Mechanical forces are key regulators of cellular behavior and function, affecting many fundamental biological processes such as cell migration, embryogenesis, immunological responses, and pathological states. Specialized force sensors and imaging techniques have been developed to quantify these otherwise invisible forces in single cells and in vivo. However, current techniques rely heavily on high-resolution microscopy and do not allow interrogation of optically dense tissue, reducing their application to 2D cell cultures and highly transparent biological tissue. Here, we introduce DEFORM, deformable microlaser force sensing, a spectroscopic technique that detects sub-nanonewton forces with unprecedented spatio-temporal resolution. DEFORM is based on the spectral analysis of laser emission from dye-doped oil microdroplets and uses the force-induced lifting of laser mode degeneracy in these droplets to detect nanometer deformations. Following validation by atomic force microscopy and development of a model that links changes in laser spectrum to applied force, DEFORM is used to measure forces in 3D and at depths of hundreds of microns within tumor spheroids and late-stage Drosophila larva. We furthermore show continuous force sensing with single-cell spatial and millisecond temporal resolution, thus paving the way for non-invasive studies of biomechanical forces in advanced stages of embryogenesis, tissue remodeling, and tumor invasion.

During locomotion, soft-bodied terrestrial animals solve complex control problems at substrate interfaces, but our understanding of how they achieve this without rigid components remains incomplete. Here, we develop new all-optical methods based on optical interference in a deformable substrate to measure ground reaction forces (GRFs) with micrometre and nanonewton precision in behaving Drosophila larvae. Combining this with a kinematic analysis of substrate-interfacing features, we shed new light onto the biomechanical control of larval locomotion. Crawling in larvae measuring ~1 mm in length involves an intricate pattern of cuticle sequestration and planting, producing GRFs of 1–7 µN. We show that larvae insert and expand denticulated, feet-like structures into substrates as they move, a process not previously observed in soft-bodied animals. These ‘protopodia’ form dynamic anchors to compensate counteracting forces. Our work provides a framework for future biomechanics research in soft-bodied animals and promises to inspire improved soft-robot design.

Mechanical forces and stiffness play key roles in the health and development of cells and tissue, but despite the physical connection between these quantities, they cannot be monitored in parallel in most cases. Here, we introduce a fully integrated microscope that combines a method for high-resolution cell force imaging (elastic resonator interference stress microscopy, ERISM) with non-contact mapping of the elastic properties of cells (via Brillouin microscopy). In order to integrate both techniques, we had to account for the strong back reflection on the surface of the microcavity used for ERISM measurements as well as the local destruction of the cavity under illumination for Brillouin microscopy measurements. Therefore, we developed an elastic optical microcavity with minimal absorption that can perform ERISM measurements without sustaining laser damage during Brillouin microscopy. Furthermore, an unequal-arm Michelson interferometer was designed to suppress the back reflection of the laser on the ERISM microcavity surface using division by amplitude interference to reduce the reflected light and enhance the Brillouin signal. We show the utility of our integrated microscope by simultaneously mapping cellular forces and Brillouin shifts in cultures of fibroblast cells.

AbstractDuring locomotion, soft-bodied terrestrial animals solve complex control problems at substrate interfaces, but our understanding of how they achieve this without rigid components remains incomplete. Here, we develop new all-optical methods based on optical interference in a deformable substrate to measure ground reaction forces (GRFs) with micrometre and nanonewton precision in behavingDrosophilalarvae. Combining this with a kinematic analysis of substrate interfacing features, we shed new light onto the biomechanical control of larval locomotion. Crawling in larvae measuring ∼1 mm in length involves an intricate pattern of cuticle sequestration and planting, producing GRFs of 1-7 µN. We show that larvae insert and expand denticulated, feet-like structures into substrates as they move, a process not previously observed in soft bodied animals. These ‘protopodia’ form dynamic anchors to compensate counteracting forces. Our work provides a framework for future biomechanics research in soft-bodied animals and promises to inspire improved soft-robot design.

Important dynamic processes in mechanobiology remain elusive due to a lack of tools to image the small cellular forces at play with sufficient speed and throughput. Here, we introduce a fast, interference-based force imaging method that uses the illumination of an elastic deformable microcavity with two rapidly alternating wavelengths to map forces. We show real-time acquisition and processing of data, obtain images of mechanical activity while scanning across a cell culture, and investigate sub-second fluctuations of the piconewton forces exerted by macrophage podosomes. We also demonstrate force imaging of beating neonatal cardiomyocytes at 100 fps which reveals mechanical aspects of spontaneous oscillatory contraction waves in between the main contraction cycles. These examples illustrate the wider potential of our technique for monitoring cellular forces with high throughput and excellent temporal resolution.

