The precise quantification of movements is critical for understanding how neurons, biomechanics, and the environment influence and give rise to animal behaviors. For organisms with skeletons and exoskeletons, these measurements are naturally made with reference to 3D joint and appendage locations. Paired with modern approaches to simultaneously record the activity of neural populations in tethered, behaving animals (Dombeck et al., 2007; Seelig et al., 2010; Chen et al., 2018), 3D joint and appendage tracking promises to accelerate the discovery of neural control principles, particularly in the genetically tractable and numerically simple nervous system of the fly, Drosophila melanogaster.

However, algorithms for reliably estimating 3D pose in such small Drosophila-sized animals have not yet been developed. Instead, multiple alternative approaches have been taken. For example, one can affix and use small markers—reflective, colored, or fluorescent particles—to identify and reconstruct keypoints from video data (Bender et al., 2010; Kain et al., 1910; Todd et al., 2017). Although this approach works well on humans (Moeslund and Granum, 2000), in smaller, Drosophila-sized animals markers likely hamper movements and are difficult to mount on sub-millimeter scale limbs. Most importantly, measurements of one or even two markers for each leg (Todd et al., 2017) cannot fully describe 3D limb kinematics. Another strategy has been to use computer vision techniques that operate without markers. However, these measurements have been restricted to 2D pose in freely behaving flies. Before the advent of deep learning, this was accomplished by matching the contours of animals seen against uniform backgrounds (Isakov et al., 2016), measuring limb tip positions using complex TIRF-based imaging approaches (Mendes et al., 2013), or measuring limb segments using active contours (Uhlmann et al., 2017). In addition to being limited to 2D rather than 3D pose, these methods are complex, time-consuming, and error-prone in the face of long data sequences, cluttered backgrounds, fast motion, and occlusions that naturally occur when animals are observed from a single 2D perspective.

As a result, in recent years the computer vision community has largely forsaken these techniques in favor of deep learning-based methods. Consequently, the efficacy of monocular 3D human pose estimation algorithms has greatly improved. This is especially true when capturing human movements for which there is enough annotated data to train deep networks effectively. Walking and upright poses are prime examples of this, and state-of-the-art algorithms (Pavlakos et al., 2017a; Tome et al., 2017; Popa et al., 2017; Moreno-noguer, 2017; Martinez et al., 2017; Mehta et al., 2017; Rogez et al., 2017; Pavlakos et al., 2017b; Zhou et al., 2017; Tekin et al., 2017; Sun et al., 2017) now deliver impressive real-time results in uncontrolled environments. Increased robustness to occlusions can be obtained by using multi-camera setups (Elhayek et al., 2015; Rhodin et al., 2016; Simon et al., 2017; Pavlakos et al., 2017b) and triangulating the 2D detections. This improves accuracy while making it possible to eliminate false detections.

These advances in 2D pose estimation have also recently been used to measure behavior in laboratory animals. For example, DeepLabCut provides a user-friendly interface to DeeperCut, a state-of-the-art human pose estimation network (Mathis et al., 2018), and LEAP (Pereira et al., 2019) can successfully track limb and appendage landmarks using a shallower network. Still, 2D pose provides an incomplete representation of animal behavior: important information can be lost due to occlusions, and movement quantification is heavily influenced by perspective.

Approaches used to translate human 2D to 3D pose have also been applied to larger animals, like lab mice and cheetahs (Nath et al., 2019), but require the use of calibration boards. These techniques cannot be easily transferred for the study of small animals like Drosophila: adult flies are approximately 2.5 mm long and precisely registering multiple camera viewpoints using traditional approaches would require the fabrication of a prohibitively small checkerboard pattern, along with the tedious labor of using a small, external calibration pattern. Moreover, flies have many appendages and joints, are translucent, and in most laboratory experiments are only illuminated using infrared light (to avoid visual stimulation)—precluding the use of color information.

To overcome these challenges, we introduce DeepFly3D, a deep learning-based software pipeline that achieves comprehensive, rapid, and reliable 3D pose estimation in tethered, behaving adult Drosophila (Figure 1, Figure 1—video 1). DeepFly3D is applied to synchronized videos acquired from multiple cameras. It first uses a state-of-the-art deep network (Newell et al., 2016) and then enforces consistency across views. This makes it possible to eliminate spurious detections, achieve high 3D accuracy, and use 3D pose errors to further fine-tune the deep network to achieve even better accuracy. To register the cameras, DeepFly3D uses a novel calibration mechanism in which the fly itself is the calibration target. During the calibration process, we also employ sparse bundle adjustment methods, as previously used for human pose estimation (Takahashi et al., 2018; Triggs et al., 2000; Puwein et al., 2014). Thus, the user does not need to manufacture a prohibitively small calibration pattern, or repeat cumbersome calibration protocols. We explain how users can modify the codebase to extend DeepFly3D for 3D pose estimation in other animals (see Materials and methods). Finally, we demonstrate that unsupervised behavioral embedding of 3D joint angle data is robust against problematic artifacts present in embeddings of 2D pose data. In short, DeepFly3D delivers 3D pose estimates reliably, accurately, and with minimal manual intervention while also providing a critical tool for automated behavioral data analysis.

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We have developed DeepFly3D, a deep learning-based 3D pose estimation system that is optimized for quantifying limb and appendage movements in tethered, behaving Drosophila. By using multiple synchronized cameras and exploiting multiview redundancy, our software delivers robust and accurate pose estimation at the sub-millimeter scale. Ultimately, we may work solely with monocular images by lifting the 2D detections (Pavlakos et al., 2017b) to 3D or by directly regressing to 3D (Tekin et al., 2017) as has been achieved in human pose estimation studies. Our approach relies on supervised deep learning to train a neural network that detects 2D joint locations in individual camera images. Importantly, our network becomes increasingly competent as it runs: By leveraging the redundancy inherent to a multiple-camera setup, we iteratively reproject 3D pose to automatically detect and correct 2D errors, and then use these corrections to further train the network without user intervention.

None of the techniques we have put together—an approach for multiple-camera calibration that uses the animal itself rather than an external apparatus, an iterative approach to inferring 3D pose using graphical models as well as optimization based on dynamic programming and belief propagation, and a graphical user interface and active learning policy for interacting with, annotating, and correcting 3D pose data—are fly-specific. They could easily be adapted to other limbed animals, from mice to primates and humans. The only thing that would have to change significantly are the dimensions of the experimental setup. This would remove the need to deal with the very small scales Drosophila requires and would, in practice, make pose estimation easier. In the Materials and methods section, we explain in detail how organism-specific features of DeepFly3D—bone segment length, number of legs, and camera focal distance—can be modified to study, for example, humans, primates, rodents, or other insects.

