Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

Distributed and Localized Dynamics Emerge in the Mouse Neocortex during Reach-to-Grasp Behavior.

A long-standing question in systems neuroscience is to what extent task-relevant features of neocortical processing are localized or distributed. Coordinated activity across the neocortex has been recently shown to drive complex behavior in the mouse, while activity in selected areas is canonically associated with specific functions (e.g., movements in the case of the motor cortex). Reach-to-grasp (RtG) movements are known to be dependent on motor circuits of the neocortex; however, the global activity of the neocortex during these movements has been largely unexplored in the mouse. Here, we characterized, using wide-field calcium imaging, these neocortex-wide dynamics in mice of either sex engaging in an RtG task. We demonstrate that, beyond motor regions, several areas, such as the visual and the retrosplenial cortices, also increase their activity levels during successful RtGs, and homologous regions across the ipsilateral hemisphere are also involved. Functional connectivity among neocortical areas increases transiently around movement onset and decreases during movement. Despite this global phenomenon, neural activity levels correlate with kinematics measures of successful RtGs in sensorimotor areas only. Our findings establish that distributed and localized neocortical dynamics co-orchestrate efficient control of complex movements.SIGNIFICANCE STATEMENT Mammals rely on reaching and grasping movements for fine-scale interactions with the physical world. In the mouse, the motor cortex is critical for the execution of such behavior, yet little is known about the activity patterns across neocortical areas. Using the mesoscale-level networks as a model of cortical processing, we investigated the hypothesis that areas beyond the motor regions could participate in RtG planning and execution, and indeed a large network of areas is involved while performing RtGs. Movement kinematics correlates mostly with neural activity in sensorimotor areas. By demonstrating that distributed and localized neocortical dynamics for the execution of fine movements coexist in the mouse neocortex during RtG, we offer an unprecedented view on the neocortical correlates of mammalian motor control.

Optimization of highly inclined illumination for diffraction-limited and super-resolution microscopy.

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and of how this impacts on the image quality complicates the setting of HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the effects of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 µm). Based on this, we present a simple optical solution that exploits a rectangular slit to reduce the inclined beam thickness down to 2.6 µm while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

Studying the trafficking of labeled trodusquemine and its application as nerve marker for light-sheet and expansion microscopy.

Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.

Imaging Approaches to Investigate Pathophysiological Mechanisms of Brain Disease in Zebrafish.

Zebrafish has become an essential model organism in modern biomedical research. Owing to its distinctive features and high grade of genomic homology with humans, it is increasingly employed to model diverse neurological disorders, both through genetic and pharmacological intervention. The use of this vertebrate model has recently enhanced research efforts, both in the optical technology and in the bioengineering fields, aiming at developing novel tools for high spatiotemporal resolution imaging. Indeed, the ever-increasing use of imaging methods, often combined with fluorescent reporters or tags, enable a unique chance for translational neuroscience research at different levels, ranging from behavior (whole-organism) to functional aspects (whole-brain) and down to structural features (cellular and subcellular). In this work, we present a review of the imaging approaches employed to investigate pathophysiological mechanisms underlying functional, structural, and behavioral alterations of human neurological diseases modeled in zebrafish.

A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy.

The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence nuclear and eosin Y staining that enabled us to obtain hematoxylin and eosin pseudo-coloring comparable with the gold standard H&E analysis. The computational reconstructions obtained with 3D optical imaging can be analyzed by a pathologist without any specific training in volumetric microscopy, paving the way for new biomedical applications in clinical pathology.

Structural and Biochemical Changes in Pericardium upon Genipin Cross-Linking Investigated Using Nondestructive and Label-Free Imaging Techniques.

Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

Characterization of substituted piperazines able to reverse MDR in Escherichia coli strains overexpressing resistance-nodulation-cell division (RND) efflux pumps.

BACKGROUND: MDR in bacteria is threatening to public health. Overexpression of efflux pumps is an important cause of MDR. The co-administration of antimicrobial drugs and efflux pump inhibitors (EPIs) is a promising approach to address the problem of MDR. OBJECTIVES: To identify new putative EPIs and to characterize their mechanisms of action. METHODS: The effects of four selected piperazine derivatives on resistance-nodulation-cell division (RND) pumps was evaluated in Escherichia coli strains overexpressing or not expressing RND pumps by assays aimed at evaluating antibiotic potentiation, membrane functionality, ethidium bromide accumulation and AcrB expression. The cytotoxicity of selected piperazines towards primary cultures of human dermal fibroblasts was also investigated. RESULTS: Four molecules enhanced levofloxacin activity against strains overexpressing RND efflux pumps (AcrAB-TolC and AcrEF-TolC), but not against RND pump-deficient strains. They had little effects on membrane potential. Molecule 4 decreased, whereas the other three increased, membrane permeability compared with untreated control cells. The four molecules showed differences in the specificity of interaction with RND efflux pumps, by inactivating the transport of one or more antibiotics, and in the levels of ethidium bromide accumulation and of acrB expression inhibition. CONCLUSIONS: Piperazine derivatives are good candidates as inhibitors of RND efflux pumps. They decreased the activity of RND pumps by mixed mechanisms of action. Small structural differences among the molecules can be critical in defining their behaviour.

