Biology subjects

Harfouche, M.

Publications and source records attributed to Harfouche, M..

7 recordsLinked to original sources

bioRxiv2025

Parallelized Brightfield and Fluorescence Imaging of Organoids Using a Scalable Multi-Camera Platform

Organoid viability, maturation, and growth is commonly assayed through brightfield and fluorescence microscopy using a single objective lens. However, standard microscopic imaging systems pose significant limitations for high-throughput applications, particularly in large-scale experiments where simultaneous imaging of multiple organoids requires increased throughput. There is a strong need for systems that can capture organoid growth rapidly and consistently while minimizing disturbances to culture conditions. Here, we present a novel multi-camera array scanner (MCAS) that parallelizes imaging through the simultaneous use of 48 objective lenses and sensors, resulting in a 95% reduction in acquisition times compared to commercial high-content imagers. We demonstrate and validate this system in multiple well plate formats, in both 2D and 3D neural cell cultures, and in brightfield and fluorescence. The MCAS improves efficiency for measuring organoid growth rates, assessing responses to morphogens and drugs, and measuring viral transduction efficiency. Together, these findings establish the MCAS as a scalable and versatile imaging platform for rapid phenotyping in organoid research.

neuroscience↗

bioRxiv2025

High-Throughput Tracking of Freely Moving Drosophila Reveals Variations in Aggression and Courtship Behaviors

Aggression is a nearly universal behavior used to secure food, territory, and mates across species, including the fruit fly Drosophila melanogaster. In fruit flies, both sexes display aggression through stereotypical motor patterns. This, along with their sophisticated genetic and molecular toolkit, makes Drosophila melanogaster an excellent model for studying aggression. While male- and female-specific aggressive motor programs have been qualitatively described, automated systems for quantifying these behaviors in freely moving flies remain limited in their ability to combine high-resolution analysis with high throughput. Here, we pair a high-resolution, high-throughput imaging system (the Kestrel) with DeepLabCut pose estimation to create a pipeline that tracks multiple freely moving fly pairs and quantifies social dynamics with high fidelity. We validated body-part tracking using published benchmarks. The platform reliably reproduced a known phenotype: heightened female aggression following thermogenetic activation of cholinergic pC1 neurons in female brain. It also detected increased unilateral wing extension, a courtship display inversely related to aggression, between two males upon activating a previously uncharacterized [~]40-neuron group in the male brain. Pose-based analysis revealed locomotive differences between experimental and control groups, and subtle, genotype-specific variations in head butts and UWEs. This workflow enables high-throughput screening and mechanistic dissection of social behaviors.

neuroscience↗

bioRxiv2025

High-throughput multi-camera array microscope platform for automated 3D behavioral analysis of freely swimming zebrafish larvae

Understanding the behavioral and morphological dynamics of moving model organisms like the zebrafish larvae requires accurate, high-throughput 3D analysis. However, traditional single-view 2D video tracking fails to capture the full scope of natural 3D movements and postural dynamics. Here, we present a novel high-throughput 24-camera array microscope with a co-designed "mirrored well plate" that allows for snapshot imaging of up to 48 wells over a 118 mm x 82 mm field of view from two orthogonal directions (i.e., a top-view and side-view). Accurate 3D position estimation and tracking is achieved with an efficient machine learning algorithm that scales well to high-throughput measurements. The proposed approach automates parallelized 3D model organism behavioral analysis, providing 3D skeletal tracking, swim bladder morphological dynamics, and kinematics of up to 48 swimming zebrafish larvae at up to several hundred frames per second. The result is an efficient and scalable solution for high-throughput 3D behavioral studies with broad compatibility with standard workflows across laboratories and procedures working with pharmacology, toxicology, and neuroscience.

animal behavior and cognition↗

bioRxiv2025

High throughput machine learning pipeline to characterize larval zebrafish motor behavior

Using machine learning, we developed models that rigorously detect and classify larval zebrafish spontaneous and stimulus-evoked behaviors in various well plate formats. Zebrafish are an ideal model system for investigating the neural substrates underlying behavior due to their simple nervous system and well-documented responses to environmental stimuli. To track movement, we utilized an 8 key point pose estimation model, allowing precise capture of zebrafish kinematics. Using this kinematic data, we trained two random forest classifiers in a semi-supervised learning framework to classify various discreet behavioral outputs including stationary, scoot, turn, acoustic-startle like behavior, and visual-startle like behavior. The classifiers were trained on a manually labeled dataset, and their accuracy was validated showing high precision. To validate our machine learning models, we analyzed behavioral outputs during various stimulus evoked responses and during spontaneous behavior. For additional validation, and to show the utility of our recording and analysis pipeline, we investigated the locomotor effects of several established drugs with well-defined impacts on neurophysiology. Here we show that machine learning model development, enabled by semi-supervised learning developed classification models, provide detailed insights into the behavioral phenotypes of zebrafish, offering a powerful, high throughput method for studying neural control of behavior.