As in the past, we anticipate that the development of new technologies for quantifying behavior will open new avenues and enhance existing lines of investigation. For example, deriving 3D pose using DeepFly3D can improve the resolution of studies examining how neuronal stimulation influences animal behavior (Cande et al., 2018; McKellar et al., 2019), the precision and predictive power of efforts to define natural action sequences (Seeds et al., 2014; McKellar et al., 2019), the assessment of interventions that target models of human disease (Feany and Bender, 2000; Hewitt and Whitworth, 2017), and links between neural activity and animal behavior—when coupled with recording technologies like 2-photon microscopy (Seelig et al., 2010; Chen et al., 2018). Importantly, 3D pose improves the robustness of unsupervised behavioral classification approaches. Therefore, DeepFly3D is a critical step toward the ultimate goal of achieving fully-automated, high-fidelity behavioral data analysis.

With synchronized Drosophila video sequences from seven cameras in hand, the first task for DeepFly3D is to detect the 2D location of 38 landmarks. These 2D locations of the same landmarks seen across multiple views are then triangulated to generate 3D pose estimates. This pipeline is depicted in Figure 6. First, we will describe our deep learning-based approach to detect landmarks in images. Then, we will explain the triangulation process that yields full 3D trajectories. Finally, we will describe how we identify and correct erroneous 2D detections automatically.

Figure 6

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2D pose estimation

Deep network architecture

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We aim to detect five joints on each limb, six on the abdomen, and one on each antenna, giving a total of 38 keypoints per time instance. To achieve this, we adapted a state-of-the-art Stacked Hourglass human pose estimation network (Newell et al., 2016) by changing the input and output layers to accommodate a new input image resolution and a different number of tracked points. A single hourglass stack consists of residual bottleneck modules with max pooling, followed by up-sampling layers and skip connections. The first hourglass network begins with a convolutional layer and a pooling layer to reduce the input image size from 256 × 512 to 64 × 128 pixels. The remaining hourglass input and output tensors are 64 × 128. We used 8 stacks of hourglasses in our final implementation. The output of the network is a stack of probability maps, also known as heatmaps or confidence maps. Each probability map encodes the location of one keypoint, as the belief of the network that a given pixel contains that particular tracked point. However, probability maps do not formally define a probability distribution; their sum over all pixels does not equal 1.

2D pose training dataset

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We trained our network for 19 keypoints, resulting in the tracking of 38 points when both sides of the fly are taken into account. Determining which images to use for training purposes is critical. The intuitively simple approach—training with randomly selected images—may lead to only marginal improvements in overall network performance. This is because images for which network predictions can already be correctly made give rise to only small gradients during training. On the other hand, manually identifying images that may lead to incorrect network predictions is highly laborious. Therefore, to identify such challenging images, we exploited the redundancy of having multiple camera views (see section 3D pose correction). Outliers in individual camera images were corrected automatically using images from other cameras, and frames that still exhibited large reprojection errors on multiple camera views were selected for manual annotation and network retraining. This combination of self supervision and active learning permits faster training using a smaller manually annotated dataset (Simon et al., 2017). The full annotation and iterative training pipeline is illustrated in Figure 6. In total, 40,063 images were annotated: 5,063 were labeled manually in the first iteration, 29,000 by automatic correction, and 6,000 by manually correcting those proposed by the active learning strategy.

Deep network training procedure

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We trained our Stacked Hourglass network to regress from 256 × 512 pixel grayscale video images to multiple 64 × 128 probability maps. Specifically, during training and testing, networks output a 19 × 64 × 128 tensor; one 64 × 128 probability map per tracked point. During training, we created probability maps by embedding a 2D Gaussian with mean at the ground-truth point and 1px symmetrical extent (i.e. $\sigma =1px$) on the diagonal of the covariance matrix. We calculated the loss as the $L_2$ distance between the ground-truth and predicted probability maps. During testing, the final network prediction for a given point was the probability map pixel with maximum probability. We started with a learning rate of 0.0001 and then multiplied the learning rate by a factor of 0.1 once the loss function plateaued for more than five epochs. We used an RMSPROP optimizer for gradient descent, following the original Stacked Hourglass implementation, with a batch-size of eight images. Using 37,000 training images, the Stacked Hourglass network usually converges to a local minimum after 100 epochs (20 h on a single GPU).

Network training details

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Variations in each fly’s position across experiments are handled by the translational invariance of the convolution operation. In addition, we artificially augment training images to improve network generalization for further image variables. These variables include (i) illumination conditions – we randomly changed the brightness of images using a gamma transformation, (ii) scale – we randomly rescaled images between 0.80x - 1.20x, and (iii) rotation – we randomly rotated images and corresponding probability maps ±15°. This augmentation was enough to compensate for real differences in the size and orientation of tethered flies across experiments. Furthermore, as per general practice, the mean channel intensity was subtracted from each input image to distribute annotations symmetrically around zero. We began network training using pretrained weights from the MPII human pose dataset (Andriluka et al., 2014). This dataset consists of more than 25,000 images with 40,000 annotations, possibly with multiple ground-truth human pose labels per image. Starting with a pretrained network results in faster convergence. However, in our experience, this does not affect final network accuracy in cases with a large amount of training data. We split the dataset into 37,000 training images, 2,063 testing images, and 1,000 validation images. None of these subsets shared common images or common animals, to ensure that the network could generalize across animals, and experimental setups. 5,063 of our training images were manually annotated, and the remaining data were automatically collected using belief propagation, graphical models, and active learning, (see section 3D pose correction). Deep neural network parameters need to be trained on a dataset with manually annotated ground-truth key point positions. To initialize the network, we collected annotations using a custom multicamera annotation tool that we implemented in JavaScript using Google Firebase (Figure 7). The DeepFly3D annotation tool operates on a simple web-server, easing the distribution of annotations across users and making these annotations much easier to inspect and control.

Figure 7

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Computing hardware and software

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We trained our model on a desktop computing workstation running on an Intel Core i9-7900X CPU, 32 GB of DDR4 RAM, and a GeForce GTX 1080. With 37,000 manually and automatically labeled images, training takes nearly 20 h on a single GeForce GTX 1080 GPU. Our code is implemented with Python 3.6, Pytorch 0.4 and CUDA 9.2. Using this desktop configuration, our network can run at 100 Frames-Per-Second (FPS) using the 8-stack variant of the Hourglass network, and can run at 420 FPS using the smaller 2-stack version. Thanks to an effective initialization step, calibration takes 3–4 s. Error checking and error correction can be performed at 100 FPS and 10 FPS, respectively. Error correction is only performed in response to large reprojection errors and does not create a bottleneck in the overall speed of the pipeline.