Cortical propagation tracks functional recovery after stroke.

Stroke is a debilitating condition affecting millions of people worldwide. The development of improved rehabilitation therapies rests on finding biomarkers suitable for tracking functional damage and recovery. To achieve this goal, we perform a spatiotemporal analysis of cortical activity obtained by wide-field calcium images in mice before and after stroke. We compare spontaneous recovery with three different post-stroke rehabilitation paradigms, motor training alone, pharmacological contralesional inactivation and both combined. We identify three novel indicators that are able to track how movement-evoked global activation patterns are impaired by stroke and evolve during rehabilitation: the duration, the smoothness, and the angle of individual propagation events. Results show that, compared to pre-stroke conditions, propagation of cortical activity in the subacute phase right after stroke is slowed down and more irregular. When comparing rehabilitation paradigms, we find that mice treated with both motor training and pharmacological intervention, the only group associated with generalized recovery, manifest new propagation patterns, that are even faster and smoother than before the stroke. In conclusion, our new spatiotemporal propagation indicators could represent promising biomarkers that are able to uncover neural correlates not only of motor deficits caused by stroke but also of functional recovery during rehabilitation. In turn, these insights could pave the way towards more targeted post-stroke therapies.

Design, synthesis and evaluation of RGD peptidomimetic - Gold nanostar conjugates as M21 cell adhesion inhibitors.

Effective targeting of αvß3 integrin is of high relevance in cancer research as this protein is overexpressed on several types of tumor cells, making such receptor ideal for the development of therapeutics and of diagnostic imaging agents. In this paper, the synthesis of a novel functionalized triazole-based RGD peptidomimetic and its covalent conjugation on pegylated gold nanostars is reported. These highly stable nanoconstructs showed a multivalent effect in binding αvß3 integrin receptors and proved to inhibit M21 cell adhesion at 25 pM concentration. Thanks to their peculiar surface plasmon resonance in the "NIR transparent window", targeted gold nanostars may represent a promising agent for anticancer multi-modality treatments. 2009 Elsevier Ltd. All rights reserved.

Design and Synthesis of Novel Raman Reporters for Bioorthogonal SERS Nanoprobes Engineering.

Surface-enhanced Raman spectroscopy (SERS) exploiting Raman reporter-labeled nanoparticles (RR@NPs) represents a powerful tool for the improvement of optical bio-assays due to RRs' narrow peaks, SERS high sensitivity, and potential for multiplexing. In the present work, starting from low-cost and highly available raw materials such as cysteamine and substituted benzoic acids, novel bioorthogonal RRs, characterized by strong signal (103 counts with FWHM < 15 cm−1) in the biological Raman-silent region (>2000 cm−1), RRs are synthesized by implementing a versatile, modular, and straightforward method with high yields and requiring three steps lasting 18 h, thus overcoming the limitations of current reported procedures. The resulting RRs' chemical structure has SH-pendant groups exploited for covalent conjugation to high anisotropic gold-NPs. RR@NPs constructs work as SERS nanoprobes demonstrating high colloidal stability while retaining NPs' physical and vibrational properties, with a limit of detection down to 60 pM. RR@NPs constructs expose carboxylic moieties for further self-assembling of biomolecules (such as antibodies), conferring tagging capabilities to the SERS nanoprobes even in heterogeneous samples, as demonstrated with in vitro experiments by transmembrane proteins tagging in cell cultures. Finally, thanks to their non-overlapping spectra, we envision and preliminary prove the possibility of exploiting RR@NPs constructs simultaneously, aiming at improving current SERS-based multiplexing bioassays.

Monitoring Changes in Biochemical and Biomechanical Properties of Collagenous Tissues Using Label-Free and Nondestructive Optical Imaging Techniques.

We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 µg/mL bacterial collagenase at 37 °C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm-1. Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young's modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.

Reconstruction scheme for excitatory and inhibitory dynamics with quenched disorder: application to zebrafish imaging.

