Resolving Colliding Larvae by Fitting ASM to Random Walker-Based Pre-Segmentations.

Drosophila melanogaster is an important model organism for research in neuro- and behavioral biology. Automated studies of their locomotion are crucial to link sensory input and neural processing to motor output which has led to numerous vision-based tracking systems. However, most of these approaches share the inability to segment the contours of colliding animals causing identity losses, appearing and disappearing animals, and the absence of posture and motion related measurements during the time of the collision. We present a novel collision resolution algorithm enabling an accurate contour segmentation of multiple touching Drosophila larvae. Our algorithm utilizes an adapted active shape model (ASM) to learn a low dimensional posture space which is fitted to random-walker generated pre-segmentations. We evaluate our collision resolution algorithm using three publicly available datasets and compare it with the current state-of-the-art methods. In addition, we introduce a refined dataset enabling a segmentation evaluation on the level of pixel accuracy. The results demonstrate that our approach outperforms the state-of-the-art approaches in both accuracy and computational time. We will incorporate this algorithm into our widely used tracking program to improve the statistical strength of the behavioral quantification and allow marker-free studies of interacting Drosophila larvae.

An RJMCMC-Based Method for Tracking and Resolving Collisions of Drosophila Larvae.

Drosophila melanogaster is an important model organism for ongoing research in neuro- and behavioral biology. Especially the locomotion analysis has become an integral part of such studies and thus elaborated automated tracking systems have been proposed in the past. However, most of these approaches share the inability to precisely segment the contours of colliding animals leading to the absence of model and motion-related features during collisions. Here, we translate the task of tracking and resolving colliding animals into a filtering problem solvable by Markov Chain Monte Carlo methods and elaborate an adequate larva model. By comparing our method with state-of-the-art approaches, we demonstrate that our algorithm produces significantly better results in a fraction of time and facilitates the analysis of animal behavior during interaction in more detail.

The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia.

Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, we screened for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, our findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity.

Automatic non-invasive heartbeat quantification of Drosophila pupae.

The importance of studying model organisms such as Drosophila melanogaster has significantly increased in recent biological research. Amongst others, Drosophila can be used to study heart development and heartbeat related diseases. Here we propose a method for automatic in vivo heartbeat detection of Drosophila melanogaster pupae based on morphological structures which are recorded without any dissection using FIM imaging. Our approach is easy-to-use, has low computational costs, and enables high-throughput experiments. After automatically segmenting the heart region of the pupa in an image sequence, the heartbeat is indirectly determined based on intensity variation analysis. We have evaluated our method using 47,631 manually annotated frames from 29 image sequences recorded with different temporal and spatial resolutions which are made publicly available. We show that our algorithm is both precise since it detects more than 95% of the heartbeats correctly as well as robust since the same standardized set of parameters can be used for all sequences. The combination of FIM imaging and our algorithm enables a reliable heartbeat detection of multiple Drosophila pupae while simultaneously avoiding any time consuming preparation of the animals.

The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3 Drosophila larvae.

Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the Drosophila larva is simple enough to be tractable, yet complex enough to be interesting. It features about 10,000 neurons and is capable of various taxes, kineses and Pavlovian conditioning. All its neurons are currently being mapped into a light-microscopical atlas, and Gal4 strains are being generated to experimentally access neurons one at a time. In addition, an electron microscopic reconstruction of its nervous system seems within reach. Notably, this electron microscope-based connectome is being drafted for a stage 1 larva - because stage 1 larvae are much smaller than stage 3 larvae. However, most behaviour analyses have been performed for stage 3 larvae because their larger size makes them easier to handle and observe. It is therefore warranted to either redo the electron microscopic reconstruction for a stage 3 larva or to survey the behavioural faculties of stage 1 larvae. We provide the latter. In a community-based approach we called the Ol1mpiad, we probed stage 1 Drosophila larvae for free locomotion, feeding, responsiveness to substrate vibration, gentle and nociceptive touch, burrowing, olfactory preference and thermotaxis, light avoidance, gustatory choice of various tastants plus odour-taste associative learning, as well as light/dark-electric shock associative learning. Quantitatively, stage 1 larvae show lower scores in most tasks, arguably because of their smaller size and lower speed. Qualitatively, however, stage 1 larvae perform strikingly similar to stage 3 larvae in almost all cases. These results bolster confidence in mapping brain structure and behaviour across developmental stages.