neuroscience↗

bioRxiv2024

High-speed 4D fluorescence light field tomography of whole freely moving organisms

Volumetric fluorescence imaging techniques, such as confocal, multiphoton, light sheet, and light field microscopy, have become indispensable tools across a wide range of cellular, developmental, and neurobiological applications. However, it is difficult to scale such techniques to the large 3D fields of view (FOV), volume rates, and synchronicity requirements for high-resolution 4D imaging of freely behaving organisms. Here, we present reflective Fourier light field computed tomography (ReFLeCT), a new high-speed volumetric fluorescence computational imaging technique. ReFLeCT synchronously captures entire tomograms of multiple unrestrained, unanesthetized model organisms over multi-millimeter 3D FOVs at 120 volumes per second. In particular, we applied ReFLeCT to reconstruct 4D videos of fluorescently labeled zebrafish and Drosophila larvae, enabling us to study their heartbeat, fin and tail motion, gaze, jaw motion, and muscle contractions with nearly isotropic 3D resolution while they are freely moving. As a novel approach for snapshot tomographic capture, ReFLeCT is a major advance towards bridging the gap between current volumetric fluorescence microscopy techniques and macroscopic behavioral imaging.

animal behavior and cognition↗

bioRxiv2023

Automated, high-throughput quantification of EGFP-expressing neutrophils in zebrafish by machine learning and a highly-parallelized microscope

Normal development of the immune system is essential for overall health and disease resistance. Bony fish, such as the zebrafish (Danio rerio), possess all the major immune cell lineages as mammals and can be employed to model human host response to immune challenge. Zebrafish neutrophils, for example, are present in the transparent larvae as early as 48 hours post fertilization and have been examined in numerous infection and immunotoxicology reports. One significant advantage of the zebrafish model is the ability to affordably generate high numbers of individual larvae that can be arrayed in multi-well plates for high throughput genetic and chemical exposure screens. However, traditional workflows for imaging individual larvae have been limited to low-throughput studies using traditional microscopes and manual analyses. Using a newly developed, parallelized microscope, the Multi-Camera Array Microscope (MCAM), we have optimized a rapid, high-resolution algorithmic method to count fluorescently labeled cells in zebrafish larvae in vivo. Using transgenic zebrafish larvae, in which neutrophils express EGFP, we captured 18 gigapixels of images across a full 96-well plate, in 75 seconds, and processed the resulting datastream, counting individual fluorescent neutrophils in all individual larvae in 5 minutes. This automation is facilitated by a machine learning segmentation algorithm that defines the most in-focus view of each larva in each well after which pixel intensity thresholding and blob detection are employed to locate and count fluorescent cells. We validated this method by comparing algorithmic neutrophil counts to manual counts in larvae subjected to changes in neutrophil numbers, demonstrating the utility of this approach for high-throughput genetic and chemical screens where a change in neutrophil number is an endpoint metric. Using the MCAM we have been able to, within minutes, acquire both enough data to create an automated algorithm and execute a biological experiment with statistical significance. Finally, we present this open-source software package which allows the user to train and evaluate a custom machine learning segmentation model and use it to localize zebrafish and analyze cell counts within the segmented region of interest. This software can be modified as needed for studies involving other zebrafish cell lineages using different transgenic reporter lines and can also be adapted for studies using other amenable model species.

bioinformatics↗

bioRxiv2021

Gigapixel behavioral and neural activity imaging with a novel multi-camera array microscope

The dynamics of living organisms are organized across many spatial scales. However, current cost-effective imaging systems can measure only a subset of these scales at once. We have created a scalable multi-camera array microscope (MCAM) that enables comprehensive high-resolution recording from multiple spatial scales simultaneously, ranging from cellular structures to large-group behavioral dynamics. By collecting data from up to 96 cameras, we computationally generate gigapixel-scale images and movies with a field of view over hundreds of square centimeters at 5um sensitivity. This allows us to observe the behavior and fine anatomical features of numerous freely moving model organisms on multiple spatial scales, including larval zebrafish, fruit flies, nematodes, carpenter ants, and slime mold. The MCAM architecture allows stereoscopic tracking of the z-position of organisms using the overlapping field of view from adjacent cameras. Further, we demonstrate the ability to acquire dual color fluorescence video of multiple freely moving zebrafish, recording neural activity via ratiometric calcium imaging. Overall, the MCAM provides a powerful platform for investigating cellular and behavioral processes across a wide range of spatial scales by removing the bottlenecks imposed by single-camera image acquisition systems.

bioengineering↗