Semi-automated approaches for interrogating spatial heterogeneity of tissue samples.

Tissues are spatially orchestrated ecosystems composed of heterogeneous cell populations and non-cellular elements. Tissue components' interactions shape the biological processes that govern homeostasis and disease, thus comprehensive insights into tissues' composition are crucial for understanding their biology. Recently, advancements in the spatial biology field enabled the in-depth analyses of tissue architecture at single-cell resolution, while preserving the structural context. The increasing number of biomarkers analyzed, together with whole tissue imaging, generate datasets approaching several hundreds of gigabytes in size, which are rich sources of valuable knowledge but require investments in infrastructure and resources for extracting quantitative information. The analysis of multiplex whole-tissue images requires extensive training and experience in data analysis. Here, we showcase how a set of open-source tools can allow semi-automated image data extraction to study the spatial composition of tissues with a focus on tumor microenvironment (TME). With the use of Lunaphore COMET platform, we interrogated lung cancer specimens where we examined the expression of 20 biomarkers. Subsequently, the tissue composition was interrogated using an in-house optimized nuclei detection algorithm followed by a newly developed image artifact exclusion approach. Thereafter, the data was processed using several publicly available tools, highlighting the compatibility of COMET-derived data with currently available image analysis frameworks. In summary, we showcased an innovative semi-automated workflow that highlights the ease of adoption of multiplex imaging to explore TME composition at single-cell resolution using a simple slide in, data out approach. Our workflow is easily transferrable to various cohorts of specimens to provide a toolset for spatial cellular dissection of the tissue composition.

Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

A New Timing Error Cost Function for Binary Time Series Prediction.

The ability to make predictions is central to the artificial intelligence problem. While machine learning algorithms have difficulty in learning to predict events with hundreds of time-step dependencies, animals can learn event timing within tens of trials across a broad spectrum of time scales. This suggests strongly a need for new perspectives on the forecasting problem. This paper focuses on binary time series that can be predicted within some temporal precision. We demonstrate that the sum of squared errors (SSE) calculated at every time step is not appropriate for this problem. Next, we look at the advantages and shortcomings of using a dynamic time warping (DTW) cost function. Then, we propose the squared timing error (STE) that uses DTW on the event space and applies SSE on the timing error instead of at each time step. We evaluate all three cost functions on different types of timing errors, such as phase shift, warping, and missing events, on synthetic and real-world binary time series (heartbeats, finance, and music). The results show that STE provides more information about timing error, is differentiable, and can be computed online efficiently. Finally, we devise a gradient descent algorithm for STE on a simplified recurrent neural network. We then compare the performance of the STE-based algorithm to SSE- and logit-based gradient descent algorithms on the same network architecture. The results in real-world binary time series show that the STE algorithm generally outperforms all the other cost functions considered.

Engineered signaling centers for the spatially controlled patterning of human pluripotent stem cells.

Signaling centers, localized groups of cells that secrete morphogens, play a key role in early development and organogenesis by orchestrating spatial cell fate patterning. Here we present a microfluidic approach that exposes human pluripotent stem cell (hPSC) colonies to spatiotemporally controlled morphogen gradients generated from artificial signaling centers. In response to a localized source of bone morphogenetic protein 4 (BMP4), hPSC colonies reproducibly break their intrinsic radial symmetry to produce distinct, axially arranged differentiation domains. Counteracting sources of the BMP antagonist NOGGIN enhance this spatial control of cell fate patterning. We also show how morphogen concentration and cell density affect the BMP response and germ layer patterning. These results demonstrate that the intrinsic capacity of stem cells for self-organization can be extrinsically controlled through the use of engineered signaling centers.

Node-Pore Coded Coincidence Correction: Coulter Counters, Code Design, and Sparse Deconvolution.

We present a novel method to perform individual particle (e.g. cells or viruses) coincidence correction through joint channel design and algorithmic methods. Inspired by multiple-user communication theory, we modulate the channel response, with Node-Pore Sensing, to give each particle a binary Barker code signature. When processed with our modified successive interference cancellation method, this signature enables both the separation of coincidence particles and a high sensitivity to small particles. We identify several sources of modeling error and mitigate most effects using a data-driven self-calibration step and robust regression. Additionally, we provide simulation analysis to highlight our robustness, as well as our limitations, to these sources of stochastic system model error. Finally, we conduct experimental validation of our techniques using several encoded devices to screen a heterogeneous sample of several size particles.

BARKER-CODED NODE-PORE RESISTIVE PULSE SENSING WITH BUILT-IN COINCIDENCE CORRECTION.

A resistive pulse sensing device is able to extract quantities such as concentration and size distribution of particles, e.g. cells or microspheres, as they flow through the device's sensor region, i.e. channel, in an electrolyte solution. The dynamic range of detectable particle sizes is limited by the channel dimensions. In addition, signal interference from multiple particles transiting the channel simultaneously, i.e. coincidence event, further hinder the dynamic range. Coincidence data is often considered unusable and is discarded, reducing the throughput and introducing possible biases and errors into the distributions. Here, we propose a two-step solution. We code the channel such that the system response results in a Manchester encoded Barker-Code sequence, allowing us to take advantage of the code's pulse compression properties. We pose the parameter estimation problem as a sparse inverse problem, which enables estimation of particle sizes and velocities while resolving coincidences, and solve it with a successive interference cancellation algorithm. We introduce modifications to the algorithm to account for device fabrication variations and natural stochastic variations in flow. We demonstrate the ability to resolve coincidences and possible increases in the device's dynamic range by screening particles of different size through a Barker encoded device.

