Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy

Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.

Non-Invasive Confocal Fluorescence Imaging of Mice Beyond 1700 nm Using Superconducting Nanowire Single-Photon Detectors

Light scattering by biological tissues sets a limit to the penetration depth of high-resolution optical microscopy imaging of live mammals in vivo. An effective approach to reduce light scattering and increase imaging depth is by extending the excitation and emission wavelengths to the > 1000 nm second near-infrared (NIR-II), also called the short-wavelength infrared (SWIR) window. Here, we developed biocompatible core-shell lead sulfide/cadmium sulfide (PbS/CdS) quantum dots emitting at ~1880 nm and superconducting nanowire single photon detectors (SNSPD) for single-photon detection up to 2000 nm, enabling one-photon fluorescence imaging window in the 1700-2000 nm (NIR-IIc) range. Confocal fluorescence imaging in NIR-IIc reached an imaging depth of ~ 800 μm through intact mouse head, and enabled non-invasive imaging of inguinal lymph nodes (LNs) without any surgery. In vivo molecular imaging of high endothelial venules (HEVs) with diameter down to ~ 6.6 μm in the lymph nodes was achieved, opening the possibility of non-invasive imaging of immune trafficking in lymph nodes at the single-cell/vessel level longitudinally.

A Novel NIR Fluorescent Probe for Highly Selective Detection of Nitroreductase and Hypoxic-Tumor-Cell Imaging

Nitroreductase as a potential biomarker for aggressive tumors has received extensive attention. In this work, a novel NIR fluorescent probe for nitroreductase detection was synthesized. The probe Py-SiRh-NTR displayed excellent sensitivity and selectivity. Most importantly, the confocal fluorescence imaging demonstrated that HepG-2 cells treated with Py-SiRh-NTR under hypoxic conditions showed obvious enhanced fluorescence, which means that the NTR was overexpressed under hypoxic conditions. Moreover, the probe showed great promise that could help us to study related anticancer mechanisms research.

The initiation knot is a signaling center required for molar tooth development

ABSTRACT Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.

The Initiation Knot is a Signaling Center Required for Molar Tooth Development

SummarySignaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determine tooth patterning. Preceding the first, primary EK, a transient epithelial thickening appears whose significance remains debated. Here, using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK) that is required for the progression of tooth development. IK cell dynamics manifest the hallmarks of a signaling center; cell cycle exit, condensation, and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of high Wnt activity cells to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.

Ca2+ waves coordinate purinergic receptor–evoked integrin activation and polarization

Cells sense extracellular nucleotides through the P2Y class of purinergic G protein–coupled receptors (GPCRs), which stimulate integrin activation through signaling events, including intracellular Ca2+ mobilization. We investigated the relationship between P2Y-stimulated repetitive Ca2+ waves and fibrinogen binding to the platelet integrin αIIbβ3 (GPIIb/IIIa) through confocal fluorescence imaging of primary rat megakaryocytes. Costimulation of the receptors P2Y1 and P2Y12 generated a series of Ca2+ transients that each induced a rapid, discrete increase in fibrinogen binding. The peak and net increase of individual fibrinogen binding events correlated with the Ca2+ transient amplitude and frequency, respectively. Using BAPTA loading and selective receptor antagonists, we found that Ca2+ mobilization downstream of P2Y1 was essential for ADP-evoked fibrinogen binding, whereas P2Y12 and the kinase PI3K were also required for αIIbβ3 activation and enhanced the number of Ca2+ transients. ADP-evoked fibrinogen binding was initially uniform over the cell periphery but subsequently redistributed with a polarity that correlated with the direction of the Ca2+ waves. Polarization of αIIbβ3 may be mediated by the actin cytoskeleton, because surface-bound fibrinogen is highly immobile, and its motility was enhanced by cytoskeletal disruption. In conclusion, spatial and temporal patterns of Ca2+ increase enable fine control of αIIbβ3 activation after cellular stimulation. P2Y1-stimulated Ca2+ transients coupled to αIIbβ3 activation only in the context of P2Y12 coactivation, thereby providing an additional temporal mechanism of synergy between these Gq- and Gi-coupled GPCRs.