Accuracy analysis

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Consistent with the human pose estimation literature, we report accuracy as Percentage of Correct Keypoints (PCK) and Root Mean Squared Error (RMSE). PCK refers to the percentage of detected points lying within a specific radius from the ground-truth label. We set this threshold as 50 pixels, which is roughly one third of the 3D length of the femur. The final estimated position of each keypoint was obtained by selecting the pixel with the largest probability value on the relevant probability map. We compared DeepFly3D’s annotations with manually annotated ground-truth labels to test our model’s accuracy. For RMSE, we report the square root of average pixel distance between the prediction and the ground-truth location of the tracked point. We remove trivial points such as the body-coxa and coxa-femur—which remain relatively stationary—to fairly evaluate our algorithms and to prevent these points from dominating our accuracy measurements.

From 2D landmarks to 3D trajectories

In the previous section, we described our approach to detect 38 2D landmarks. Let ${\mathbf{𝐱}}_{c,j}\in {ℝ}^2$ denote the 2D position of landmark $j$ in the image acquired by camera $c$. For each landmark, our task is now to estimate the corresponding 3D position, ${\mathbf{𝐗}}_j\in {ℝ}^3$. To accomplish this, we used triangulation and bundle-adjustment (Hartley and Zisserman, 2000) to compute 3D locations, and we used pictorial structures (Felzenszwalb and Huttenlocher, 2005) to enforce geometric consistency and to eliminate potential errors caused by misdetections. We present these steps below.

Pinhole camera model

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The first step is to model the projection operation that relates a specific ${\mathbf{𝐗}}_j$ to its seven projections in each camera view ${\mathbf{𝐱}}_{c,j}$. To make this easier, we follow standard practice and convert all Cartesian coordinates $\left[\begin{array}{c}\hfill x_c,y_c,z_c\hfill \end{array}\right]$ to homogeneous ones $\left[\begin{array}{c}\hfill x_h,y_h,z_h,s\hfill \end{array}\right]$ such that $x_c=x_h/s$, $y_c=y_h/s$, $z_c=z_h/s$. From now on, we will assume that all points are expressed in homogeneous coordinates and omit the $h$ subscript. Assuming that these coordinates are expressed in a coordinate system whose origin is in the optical center of the camera and whose z-axis is its optical axis, the 2D image projection $\left[\begin{array}{c}\hfill u,v\hfill \end{array}\right]$ of a 3D homogeneous point $\left[\begin{array}{c}\hfill x,y,z,1\hfill \end{array}\right]$ can be written as

(1) ${\displaystyle \begin{array}{l}{\displaystyle u={\displaystyle U/W,}}\\ v{\displaystyle =V/W,}\\ {\displaystyle \left[\begin{array}{c}U\\ V\\ W\end{array}\right]{\displaystyle =\mathbf{K}\left[\begin{array}{c}x\\ y\\ z\\ 1\end{array}\right],\text{ with }\mathbf{K}=\left[\begin{array}{cccc}{f}_x& 0& c_x& 0\\ 0& f_y& c_y& 0\\ 0& 0& 1& 0\end{array}\right],}}\end{array}}$

where the 3 × 4 matrix $\mathbf{𝐊}$ is known as the intrinsic parameters matrix—scaling in the $x$ and $y$ direction and image coordinates of the principal point $c_x$ and $c_y$—that characterizes the camera settings.

In practice, the 3D points are not expressed in a camera fixed coordinate system, especially in our application where we use seven different cameras. Therefore, we use a world coordinate system that is common to all cameras. For each camera, we must therefore convert 3D coordinates expressed in this world coordinate system to camera coordinates. This requires rotating and translating the coordinates to account for the position of the camera’s optical center and its orientation. When using homogeneous coordinates, this is accomplished by multiplying the coordinate vector by a 4 × 4 extrinsic parameters matrix

(2) $\mathbf{𝐌}=\left[\begin{array}{cc}\hfill \mathbf{𝐑}\hfill & \hfill \mathbf{𝐓}\hfill \\ \hfill 0\hfill & \hfill 1\hfill \end{array}\right],$

where $\mathbf{𝐑}$ is a 3 × 3 rotation matrix and $\mathbf{𝐓}$ a 3 × 1 translation vector. Combining Equation 1 and Equation 2 yields

(3) ${\displaystyle \begin{array}{l}{\displaystyle u={\displaystyle U/W,}}\\ v{\displaystyle =V/W,}\\ {\displaystyle \left[\begin{array}{c}U\\ V\\ W\end{array}\right]{\displaystyle =\mathbf{P}\left[\begin{array}{c}x\\ y\\ z\\ 1\end{array}\right],}}\end{array}}$

where ${\displaystyle \mathbf{\text{P}}=\mathbf{\text{MK}}}$ is a 3 × 4 matrix.

Camera distortion

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The pinhole camera model described above is an idealized one. The projections of real cameras deviate from it. These deviations are referred to as distortions and must be accounted for. The most significant distortion is known as radial distortion because the error grows with the distance from the image center. For the cameras we use, radial distortion can be expressed as

(4) ${\displaystyle \begin{array}{l}{\displaystyle u_{\text{ pinhole }}{\displaystyle =u\left(1+{\mathrm{k}}_1^x{\mathrm{r}}^2+{\mathrm{k}}_2^x{\mathrm{r}}^4\right),}}\\ {\displaystyle v_{\text{ pinhole }}{\displaystyle =v\left(1+{\mathrm{k}}_1^y{\mathrm{r}}^2+{\mathrm{k}}_2^y{\mathrm{r}}^4\right),}}\end{array}}$

where $\left[\begin{array}{c}\hfill u,v\hfill \end{array}\right]$ is the actual projection of a 3D point and $\left[\begin{array}{c}\hfill u_{\text{pinhole}},v_{\text{pinhole}}\hfill \end{array}\right]$ is the one the pinhole model predicts. In other words, the four parameters $\{k_1^x,k_2^x,k_1^y,k_2^y\}$ characterize the distortion. From now on, we will therefore write the full projection as

(5) ${\displaystyle \begin{array}{ll}\mathbf{X}{\displaystyle =}& \pi (\mathbf{x})=f_d(f_p(\mathbf{x})),\\ \mathbf{X}=& [x,y,z],\\ \mathbf{X}=& [u,v],\end{array}}$

where $f_p$ denotes the ideal pinhole projection of Equation 3 and $f_d$ the correction of Equation 4.