An inverse procedure is developed and tested to recover functional and structural information from global signals of brains activity. The method assumes a leaky-integrate and fire model with excitatory and inhibitory neurons, coupled via a directed network. Neurons are endowed with a heterogenous current value, which sets their associated dynamical regime. By making use of a heterogenous mean-field approximation, the method seeks to reconstructing from global activity patterns the distribution of in-coming degrees, for both excitatory and inhibitory neurons, as well as the distribution of the assigned currents. The proposed inverse scheme is first validated against synthetic data. Then, time-lapse acquisitions of a zebrafish larva recorded with a two-photon light sheet microscope are used as an input to the reconstruction algorithm. A power law distribution of the in-coming connectivity of the excitatory neurons is found. Local degree distributions are also computed by segmenting the whole brain in sub-regions traced from annotated atlas.

Development of Light-Responsive Liquid Crystalline Elastomers to Assist Cardiac Contraction.

RATIONALE: Despite major advances in cardiovascular medicine, heart disease remains a leading cause of death worldwide. However, the field of tissue engineering has been growing exponentially in the last decade and restoring heart functionality is now an affordable target; yet, new materials are still needed for effectively provide rapid and long-lasting interventions. Liquid crystalline elastomers (LCEs) are biocompatible polymers able to reversibly change shape in response to a given stimulus and generate movement. Once stimulated, LCEs can produce tension or movement like a muscle. However, so far their application in biology was limited by slow response times and a modest possibility to modulate tension levels during activation. OBJECTIVE: To develop suitable LCE-based materials to assist cardiac contraction. METHODS AND RESULTS: Thanks to a quick, simple, and versatile synthetic approach, a palette of biocompatible acrylate-based light-responsive LCEs with different molecular composition was prepared and mechanically characterized. Out of this, the more compliant one was selected. This material was able to contract for some weeks when activated with very low light intensity within a physiological environment. Its contraction was modulated in terms of light intensity, stimulation frequency, and ton/toff ratio to fit different contraction amplitude/time courses, including those of the human heart. Finally, LCE strips were mounted in parallel with cardiac trabeculae, and we demonstrated their ability to improve muscular systolic function, with no impact on diastolic properties. CONCLUSIONS: Our results indicated LCEs are promising in assisting cardiac mechanical function and developing a new generation of contraction assist devices.

Colored visual stimuli evoke spectrally tuned neuronal responses across the central nervous system of zebrafish larvae.

BACKGROUND: Visually guided behaviors such as optomotor and optokinetic responses, phototaxis, and prey capture are crucial for survival in zebrafish and become apparent after just a few days of development. Color vision, which in zebrafish is based on a spatially anisotropic tetrachromatic retina, provides an additional important component of world representation driving fundamental larval behaviors. However, little is known about the central nervous system (CNS) circuitry underlying color vision processing downstream of the retina, and its activity correlates with behavior. Here, we used the transparent larva of zebrafish to image CNS neurons and their activity in response to colored visual stimuli. RESULTS: To investigate the processing of chromatic information in the zebrafish larva brain, we mapped with cellular resolution, spectrally responsive neurons in the larva encephalon and spinal cord. We employed the genetically encoded calcium indicator GCaMP6s and two-photon microscopy to image the neuronal activity while performing visual stimulation with spectrally distinct stimuli at wavelengths matching the absorption peaks of the four zebrafish cone types. We observed the presence of a high number of wavelength-selective neurons not only in the optic tectum, but also in all other regions of the CNS, demonstrating that the circuitry involved in processing spectral information and producing color-selective responses extends to the whole CNS. CONCLUSIONS: Our measurements provide a map of neurons involved in color-driven responses, revealing that spectral information spreads in all regions of the CNS. This suggests the underlying complexity of the circuits involved and opens the way to their detailed future investigation.

Simultaneous fluorescence lifetime and Raman fiber-based mapping of tissues.

We report the development of a novel, to the best of our knowledge, fiber-based system to realize coregistered simultaneous acquisition of fluorescence lifetime (FL) data and Raman spectra from the same area. FL measurements by means of time-correlated single photon counting are realized with periodic out-of-phase external illumination of the field of view, enabling acquisition of data under bright illumination of the specimen. Raman measurements in the near-infrared are realized asynchronously. We present a detailed characterization of this technique and validate its potential to report intrinsic contrast. Fiber-based FL and Raman maps report complementary structural, compositional, and molecular contrast in biological tissues with diverse compositional features.

Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching.

