Multispectral imaging and time-resolved imaging are two common acquisition schemes in fluorescence microscopy, and their combination can be beneficial to increase specificity. The multidimensionality of the dataset (space, time, and spectrum) introduces some challenges, such as the acquisition of big datasets and long measurement times. In this work, we present a time-resolved multispectral fluorescence microscopy system with a short measurement time, achieved by exploiting Compressive Sensing (CS) based on the Single-Pixel Camera (SPC) scheme. Data Fusion (DF) with a high-resolution camera allows us to tackle the problem of low spatial resolution, typical of SPC. The combined use of SPC, CS, and DF, in which hardware and algorithms are integrated, represents a computational imaging framework to reduce the number of measurements while preserving the information content. This approach has been exploited to demonstrate a zoom feature without moving the optical system. We describe and characterize the system in terms of spatial, spectral, and temporal properties, along with validation on a cellular sample.

Bio-hybrid actuators are devices based on muscle cells or tissues, designed to exploit the force generated by the biological element to execute an action or a movement. Typically, cells and tissue control are achieved electrically by using metal electrodes implanted in living elements. In this Perspective, we focus our attention on an alternative stimulation approach based on light. We highlight the potential of light stimulation in terms of performance and discuss the comparison with classical electrical stimulation. Finally, we discuss future challenges and possible applications of light-controlled actuators.

Light is recognized as an accurate and noninvasive tool for stimulating excitable cells. Here, we report on a non-genetic approach based on organic molecular phototransducers that allows wiring- and electrode-free tissue modulation. As a proof of concept, we show photostimulation of an in vitro cardiac microphysiological model mediated by an amphiphilic azobenzene compound that preferentially dwells in the cell membrane. Exploiting this optical based stimulation technology could be a disruptive approach for highly resolved cardiac tissue stimulation.

Non-genetic photostimulation, which allows for control over cellular activity via the use of cell-targeting phototransducers, is widely used nowadays to study and modulate/restore biological functions. This approach relies on non-covalent interactions between the phototransducer and the cell membrane, thus implying that cell conditions and membrane status can dictate the effectiveness of the method. For instance, although immortalized cell lines are traditionally used in photostimulation experiments, it has been demonstrated that the number of passages they undergo is correlated to the worsening of cell conditions. In principle, this could impact cell responsivity against exogenous stressors, including photostimulation. However, these aspects have usually been neglected in previous experiments. In this work, we investigated whether cell passages could affect membrane properties (such as polarity and fluidity). We applied optical spectroscopy and electrophysiological measurements in two different biological models: (i) an epithelial immortalized cell line (HEK-293T cells) and (ii) liposomes. Different numbers of cell passages were compared to a different morphology in the liposome membrane. We demonstrated that cell membranes show a significant decrease in ordered domains upon increasing the passage number. Furthermore, we observed that cell responsivity against external stressors is markedly different between aged and non-aged cells. Firstly, we noted that the thermal-disordering effect that is usually observed in membranes is more evident in aged cells than in non-aged ones. We then set up a photostimulation experiment by using a membrane-targeted azobenzene as a phototransducer (Ziapin2). As an example of a functional consequence of such a condition, we showed that the rate of isomerization of an intramembrane molecular transducer is significantly impaired in aged cells. The reduction in the photoisomerization rate translates in cells with a sustained reduction of the Ziapin2-related hyperpolarization of the membrane potential and an overall increase in the molecule fluorescence. Overall, our results suggest that membrane stimulation strongly depends on membrane order, highlighting the importance of cell passage during the characterization of the stimulation tools. This study can shine light on the correlation between aging and the development of diseases driven by membrane degradation as well as on the different cell responsivities against external stressors, such as temperature and photostimulation.