FIMTrack: An open source tracking and locomotion analysis software for small animals.

Imaging and analyzing the locomotion behavior of small animals such as Drosophila larvae or C. elegans worms has become an integral subject of biological research. In the past we have introduced FIM, a novel imaging system feasible to extract high contrast images. This system in combination with the associated tracking software FIMTrack is already used by many groups all over the world. However, so far there has not been an in-depth discussion of the technical aspects. Here we elaborate on the implementation details of FIMTrack and give an in-depth explanation of the used algorithms. Among others, the software offers several tracking strategies to cover a wide range of different model organisms, locomotion types, and camera properties. Furthermore, the software facilitates stimuli-based analysis in combination with built-in manual tracking and correction functionalities. All features are integrated in an easy-to-use graphical user interface. To demonstrate the potential of FIMTrack we provide an evaluation of its accuracy using manually labeled data. The source code is available under the GNU GPLv3 at https://github.com/i-git/FIMTrack and pre-compiled binaries for Windows and Mac are available at http://fim.uni-muenster.de.

FIM$

In vivo whole-body imaging of small animals plays an important role for biomedical studies. In particular, animals like the fruit fly Drosophila melanogaster or the nematode Caenorhabditis elegans are popular model organisms for preclinical research since they offer sophisticated genetic tool-kits. Recording these translucent animals with high contrast in a large arena is however not trivial. Furthermore, fluorescent proteins are widely used to mark cells in vivo and report their functions. This paper introduces a novel optical imaging technique called FIM2c enabling simultaneous detection of the animals posture and movement as well as fluorescent markers like green fluorescent protein (GFP). FIM2c utilizes frustrated total internal reflection of two distinct wavelengths and captures both, reflected and emitted light. The resultant two-color high-contrast images are superb compared to other imaging systems for larvae or worms. This multipurpose method enables a large variety of different experimental approaches. For example, FIM2c can be used to image GFP positive cells/tissues/animals and supports the integration of fluorescent tracers into multitarget tracking paradigms. Moreover, optogenetic tools can be applied in large-scale behavioral analysis to manipulate and study neuronal functions. To demonstrate the benefit of our system, we use FIM2c to resolve colliding larvae in a high-throughput approach, which was impossible given the existing tools. Finally, we present a comprehensive database including images and locomotion features of more than 1300 resolved collisions available for the community. In conclusion, FIM2c is a versatile tool for advanced imaging and locomotion analysis for a variety of different model organisms.

An FIM-Based Long-Term In-Vial Monitoring System for Drosophila Larvae.

Drosophila larvae are an insightful model and the automated analysis of their behavior is an integral readout in behavioral biology. Current tracking systems, however, entail a disturbance of the animals, are labor-intensive, and cannot be easily used for long-term monitoring purposes. Here, we present a novel monitoring system for Drosophila larvae, which allows us to analyze the animals in cylindrical culture vials. By utilizing the frustrated total internal reflection in combination with a multi-camera/microcomputer setup, we image the complete housing vial surface and, thus, the larvae for days. We introduce a calibration scheme to stitch the images from the multi-camera system and unfold arbitrary cylindrical surfaces to support different vials. As a result, imaging and analysis of a whole population can be done implicitly. For the first time, this allows us to extract long-term activity quantities of larvae without disturbing the animals. We demonstrate the capabilities of this new setup by automatically quantifying the activity of multiple larvae moving in a vial. The accuracy of the system and the spatio-temporal resolution are sufficient to obtain motion trajectories and higher level features, such as body bending. This new setup can be used for in-vial activity monitoring and behavioral analysis and is capable of gathering millions of data points without both disturbing the animals and increasing labor time. In total, we have analyzed 107 671 frames resulting in 8650 trajectories, which are longer than 30 s, and obtained more than 4.2 × 106 measurements.