Triangulation

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We can associate to each of the seven cameras a projection function ${\pi }_c$ like the one in Equation 5, where $c$ is the camera number. Given a 3D point and its projections ${\mathbf{𝐱}}_c$ in the images, its 3D coordinates can be estimated by minimizing the reprojection error

(6) ${\displaystyle \underset{\mathbf{X}\in {\mathbb{R}}^4}{\mathrm{a}\mathrm{r}\mathrm{g}\mathrm{m}\mathrm{i}\mathrm{n}}\sum _{c=1}^7{e}_c\Vert {\pi }_c(\mathbf{X})-{\mathbf{x}}_c{\Vert }^2,}$

where $e_c$ is one if the point was visible in image $c$ and zero otherwise. In the absence of camera distortion, that is, when the projection $\pi$ is a purely linear operation in homogeneous coordinates, this can be done for any number of cameras by solving a Singular Value Decomposition (SVD) problem (Hartley and Zisserman, 2000). In the presence of distortions, we replace the observed $u$ and $v$ coordinates of the projections by the corresponding $u_{\text{pinhole}}$ and $u_{\text{pinhole}}$ values of Equation 5 before performing the SVD.

Camera calibration

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Triangulating as described above requires knowing the projection matrices ${\mathbf{𝐏}}_c$ of Equation 3 for each camera $c$, corresponding distortion parameters $\{k_1^x,k_2^x,k_1^y,k_2^y\}$ of Equation 4, together with the intrinsic parameters of focal length and principal point offset. In practice, we use the focal length and principal point offset provided by the manufacturer and estimate the remaining parameters automatically: the three translations and three rotations for each camera that define the corresponding matrix $\mathbf{𝐌}$ of extrinsic parameters along with the distortion parameters.

To avoid having to design the exceedingly small calibration pattern that more traditional methods use to estimate these parameters, we use the fly itself as calibration pattern and minimize the reprojection error of Equation 6 for all joints simultaneously while allowing the camera parameters to also change. In other words we look for

(7) ${\displaystyle \begin{array}{ll}& \mathrm{a}\mathrm{r}\mathrm{g}\mathrm{m}\mathrm{i}\mathrm{n}\sum _{c=1}^7\sum _{j=1}^m{e}_{c,j}\rho ({\pi }_c({\mathbf{X}}_j)-{\mathbf{x}}_{c,j}),\\ & {\pi }_{c_{1\le c\le 7}}\\ & {{\mathbf{X}}_{\mathbf{j}}}_{1\le j\le m}\end{array}}$

where ${\mathbf{𝐗}}_{\mathbf{𝐣}}$ and ${\mathbf{𝐱}}_{c,j}$ are the 3D locations and 2D projections of the landmarks introduced above and $\rho$ denotes the Huber loss. Equation 7 is known as bundle-adjustment (Hartley and Zisserman, 2000). Huber loss is defined as

${\displaystyle {\rho }_{\delta }(a)=\{\begin{array}{ll}\frac{1}{2}{a}^2& \text{ for }|a|\le \delta \\ \delta \left(|a|-\frac{1}{2}\delta \right)& \text{ otherwise }\end{array}.}$

Replacing the squared loss by the Huber loss makes our approach more robust to erroneous detections ${\mathbf{𝐱}}_{c,j}$. We empirically set $\delta$ to 20 pixels. Note that we perform this minimization with respect to ten degrees-of-freedom per camera: three translations, three rotations, and four distortions.

For this optimization to work properly, we need to initialize these 10 parameters and we need to reduce the number of outliers. To achieve this, the initial distortion parameters are set to zero. We also produce initial estimates for the three rotation and three translation parameters by measuring the distances between adjacent cameras and their relative orientations. To initialize the rotation and translation vectors, we measure the distance and the angle between adjacent cameras, from which we infer rough initial estimates. Finally, we rely on epipolar geometry (Hartley and Zisserman, 2000) to automate outlier rejection. Because the cameras form a rough circle and look inward, the epipolar lines are close to being horizontal Figure 8A. Thus, corresponding 2D projections must belong to the same image rows, or at most a few pixels higher or lower. In practice, this means checking if all 2D predictions lie in nearly the same rows and discarding a priori those that do not.

Figure 8

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3D pose correction

The triangulation procedure described above can produce erroneous results when the 2D estimates of landmarks are wrong. Additionally, it may result in implausible 3D poses for the entire animal because it treats each joint independently. To enforce more global geometric constraints, we rely on pictorial structures (Felzenszwalb and Huttenlocher, 2005) as described in Figure 9. Pictorial structures encode the relationship between a set of variables (in this case the 3D location of separate tracked points) in a probabilistic setting using a graphical model. This makes it possible to consider multiple 2D locations ${\mathbf{𝐱}}_{c,j}$ for each landmark ${\mathbf{𝐗}}_c$ instead of only one. This increases the likelihood of finding the true 3D pose.

Figure 9

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Generating multiple candidates

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Instead of selecting landmarks as the locations with the maximum probability in maps output by our Stacked Hourglass network, we generate multiple candidate 2D landmark locations $x_{c,j}$. From each probability map, we select 10 local probability maxima that are at least one pixel apart from one another. Then, we generate 3D candidates by triangulating 2D candidates in every tuple of cameras. Because a single point is visible from at most four cameras, this results in at most ${\displaystyle \left(\begin{array}{c}4\\ 2\end{array}\right)\times {10}^2}$ candidates for each tracked point.