In any living cell, genome maintenance is carried out by DNA-binding proteins that recognize specific sequences among a vast amount of DNA. This includes fundamental processes such as DNA replication, DNA repair, and gene expression and regulation. Here, we study the mechanism of DNA target search by a single lac repressor protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and few base-pair resolution technique based on laser tweezers. We measure 1D-diffusion of proteins on DNA at physiological salt concentrations with 20 bp resolution and find that sliding of LacI along DNA is sequence dependent. We show that only allosterically activated LacI slides along non-specific DNA sequences during target search, whereas the inhibited conformation does not support sliding and weakly interacts with DNA. Moreover, we find that LacI undergoes a load-dependent conformational change when it switches between sliding and strong binding to the target sequence. Our data reveal how DNA sequence and molecular switching regulate LacI target search process and provide a comprehensive model of facilitated diffusion for LacI.

Cardiac sympathetic innervation network shapes the myocardium by locally controlling cardiomyocyte size through the cellular proteolytic machinery.

KEY POINTS: The heart is innervated by a dense sympathetic neuron network which, in the short term, controls chronotropy and inotropy and, in the long term, regulates cardiomyocyte size. Acute neurogenic control of heart rate is achieved locally through direct neuro-cardiac coupling at specific junctional sites (neuro-cardiac junctions). The ventricular sympathetic network topology is well-defined and characteristic for each mammalian species. In the present study, we used cell size regulation to determine whether long-term modulation of cardiac structure is achieved via direct sympatho-cardiac coupling. Local density of cardiac innervation correlated with cell size throughout the myocardial walls in all mammalian species analysed, including humans. The data obtained suggest that constitutive neurogenic control of cardiomyocyte trophism occurs through direct intercellular signalling at neuro-cardiac junctions. ABSTRACT: It is widely appreciated that sympathetic stimulation of the heart involves a sharp increase in beating rate and significant enhancement of contractility. We have previously shown that, in addition to these evident functions, sympathetic neurons (SNs) also provide trophic input to cardiomyocytes (CMs), regulating cell and organ size. More recently, we have demonstrated that cardiac neurons establish direct interactions with CMs, allowing neuro-cardiac communication to occur locally, with a 'quasi-synaptic' mechanism. Based on the evidence that cardiac SNs are unevenly distributed throughout the myocardial walls, we investigated the hypothesis that CM size distribution reflects the topology of neuronal density. In vitro analyses of SN/CM co-cultures, ex vivo confocal and multiphoton imaging in clarified hearts, and biochemical and molecular approaches were employed, in both rodent and human heart biopsies. In line with the trophic effect of SNs, and with local neuro-cardiac communication, CMs, directly contacted by SNs in co-cultures, were larger than the non-targeted ones. This property reflects the distribution of CM size throughout the ventricles of intact mouse heart, in which cells in the outer myocardial layers, which were contacted by more neuronal processes, were larger than those in the less innervated subendocardial region. Such differences disappeared upon genetic or pharmacological interference with the trophic SN/CM signalling axis. Remarkably, CM size followed the SN distribution pattern in other mammals, including humans. Our data suggest that both the acute and chronic influence of SNs on cardiac function and structure is enacted as a result of the establishment of specific intercellular neuro-cardiac junctions.

Multispectral Depth-Resolved Fluorescence Lifetime Spectroscopy Using SPAD Array Detectors and Fiber Probes.

Single Photon Avalanche Diode (SPAD) arrays are increasingly exploited and have demonstrated potential in biochemical and biomedical research, both for imaging and single-point spectroscopy applications. In this study, we explore the application of SPADs together with fiber-optic-based delivery and collection geometry to realize fast and simultaneous single-point time-, spectral-, and depth-resolved fluorescence measurements at 375 nm excitation light. Spectral information is encoded across the columns of the array through grating-based dispersion, while depth information is encoded across the rows thanks to a linear arrangement of probe collecting fibers. The initial characterization and validation were realized against layered fluorescent agarose-based phantoms. To verify the practicality and feasibility of this approach in biological specimens, we measured the fluorescence signature of formalin-fixed rabbit aorta samples derived from an animal model of atherosclerosis. The initial results demonstrate that this detection configuration can report fluorescence spectral and lifetime contrast originating at different depths within the specimens. We believe that our optical scheme, based on SPAD array detectors and fiber-optic probes, constitute a powerful and versatile approach for the deployment of multidimensional fluorescence spectroscopy in clinical applications where information from deeper tissue layers is important for diagnosis.

PLANT: A Method for Detecting Changes of Slope in Noisy Trajectories.

Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.