Interactions among Drosophila larvae before and during collision.

In populations of Drosophila larvae, both, an aggregation and a dispersal behavior can be observed. However, the mechanisms coordinating larval locomotion in respect to other animals, especially in close proximity and during/after physical contacts are currently only little understood. Here we test whether relevant information is perceived before or during larva-larva contacts, analyze its influence on behavior and ask whether larvae avoid or pursue collisions. Employing frustrated total internal reflection-based imaging (FIM) we first found that larvae visually detect other moving larvae in a narrow perceptive field and respond with characteristic escape reactions. To decipher larval locomotion not only before but also during the collision we utilized a two color FIM approach (FIM(2c)), which allowed to faithfully extract the posture and motion of colliding animals. We show that during collision, larval locomotion freezes and sensory information is sampled during a KISS phase (german: Kollisions Induziertes Stopp Syndrom or english: collision induced stop syndrome). Interestingly, larvae react differently to living, dead or artificial larvae, discriminate other Drosophila species and have an increased bending probability for a short period after the collision terminates. Thus, Drosophila larvae evolved means to specify behaviors in response to other larvae.

Quantifying subtle locomotion phenotypes of Drosophila larvae using internal structures based on FIM images.

Quantitative analysis of behavioral traits requires precise image acquisition and sophisticated image analysis to detect subtle locomotion phenotypes. In the past, we have established Frustrated Total Internal Reflection (FTIR) to improve the measurability of small animals like insects. This FTIR-based Imaging Method (FIM) results in an excellent foreground/background contrast and even internal organs and other structures are visible without any complicated imaging or labeling techniques. For example, the trachea and muscle organizations are detectable in FIM images. Here these morphological details are incorporated into phenotyping by performing cluster analysis using histogram-based statistics for the first time. We demonstrate that FIM enables the precise quantification of locomotion features namely rolling behavior or muscle contractions. Both were impossible to quantify automatically before. This approach extends the range of FIM applications by enabling advanced automatic phenotyping for particular locomotion patterns.

The Drosophila FHOD1-like formin Knittrig acts through Rok to promote stress fiber formation and directed macrophage migration during the cellular immune response.

A tight spatiotemporal control of actin polymerization is important for many cellular processes that shape cells into a multicellular organism. The formation of unbranched F-actin is induced by several members of the formin family. Drosophila encodes six formin genes, representing six of the seven known mammalian subclasses. Knittrig, the Drosophila homolog of mammalian FHOD1, is specifically expressed in the developing central nervous system midline glia, the trachea, the wing and in macrophages. knittrig mutants exhibit mild tracheal defects but survive until late pupal stages and mainly die as pharate adult flies. knittrig mutant macrophages are smaller and show reduced cell spreading and cell migration in in vivo wounding experiments. Rescue experiments further demonstrate a cell-autonomous function of Knittrig in regulating actin dynamics and cell migration. Knittrig localizes at the rear of migrating macrophages in vivo, suggesting a cellular requirement of Knittrig in the retraction of the trailing edge. Supporting this notion, we found that Knittrig is a target of the Rho-dependent kinase Rok. Co-expression with Rok or expression of an activated form of Knittrig induces actin stress fibers in macrophages and in epithelial tissues. Thus, we propose a model in which Rok-induced phosphorylation of residues within the basic region mediates the activation of Knittrig in controlling macrophage migration.

FIM imaging and FIMtrack: two new tools allowing high-throughput and cost effective locomotion analysis.

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.