Choosing the best candidates

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To identify the best subset of resulting 3D locations, we introduce the probability distribution $P(L|I,\theta )$ that assigns a probability to each solution $L$, consisting of 38 sets of 2D points observed from each camera. Our goal is then to find the most likely one. More formally, $P$ represents the likelihood of a set of tracked points $L$, given the images, model parameters, camera calibration, and geometric constraints. In our formulation, $I$ denotes the seven camera images $I={\{I_c\}}_{1\le c\le 7}$ and $\theta$ represents the set of projection functions ${\pi }_c$ for camera $c$ along with a set of length distributions $S_{i,j}$ between each pair of points $i$ and $j$ that are connected by a limb. $L$ consists of a set of tracked points ${\{L_i\}}_{1\le i\le n}$, where each $L_i$ describes a set of 2D observations $l_{i,c}$ from multiple camera views. These are used to triangulate the corresponding 3D point locations $\overline{l_i}$. If the set of 2D observations is incomplete, as some points are totally occluded in some camera views, we triangulate the 3D point ${\overline{l}}_i$ using the available ones and replace the missing observations by projecting the recovered 3D positions into the images, ${\pi }_c({\overline{l}}_i)$ in Equation 3. In the end, we aim to find the solution $\widehat{L}=\underset{L}{argmax}P(L|I,\theta ).$ This is known as Maximum a Posteriori (MAP) estimation. Using Bayes rule, we write

(8) $P(L|I,\theta )\propto P(I|L,\theta )P(L|\theta ),$

where the two terms can be computed separately. We compute $P(I|J,\theta )$ using the probability maps $H_{j,c}$ generated by the Stacked Hourglass network for the tracked point $j$ for camera $c$. For a single joint $j$ seen by camera $c$, we model the likelihood of observing that particular point using $P(H_{j,c}|l_{j,c})$, which can be directly read from the probability maps as the pixel intensity. Ignoring the dependency between the cameras, we write the overall likelihood as the product of the individual likelihood terms

(9) $P(I|L,\theta )=P(H|L)\propto \prod _{i=1}^n\prod _{c=1}^{7}P(H_{j,c}|l_{i,c}),$

which can be read directly from the probability maps as pixel intensities and represent the network’s confidence that a particular keypoint is located at a particular pixel. When a point is not visible from a particular camera, we assume the probability map only contains a constant non-zero probability, which does not affect the final solution. We express $P(L|\theta )$ as

(10) $P(L|\theta )=P(L|\pi ,S)=\prod _{(i,j)\in E}P({\overline{l}}_i,{\overline{l}}_j|S_{i,j})\prod _{j=1}^n\prod _{c=1}^7{e}_{c,j}{\parallel {\pi }_c({\overline{l}}_j)-l_{c,j}\parallel }_2^{-1},$

where pairwise dependencies $P({\overline{l}}_i,{\overline{l}}_j|S_{i,j})$ between two variables respect the segment length constraint when the variables are connected by a limb. The length of segments defined by pairs of connected 3D points follows a normal distribution. Specifically, we model $P({\overline{l}}_i,{\overline{l}}_j|S_{i,j})$ as $S_{i,j}({\overline{l}}_i,{\overline{l}}_j)=𝒩(\parallel {\overline{l}}_i-{\overline{l}}_j\parallel -{\mu }_{i,j},{\sigma }_{i,j})$. We model the reprojection error for a particular point $j$ as ${\prod }_{c=1}^7{e}_{c,j}{\parallel {\pi }_c({\overline{l}}_j)-l_{c,j}\parallel }_2^{-1}$ which is set to zero using the variable $e_{c,j}$ denoting the visibility of the point $j$ from camera $c$. If a 2D observation for a particular camera is manually set by a user with the DeepFly3D GUI, we take it to be the only possible candidate for that particular image and we set $P(L_j|H)$ to 1, where $j$ denotes the manually assigned pixel location.

Solving the MAP problem using the Max-Sum algorithm

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For general graphs, MAP estimation with pairwise dependencies is NP-hard and therefore intractable. However, in the specific case of non-cyclical graphs, it is possible to solve the inference problem using belief propagation (Bishop, 2006). Since the fly’s skeleton has a root and contains no loops, we can use a message passing approach (Felzenszwalb and Huttenlocher, 2005). It is closely related to Viterbi recurrence and propagates the unary probabilities $P(L_j|L_i)$ between the edges of the graph starting from the root and ending at the leaf nodes. This first propagation ends with the computation of the marginal distribution for the leaf node variables. During the subsequent backward iteration, as $P(L_j)$ for leaf node is computed, the point $L_j$ with maximum posterior probability is selected in $O(k)$ time, where $k$ is the upper bound on the number of proposals for a single tracked point. Next, the distribution $P(L_i|L_j)$ is calculated, adjacent nodes for the leaf node. Continuing this process on all the remaining points results in a MAP solution for the overall distribution $P(L)$, as shown in Figure 9, with overall $O(k^2)$ computational complexity.

Learning the parameters

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We learn the parameters for the set of pairwise distributions $S_{i,j}$ using a maximum likelihood process and assuming the distributions to be Gaussian. We model the segment length $S_{i,j}$ as the euclidean distance between the points ${\overline{l}}_j$ and ${\overline{l}}_j$. We then solve for $\underset{S}{argmax}P(S|L,\theta )$, assuming segments have a Gaussian distribution resulting from the Gaussian noise in point observations $L$. This gives us the mean and variance, defining each distribution $S_{i.j}$. We exclude the same points that we removed from the calibration procedure, that exhibit high reprojection error.

In practice, we observe a large variance for pretarsus values (Figure 10). This is because occlusions occasionally shorten visible tarsal segments. To eliminate the resulting bias, we treat these limbs differently from the others and model the distribution of tibia-tarsus and tarsus-tip points as a Beta distribution, with parameters found using a similar Maximum Likelihood Estimator (MLE) formulation. Assuming the observation errors to be Gaussian and zero-centered, the bundle adjustment procedure can also be understood as an MLE of the calibration parameters (Triggs et al., 2000). Therefore, the entire set of parameters for the formulation can be learned using MLE. Thus, prior information about potentially occluded targets can be used to guide inference. For example, in a head-fixed rodent, the left eye may not always be visible from the right-side of the animal. This information can be incorporated into DeepFly3D’s inference system in the file, skeleton.py, by editing the function camera_see_joint. Afterwards, predictions from occluded cameras will not be used to triangulate a given 3D point. If no such information is provided, every prediction will be used to triangulate a given 3D point.

Figure 10

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The pictorial structure formulation can be further expanded using temporal information, penalizing large movements of a single tracked point between two consecutive frames. However, we abstained from using temporal information more extensively for several reasons. First, temporal dependencies would introduce loops in our pictorial structures, thus making exact inference NP-hard as discussed above. This can be handled using loopy belief propagation algorithms (Murphy et al., 1999) but requires multiple message passing rounds, which prevents real-time inference without any theoretical guarantee of optimal inference. Second, the rapidity of Drosophila limb movements makes it hard to assign temporal constraints, even with fast video recording. Finally, we empirically observed that the current formulation, enforcing structured poses in a single temporal frame, already eliminates an overwhelming majority of false-positives inferred during the pose estimation stage of the algorithm.

