Ex vivo-generated red blood cells are a promising resource for future safe blood products, manufactured independently of voluntary blood donations. The physiological process of terminal maturation from spheroid reticulocytes to biconcave erythrocytes has not been accomplished yet. A better biomechanical characterization of cultured red blood cells (cRBCs) will be of utmost interest for manufacturer approval and therapeutic application. Here, we introduce a novel optical tweezer (OT) approach to measure the deformation and elasticity of single cells trapped away from the coverslip. To investigate membrane properties dependent on membrane lipid content, two culture conditions of cRBCs were investigated, cRBC^Plasma with plasma and cRBC^HPL supplemented with human platelet lysate. Biomechanical characterization of cells under optical forces proves the similar features of native RBCs and cRBC^HPL, and different characteristics for cRBC^Plasma. To confirm these results, we also applied a second technique, digital holographic microscopy (DHM), for cells laid on the surface. OT and DHM provided related results in terms of cell deformation and membrane fluctuations, allowing a reliable discrimination between cultured and native red blood cells. The two techniques are compared and discussed in terms of application and complementarity.

The postsynaptic density component PSD-95 undergoes activity-dependent plasticity mechanisms that rely on protein synthesis and structural remodeling. How synaptic activity can influence these dynamics at the single synapse level remains unclear. Here we combine genome-editing, pulse-chase experiments, STED and 3D MINFLUX nanoscopy on hippocampal neuronal cultures to study the integration of newly translated PSD-95 molecules at postsynaptic sites and their rearrangement within individual clusters at near-molecular resolution. We show that the amount of newly translated PSD-95 recruited to individual synapses scales with synaptic size, and modulates in a bidirectional manner, resulting in less new protein following excitatory and more new protein following inhibitory stimulation. Furthermore, we show that within synaptic clusters PSD-95 has a dispersed organization that is largely robust to long-lasting changes in activity. Altogether, this work sheds new light on the mechanisms underlying plasticity at the single synapse level, adding previously inaccessible information.

Neurofilaments are one of the main cytoskeletal components in neurons; they can be found in the form of oligomers at pre- and postsynapses. How their presence is regulated at the postsynapse remains largely unclear. Here we systematically quantified, by immunolabeling, the occurrence of the neurofilament isoform triplet neurofilament light (NFL), medium (NFM), and heavy (NFH) at the postsynapse using STED nanoscopy together with markers of synaptic strength and activity. Our data show that, within dendritic spines, neurofilament isoforms rarely colocalize with each other and that they are present to different extents, with NFL being the most abundant isoform. The amount of the three isoforms correlates with markers of postsynaptic strength and presynaptic activity to varying degrees: NFL shows the highest correlation to both synaptic traits, suggesting its involvement in synaptic response, while NFM exhibits the lowest correlations. By quantifying the presence of neurofilaments at the postsynapse within the context of the synaptic status, this work sheds new light on the regulation of synaptic neurofilaments and their possible contribution to synaptopathies.

The connection between cytoskeleton alterations and diseases is well known and has stimulated research on cell mechanics, aiming to develop reliable biomarkers. In this study, we present results on rheological, adhesion, and morphological properties of primary rat cardiac fibroblasts, the cytoskeleton of which was altered by treatment with cytochalasin D (Cyt-D) and nocodazole (Noc), respectively. We used two complementary techniques: quartz crystal microbalance (QCM) and digital holographic microscopy (DHM). Qualitative data on cell viscoelasticity and adhesion changes at the cell–substrate near-interface layer were obtained with QCM, while DHM allowed the measurement of morphological changes due to the cytoskeletal alterations. A rapid effect of Cyt-D was observed, leading to a reduction in cell viscosity, loss of adhesion, and cell rounding, often followed by detachment from the surface. Noc treatment, instead, induced slower but continuous variations in the rheological behavior for four hours of treatment. The higher vibrational energy dissipation reflected the cell’s ability to maintain a stable attachment to the substrate, while a cytoskeletal rearrangement occurs. In fact, along with the complete disaggregation of microtubules at prolonged drug exposure, a compensatory effect of actin polymerization emerged, with increased stress fiber formation.

Urinary tract infections are among the most frequent infectious diseases and require screening a great amount of urine samples from patients. However, a high percentage of samples result as negative after urine culture plate tests (CPTs), demanding a simple and fast preliminary technique to screen out the negative samples. We propose a digital holographic microscopy (DHM) method to inspect fresh urine samples flowing in a glass capillary for 3 min, recording holograms at 2 frames per second. After digital reconstruction, bacteria, white and red blood cells, epithelial cells and crystals were identified and counted, and the samples were classified as negative or positive according to clinical cutoff values. Taking the CPT as reference, we processed 180 urine samples and compared the results with those of urine flow cytometry (UFC). Using standard evaluation metrics for our screening test, we found a similar performance for DHM and UFC, indicating DHM as a suitable and fast screening technique retaining several advantages. As a benefit of DHM, the technique is label-free and does not require sample preparation. Moreover, the phase and amplitude images of the cells and other particles present in urine are digitally recorded and can serve for further investigation afterwards.

The cochlea encodes sounds into neural signals at the synapses of inner hair cells (IHCs) and spiral ganglion neurons (SGNs) with remarkable fidelity. To achieve high rates of precise synaptic transmission over long periods, IHCs use ribbon-type active zones (AZs). To understand synaptic sound encoding, we need to decipher the underlying molecular topography of these synapses, which has remained challenging because of technological limitations. Here, we applied three-dimensional minimal flux optical nanoscopy to mouse IHC-SGN synapses to chart the positions of key pre- and postsynaptic proteins with single-digit nanometer resolution. We demonstrate that nanoclusters of ion channels and their interacting proteins govern the topography of AZs and postsynaptic densities (PSDs). We count synaptic proteins and their nanoclusters and determine their spatial organization, feeding into computational modeling of AZ function. In conclusion, this study reveals a nanocluster-based molecular AZ and PSD topography, likely serving as functional modules in synaptic sound encoding.