2B fluorescent protein fused with the celluloseand collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable for scaffold-based biosensing of Ca 2+ in proximity of live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one-and twophoton excitation modes. Furthermore, the donor fluorescence lifetime of ColBD-Twitch displays response to [Ca 2+ ] over a range of ~2-2.5 ns, making it attractive biosensor for multiplexed FLIM microscopy assays. To evaluate performance of this biosensor in physiological measurements, we applied ColBD-Twitch to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca 2+ upon chelation with EGTA. When we combined it with spectrally resolved FLIM of lipid droplets using Nile Red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the extracellular Ca 2+ depletion, suggesting that intestinal epithelium can respond to and compensate such treatment. Altogether, our results demonstrate ColBD-Twitch as a prospective Ca 2+ sensor for multiplexed FLIM analysis in a complex 3D tissue environment. IntroductionExtracellular cues become increasingly important to understand tissue organization and development. For instance, building-up the tissue from the stem cell-derived organoids demands a deep understanding of the timedependent changes of biophysical parameters experienced during growth and differentiation [1][2][3]. In addition to the physical tension [4] and the extracellular matrix itself [5], changing cell density, composition and organization can also affect gradients of O2 (hypoxia) [6,7], distribution and diffusion of reactive oxygen species, peptides and growth factors, pH and other ions such as Ca 2+ [8,9]. A number of reports have indicated the role of extracellular Ca 2+ as one the primary signals that influence cell function [10]: thus, regulation of extracellular Ca 2+ have been shown to be relevant to gastrointestinal tract function [11,12], bone marrow [13], brain and CNS function [14, 15], smooth and skeletal muscles [16], wound healing [17], plasma membrane repair [18], engineered organoid-like tissues [19] and microbial biofilm formation [20]. Every mammalian cell relies on the Ca 2+ homeostasis, which in turn is tightly regulated through the activities of channels, ATPases, Na + / Ca 2+ exchanger, cytosolic Ca 2+ -binding proteins and various depots such as endoplasmic reticulum, secretory vesicles and mitochondria [21][22][23][24]. Calcium influx and efflux processes thr...