Modifying DeepFly3D to study other animals

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DeepFly3D does not assume a circular camera arrangement or that there is one degree of freedom in the camera network. Therefore, it could easily be adapted for 3D pose estimation in other animals, ranging from rodents to primates and humans. We illustrate this flexibility by using DeepFly3D to capture human 3D pose in the Human 3.6M Dataset (http://vision.imar.ro/human3.6m/description.php) very popular, publicly available computer vision benchmarking dataset generated using four synchronized cameras (Ionescu et al., 2014; Ionescu et al., 2011) (Figure 11).

Figure 11

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Generally, for any new dataset, the user first needs to provide an initial set of manual annotations. The user would describe the number of tracked points and their relationships to one another in a python setup file. Then, in a configuration file, the user specifies the number of cameras along with the resolutions of input images and output probability maps. DeepFly3D will then use these initial manual annotations to (i) train the 2D Stacked Hourglass network, (ii) perform camera calibration without an external calibration pattern, (iii) learn the epipolar geometry to perform outlier detection, and (iv) learn the segment length distributions $S_{i,j}$. After this initial bootstrapping, DeepFly3D can be then used with pictorial structures and active learning to iteratively improve pose estimation accuracy.

The initial manual annotations can be performed using the DeepFly3D Annotation GUI. Afterwards, these annotations can be downloaded from the Annotation GUI as a CSV file using the Save button (Figure 7). Once the CSV file is placed in the images folder, DeepFly3D will automatically read and display the annotations. To train the Stacked Hourglass network, use the csv-path flag while running pose2d.py (found in deepfly/pose2d/). DeepFly3D will then train the Stacked Hourglass network by performing transfer learning using the large MPII dataset and the smaller set of user manual annotations.

To perform camera calibration, the user should select the Calibration button on the GUI Figure 12. DeepFly3D will then perform bundle adjustment (Equation 7) and save the camera parameters in calibration.pickle (found in the images folder). The path of this file should then be added to Config.py to initialize calibration. These initial calibration parameters will then be used in further experiments for fast and accurate convergence. If the number of annotations is insufficient for accurate calibration, or if bundle adjustment is converging too slowly, an initial rough estimate of the camera locations can be set in Config.py. As long as a calibration is set in Config.py, DeepFly3D will use it as a projection matrix to calculate the epipolar geometry between cameras. This step is necessary to perform outlier detection on further calibration operations.

Figure 12

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DeepFly3D will also learn the distribution $S_{i,j}$, whose non-zero entries are found in skeleton.py. One can easily calculate these segment length distribution parameters using the functions provided with DeepFly3D. CameraNetwork class (found under deepfly/GUI/), will then automatically load the points and calibration parameters from the images folder. The function CameraNetwork.triangulate will convert 2D annotation points into 3D points using the calibration parameters. The $S_{i,j}$ parameters can then be saved using the pickle library (the save path can be set in Config.py). The calcBoneParams method will then output the segment lengths’ mean and variance. These values will then be used with pictorial structures (Equation 8).

We provide further technical details for how to adapt DeepFly3D to other multi-view datasets online (https://github.com/NeLy-EPFL/DeepFly3D [Günel et al., 2019] copy archived at https://github.com/elifesciences-publications/DeepFly3D).

Experimental setup

We positioned seven Basler acA1920-155um cameras (FUJIFILM AG, Niederhaslistrasse, Switzerland) 94 mm away from the tethered fly, resulting in a circular camera network with the animal in the center (Figure 13). We acquired 960 × 480 pixel video data at 100 FPS under 850 nm infrared ring light illumination (Stemmer Imaging, Pfäffikon Switzerland). Cameras were mounted with 94 mm W.D./1.00 x InfiniStix lenses (Infinity Photo-Optical GmbH, Göttingen). Optogenetic stimulation LED light was filtered out using 700 nm longpass optical filters (Edmund Optics, York UK). Each camera’s depth of field was increased using 5.8 mm aperture retainers (Infinity Photo-Optical GmbH). To automate the timing of optogenetic LED stimulation and camera acquisition triggering, we use an Arduino (Arduino, Sommerville, MA) and custom software written using the Basler camera API.

Figure 13

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We assessed the optimal number of cameras for DeepFly3D and concluded that increasing the number of cameras increases accuracy by stabilizing triangulation. Specifically, we observed the following. (i) Calibration is not a significant source of error: calibrating with fewer than seven cameras does not dramatically increase estimation error. (ii) Having more cameras improves triangulation. Reducing the number of cameras down to four, even having calibrated with seven cameras, results in an increase of 0.05 mm triangulation error. This may be because the camera views are sufficiently different, having largely non-overlapping 2D-detection failure cases. Thus, the redundancy provided by having more cameras mitigates detection errors by finding a 3D pose that is consistent across at least two camera views.

Drosophila transgenic lines

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UAS-CsChrimson (Klapoetke et al., 2014) animals were obtained from the Bloomington Stock Center (Stock $\mathrm{\#}55135$). MDN-1-Gal4 (Bidaye et al., 2014) (VT44845-DBD; VT50660-AD) was provided by B. Dickson (Janelia Research Campus, Ashburn). aDN-Gal4 (Hampel et al., 2015)(R76F12-AD; R18C11-DBD), was provided by J. Simpson (University of California, Santa Barbara). Wild-type, PR animals were provided by M. Dickinson (California Institute of Technology, Pasadena).

Optogenetic stimulation experiments

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Experiments were performed in the late morning or early afternoon Zeitgeber time (Z.T.), inside a dark imaging chamber. An adult female animal 2–3 days-post-eclosion (dpe), was mounted onto a custom stage (Chen et al., 2018) and allowed to acclimate for 5 min on an air-supported spherical treadmill (Chen et al., 2018). Optogenetic stimulation was performed using a 617 nm LED (Thorlabs, Newton, NJ) pointed at the dorsal thorax through a hole in the stage, and focused with a lens (LA1951, 01" f = 25.4 mm, Thorlabs, Newton, NJ). Tethered flies were otherwise allowed to behave spontaneously. Data were acquired in 9 s epochs: 2 s baseline, 5 s with optogenetic illumination, and 2 s without stimulation. Individual flies were recorded for five trials each, with one-minute intervals. Data were excluded from analysis if flies pushed their abdomens onto the spherical treadmill—interfering with limb movements—or if flies struggled during optogenetic stimulation, pushing their forelimbs onto the stage for prolonged periods of time.