Central pattern generators (CPGs) are neural networks that produce rhythmic motor activity in the absence of sensory input. CPGs produce 'fictive' behaviours in vitro which parallel activity seen in intact animals. CPG networks have been identified in a wide variety of model organisms and have been shown to be critical for generating rhythmic behaviours such as swimming, walking, chewing and breathing. Work with CPG preparations has led to fundamental advances in neuroscience; however, most CPG preparations involve intensive dissections and require sophisticated electrophysiology equipment, making export to teaching laboratories problematic. Here we present an integrated approach for bringing the study of locomotor CPGs in Drosophila larvae into teaching laboratories. First, we present freely available genetic constructs that enable educators to express genetically encoded calcium indicators in cells of interest in the larval central nervous system. Next, we describe how to isolate the larval central nervous system and prepare it for live imaging. We then show how to modify standard compound microscopes to enable fluorescent imaging using 3D printed materials and inexpensive optical components. Finally, we show how to use the free image analysis programme ImageJ and freely available features in the signal analysis programme DataView to analyse rhythmic CPG activity in the larval CNS. Comparison of results to those obtained on research equipment shows that signal-to-noise levels are comparable and core features of larval CPG activity can be observed. Overall, this work shows the viability of exporting live imaging experiments to low cost environments and paves the way for new teaching laboratory exercises revolving around optical imaging of CPG activity.

Organic light-emitting diodes (OLEDs) possess many unique properties such as large-area emission, fabrication on flexible plastic films, adjustment of the emission spectrum and angular distribution by chemical and optical means, and structuring to high-density arrays with millions of pixels. While especially the latter contributed to commercialization of OLED displays in consumer electronics, the properties of OLEDs also make them highly attractive for biological applications. In this contribution, we present fluorescent blue OLEDs with electrically doped charge transport layers that reach more than 100,000 cd/m² at 5 V. We applied the light source in optogenetics to stimulate primary mouse hippocampal neurons and to evoke sensory response in Drosophila melanogaster larvae using micro-structured OLED pixels. Furthermore, by applying a distributed Bragg reflector, we narrowed the OLED spectrum down in order to enable imaging of neuronal activity using genetically encoded calcium indicators. Finally, we discuss our recent efforts on providing stable encapsulation of flexible OLEDs. These examples show how OLEDs may outperform traditional light sources applied in biophotonics by enabling conformable, bio-compatible, and bright illumination with unprecedented resolution.

Fluorescence imaging is an indispensable tool in biology, with applications ranging from single-cell to whole-animal studies and with live mapping of neuronal activity currently receiving particular attention. To enable fluorescence imaging at cellular scale in freely moving animals, miniaturized microscopes and lensless imagers are developed that can be implanted in a minimally invasive fashion; but the rigidity, size, and potential toxicity of the involved light sources remain a challenge. Here, narrowband organic light-emitting diodes (OLEDs) are developed and used for fluorescence imaging of live cells and for mapping of neuronal activity in Drosophila melanogaster via genetically encoded Ca2+ indicators. In order to avoid spectral overlap with fluorescence from the sample, distributed Bragg reflectors are integrated onto the OLEDs to block their long-wavelength emission tail, which enables an image contrast comparable to conventional, much bulkier mercury light sources. As OLEDs can be fabricated on mechanically flexible substrates and structured into arrays of cell-sized pixels, this work opens a new pathway for the development of implantable light sources that enable functional imaging and sensing in freely moving animals.

This study employed the delayed judgment-of-learning (JOL) paradigm to investigate the content of metacognitive judgments; after studying cue-target word-pairs, participants predicted their ability to remember targets on a future memory test (cued recognition in Experiments 1 and 2 and cued recall in Experiment 3). In Experiment 1 and the confidence JOL group of Experiment 3, participants used a commonly employed 6-point numeric confidence JOL scale (0–20–40–60–80–100%). In Experiment 2 and the binary JOL group of Experiment 3 participants first made a binary yes/no JOL prediction followed by a 3-point verbal confidence judgment (sure-maybe-guess). In all experiments, on a subset of trials, participants gave a written justification of why they gave that specific JOL response. We used natural language processing techniques (latent semantic analysis and word frequency [n-gram] analysis) to characterize the content of the written justifications and to capture what types of evidence evaluation uniquely separate one JOL response type from others. We also used a machine learning classification algorithm (support vector machine [SVM]) to quantify the extent to which any two JOL responses differed from each other. We found that: (i) participants can justify and explain their JOLs; (ii) these justifications reference cue familiarity and target accessibility and so are particularly consistent with the two-stage metacognitive model; and (iii) JOL confidence judgements do not correspond to yes/no responses in the manner typically assumed within the literature (i.e. 0–40% interpreted as no predictions).