Conditioning and time representation in long short-term memory networks.

Dopaminergic models based on the temporal-difference learning algorithm usually do not differentiate trace from delay conditioning. Instead, they use a fixed temporal representation of elapsed time since conditioned stimulus onset. Recently, a new model was proposed in which timing is learned within a long short-term memory (LSTM) artificial neural network representing the cerebral cortex (Rivest et al. in J Comput Neurosci 28(1):107-130, 2010). In this paper, that model's ability to reproduce and explain relevant data, as well as its ability to make interesting new predictions, are evaluated. The model reveals a strikingly different temporal representation between trace and delay conditioning since trace conditioning requires working memory to remember the past conditioned stimulus while delay conditioning does not. On the other hand, the model predicts no important difference in DA responses between those two conditions when trained on one conditioning paradigm and tested on the other. The model predicts that in trace conditioning, animal timing starts with the conditioned stimulus offset as opposed to its onset. In classical conditioning, it predicts that if the conditioned stimulus does not disappear after the reward, the animal may expect a second reward. Finally, the last simulation reveals that the buildup of activity of some units in the networks can adapt to new delays by adjusting their rate of integration. Most importantly, the paper shows that it is possible, with the proposed architecture, to acquire discharge patterns similar to those observed in dopaminergic neurons and in the cerebral cortex on those tasks simply by minimizing a predictive cost function.

An adaptive drift-diffusion model of interval timing dynamics.

Animals readily learn the timing between salient events. They can even adapt their timed responding to rapidly changing intervals, sometimes as quickly as a single trial. Recently, drift-diffusion models-widely used to model response times in decision making-have been extended with new learning rules that allow them to accommodate steady-state interval timing, including scalar timing and timescale invariance. These time-adaptive drift-diffusion models (TDDMs) work by accumulating evidence of elapsing time through their drift rate, thereby encoding the to-be-timed interval. One outstanding challenge for these models lies in the dynamics of interval timing-when the to-be-timed intervals are non-stationary. On these schedules, animals often fail to exhibit strict timescale invariance, as expected by the TDDMs and most other timing models. Here, we introduce a simple extension to these TDDMs, where the response threshold is a linear function of the observed event rate. This new model compares favorably against the basic TDDMs and the multiple-time-scale (MTS) habituation model when evaluated against three published datasets on timing dynamics in pigeons. Our results suggest that the threshold for triggering responding in interval timing changes as a function of recent intervals.

Alternative time representation in dopamine models.

Dopaminergic neuron activity has been modeled during learning and appetitive behavior, most commonly using the temporal-difference (TD) algorithm. However, a proper representation of elapsed time and of the exact task is usually required for the model to work. Most models use timing elements such as delay-line representations of time that are not biologically realistic for intervals in the range of seconds. The interval-timing literature provides several alternatives. One of them is that timing could emerge from general network dynamics, instead of coming from a dedicated circuit. Here, we present a general rate-based learning model based on long short-term memory (LSTM) networks that learns a time representation when needed. Using a naïve network learning its environment in conjunction with TD, we reproduce dopamine activity in appetitive trace conditioning with a constant CS-US interval, including probe trials with unexpected delays. The proposed model learns a representation of the environment dynamics in an adaptive biologically plausible framework, without recourse to delay lines or other special-purpose circuits. Instead, the model predicts that the task-dependent representation of time is learned by experience, is encoded in ramp-like changes in single-neuron activity distributed across small neural networks, and reflects a temporal integration mechanism resulting from the inherent dynamics of recurrent loops within the network. The model also reproduces the known finding that trace conditioning is more difficult than delay conditioning and that the learned representation of the task can be highly dependent on the types of trials experienced during training. Finally, it suggests that the phasic dopaminergic signal could facilitate learning in the cortex.

Spiking neurons, dopamine, and plasticity: timing is everything, but concentration also matters.

While both dopamine (DA) fluctuations and spike-timing-dependent plasticity (STDP) are known to influence long-term corticostriatal plasticity, little attention has been devoted to the interaction between these two fundamental mechanisms. Here, a theoretical framework is proposed to account for experimental results specifying the role of presynaptic activation, postsynaptic activation, and concentrations of extracellular DA in synaptic plasticity. Our starting point was an explicitly-implemented multiplicative rule linking STDP to Michaelis-Menton equations that models the dynamics of extracellular DA fluctuations. This rule captures a wide range of results on conditions leading to long-term potentiation and depression in simulations that manipulate the frequency of induced corticostriatal stimulation and DA release. A well-documented biphasic function relating DA concentrations to synaptic plasticity emerges naturally from simulations involving a multiplicative rule linking DA and neural activity. This biphasic function is found consistently across different neural coding schemes employed (voltage-based vs. spike-based models). By comparison, an additive rule fails to capture these results. The proposed framework is the first to generate testable predictions on the dual influence of DA concentrations and STDP on long-term plasticity, suggesting a way in which the biphasic influence of DA concentrations can modulate the direction and magnitude of change induced by STDP, and raising the possibility that DA concentrations may inverse the LTP/LTD components of the STDP rule.