All data generated and analyzed during this study are included in the DeepFly3D GitHub site: https://github.com/NeLy-EPFL/DeepFly3D (copy archived at https://github.com/elifesciences-publications/DeepFly3D) and in the Harvard Dataverse.

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Thank you for submitting your article "DeepFly3D, a deep learning-based approach for 3D limb and appendage tracking in tethered, adult Drosophila" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ronald Calabrese as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Josh W Shaevitz (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Both reviewers agreed that this tools and resources article can potentially be of use to the field. By automating pose tracking a large amount of data can be extracted from experiments, enabling new scientific questions to be addressed.

However, a number of key issues with the current study were identified and outlined in the reviewer comments below. In summary, the current manuscript:

1) Lacks a comprehensive discussion of how experimental choices, e.g. camera number, affect tracking error

2) Reports a very large tracking error using a much larger training set than current state-of-the-art 2D tracking algorithms. This needs to be accounted for.

3) Lacks some key references in the field and comparison with other approaches.

Reviewer #1:

Günel et al. report the development of a new toolkit to perform 3D pose estimation on tethered flies. While the toolkit is certainly bound to find utility, there are not major computational advances or insights within the work. While deep learning approaches for animal pose estimation is a "young field" in neuroscience/ethology (i.e. (Pereira et al., 2019, Mathis et al., 2018, Nath et al., 2019), it would be important for the authors to clarify their contribution in light of this. Namely, the authors should clearly discuss the major points that potential users care about: (1) accuracy of the tool, (2) amount of data required to train networks, if required, (3) speed, and (4) usability.

While DeepFly3D provides very nice user interfaces for the toolkit, it does suffer in the other three main categories.

1) Accuracy. The authors report a 13.9-pixel error on 480 by 920 images, and consider the PCK to be 50 pixels, which is nearly as long as a leg segment – not the width! I would argue this is far too lenient of a threshold. Additionally, two points the authors need to address (1) The RMSE would be more meaningful with an estimate of the human labeling variability (i.e. what is the best the network could do?), and (2) why, given so much training data, is the performance not as accurate as other approaches (LEAP, DeepLabCut)?

i.e. in reference to:

– "… 480× 960 pixels. The test dataset included challenging frames and occasional motion blur to increase the difficulty of pose estimation. For training, we used a final training dataset of 37,000 frames, an overwhelming majority of which were first automatically corrected using pictorial structures. On test data, we achieved a Root Mean Square Error (RMSE) of 13.9 pixels"

– The error is quite a bit larger than other animal pose estimation approaches, i.e. on 480x640 on 3D freely moving flies DeepLabCut used ~560 frames with ~4-pixel error on 682 × 540 frames. LEAP: ~500 images to achieve ~3-pixel error on 192 × 192-pixel frames with flies. While I appreciate that a direct comparison is not fully straightforward, and the authors do not use "easy" points to compute the pixel error, the authors absolutely should discuss/address this point.

2) The amount of data. As this network was trained on ~37K images, it is likely expected that the end-user will use these authors pre-trained network or the human pre-trained network. Namely, creating a large dataset seems infeasible when other options exist that require much less data. Namely, DeepLabCut reports 50-200 for most applications, and LEAP requires only ~500-1,500, which is nearly a factor of 10 less images to match similar performance (I understand that 2K are hand-labeled, while the rest are not, but even that is a 4X increase over the best method, i.e. DeepLabCut). Thus, the amount of training data for the performance is very high, given current alternatives. These limitations should be discussed.

3) Speed. There is no report of the speed of the network, thus the authors should benchmark the performance on video analysis speed (only network training time is reported).

4) Since the authors needed a lot of labeled data, making this a most useful as a specialized network for tethered flies, i.e. much like OpenPose has specific networks for hands (which they could clarify). Therefore, if they provide the network weights they should show the network is useable for other labs data.

5) 3D pose in the literature. The authors do not discuss Nath et al., which is a 3D extension of DeepLabCut (bioRxiv Nov 2018, and Nature Protocols, 2019) and given it's a small field, the authors should not claim theirs is the first to do 3D pose. Beyond deep learning multiple 3D pose estimation papers exist in Neuroscience (ie. Mimica et al., 2018 – Efficient cortical coding of 3D posture in freely behaving rats).

Therefore language like this should be modified:

"However, algorithms for reliably estimating 3D pose in such small animals have not yet been developed.…"

"However, these measurements have been restricted to 2D pose in freely behaving animals"

"We demonstrate more accurate unsupervised behavioral embedding using 3D joint angles rather than commonly used 2D pose data. Thus, DeepFly3D enables the automated acquisition of behavioral measurements at an unprecedented level of resolution for a variety of biological application"

(also here "resolution" should be edited; it is not that DeepFly3D is more accurate than other alternatives.)

However, I do think these authors have a nice solution, so I do find it very fair that they state they have an alternative 3D approach, (and can highlight the advantages) solution to multi-camera estimation in body- or head- fixed animals. However, again, sparse bundle adjustment is not a new technique, so the authors should discuss this in light of the literature.

6) "Here we present DeepFly3D, a software that infers the 3D pose of tethered, adult Drosophila-or other animals-using multiple camera images" – they don't quantify performance for other animals, so this should be dropped from the Abstract.

7) "DeepLabCut provides a user-friendly interface to DeepCut, a state-of-the-art human pose estimation network" (DeepCut should be DeeperCut, which is different than DeepCut).

8) "Calibration without targets" is misleading. The authors do calibrate; they just use the rigid body of the fly that has set distances per limb segment. To note, this isn't always doable for other animals/videos, again another argument to drop the random "this can be used for anything" language…

Reviewer #2:

The manuscript by Gunel et al. describes a new tool for 3D pose tracking that automatically combines multiple 2D images from different viewing angles while also learning the camera model. There has been a flurry of recent papers that use deep neural networks to track pose in 2D images. This manuscript improves on these by generating 3D positions for each body part that is tracked using a combination of various algorithms that have been developed for human pose tracking. In addition, the authors provide a simple GUI that allows for part labeling and prediction correction. Overall the paper is well written and the most aspects of the system described and justified.

My only major issue is that the experimental setup is not discussed or motivated. In particular, I would like to see more information about the choices/requirements for the number of cameras and other considerations. Why do the authors use 7 cameras? I assume that with fewer cameras you get more error/occlusions/etc. Can you investigate these effects quantitatively? How should a researcher choose the number of cameras (recording from 7 cameras in synch is difficult)?

In addition, I would suggest that the authors show the distribution of 3D localization errors. Is this isotropic? Is it the same for all body parts (the Discussion subsection “Learning the parameters” suggests not)?

1) Lacks a comprehensive discussion of how experimental choices, e.g. camera number, affect tracking error

2) Reports a very large tracking error using a much larger training set than current state-of-the-art 2D tracking algorithms. This needs to be accounted for.

3) Lacks some key references in the field and comparison with other approaches.

Reviewer #1:

[…] While DeepFly3D provides very nice user interfaces for the toolkit, it does suffer in the other three main categories.

1) Accuracy. The authors report a 13.9-pixel error on 480 by 920 images, and consider the PCK to be 50 pixels, which is nearly as long as a leg segment – not the width! I would argue this is far too lenient of a threshold.

We illustrate the relative sizes of the error threshold (50 pixels) and limb segment lengths in Author response image 1A. We set the threshold to be approximately one third the length of femur (erroneously called the “femur tibia segment” in the initial submission), which is approximately 150 pixels. We apologize for any confusion causing the reviewer to interpret this as the width and have adjusted the text accordingly. We now state: “We set this threshold as 50 pixels, which is roughly one third of the 3D length of the femur.” If we reduce our threshold to 30 or 20 pixels, we still achieve 95% or 89% accuracy, respectively. (Author response image 1B).

Author response image 1

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Additionally, two points the authors need to address (1) The RMSE would be more meaningful with an estimate of the human labeling variability (i.e. what is the best the network could do?)

We agree with the reviewer, and have now added this important measurement by calculating the accuracy of human manual-annotations as a baseline for interpreting the quality of our network predictions. We randomly selected 210 images and had a new expert manually annotate them. We observed a RMSE of 12.4 pixels. Therefore, our Network Annotation / Manual Annotation ratio of 1.12 (13.9 pixels / 12.4 pixels) is similar to the ratio reported by Mathis et al., 2018: 1.07 (2.88 pixels / 2.69 pixels) (pg. 1282 the last paragraph, and pg. 1284 the first paragraph). However, this ratio is reported for a mouse dataset, and human variability for Drosophila is not reported. We now include this information in the revised manuscript: “Compared with a ground truth RMSE of 12.4 pixels – via manual annotation of 210 images by a new human expert – our Network Annotation / Manual Annotation ratio of 1.12 (13.9 pixels / 12.4 pixels) is similar to another state-of-the-art network (Mathis et al., 2018): 1.07 (2.88 pixels / 2.69 pixels).”

[…] and (2) why, given so much training data, is the performance not as accurate as other approaches (LEAP, DeepLabCut)?

i.e. in reference to:

– "… 480× 960 pixels. The test dataset included challenging frames and occasional motion blur to increase the difficulty of pose estimation. For training, we used a final training dataset of 37,000 frames, an overwhelming majority of which were first automatically corrected using pictorial structures. On test data, we achieved a Root Mean Square Error (RMSE) of 13.9 pixels"

– The error is quite a bit larger than other animal pose estimation approaches, i.e. on 480x640 on 3D freely moving flies DeepLabCut used ~560 frames with ~4-pixel error on 682 × 540 frames. LEAP: ~500 images to achieve ~3-pixel error on 192 × 192-pixel frames with flies. While I appreciate that a direct comparison is not fully straightforward, and the authors do not use "easy" points to compute the pixel error, the authors absolutely should discuss/address this point.

We understand that a 13.9-pixel error may seem large when compared with other pose estimation approaches. However, as the reviewer appreciates, making a direct comparison between datasets is not fully straightforward and we believe this misconception can be explained by several differences between the various datasets:

Comparison with the LEAP dataset:

1) The ~3-pixel error reported for 192x192-pixel images in Pereira et al., 2019 (LEAP) corresponds to a 11.9-pixel≅3/2(480/192)2+(960/192)2 error for the 480x960 pixel images used in our study if we similarly rescale pose predictions and ground truth annotations. See the Appendix below for a more detailed calculation of the scaling coefficient.

2) The LEAP dataset is relatively highly preprocessed compared with our dataset: images are rotated, aligned, and thresholded to generate a featureless, static background. By contrast, our data are raw, without any further preprocessing, and therefore harder for performing pose estimation. Our multiview dataset does not lend itself as readily to preprocessing, due to the more dynamic background including a rotating spherical treadmill and legs moving on the opposite side of the body.

Comparison with the DeepLabCut dataset:

1) Although Mathis et al., 2018 perform pose estimation on larger images (682x540 pixels), flies occupy a much smaller region in each image, (~340x135 pixels when flies are parallel to the image plane and take up the largest image area). By contrast, in our dataset, flies occupy nearly the entire image (~840x320 pixels). Thus, scaling the reported error for DeepLabCut (4 pixels for 340x135 pixel images) to 840x320 pixel images results in an error of 10.1 pixels ≅4.17/2(320/135)2+(840/340)2. This value is much closer to the 13.9 pixel error that we report for DeepFly3D.

2) Mathis et al., 2018 principally illustrates the tracking of landmarks on the fly’s abdomen and head (with only one landmark on the leg). These larger body parts experience smaller deformations and perform less rapid, complex movements than the legs.

These calculations are approximate but we intend for them to provide context for the error values we report in our manuscript. In short, we believe the seemingly large tracking error of DeepFly3D is (i) due to the scale of the images (rescaled pixel-wise error will grow with increasing image size) and (ii) due to the more visually complex nature of our dataset. It should be stressed that the 2D pose module of DeepFly3D – our Stacked Hourglass network – can be replaced with any other network. This includes the networks from Mathis et al., 2018, and Pereira et al., 2019, or any other pose estimation approach as long as the network outputs pixel-wise conditional probabilities.

The relative strengths of the Stacked Hourglass network and DeepLabCut have also been discussed in a recent preprint (Graving et al., 2019; Appendix 0 Figure 7). They show that, although DeepLabCut is more accurate than the Stacked Hourglass network (15% less error on a Drosophila dataset with the same number of annotations, ~2 pixel MAE versus ~2.3 pixel MAE using the same amount of annotation), the Stacked Hourglass network is much faster than DeepLabCut (4x on average). We believe this speed-accuracy trade-off is necessary to process 700 images produced per second in our system. Similarly, LEAP is significantly less accurate than both DeepLabCut and the Stacked Hourglass network on many different datasets (Graving et al., 2019; Appendix 0 Figure 7).

2) The amount of data. As this network was trained on ~37K images, it is likely expected that the end-user will use these authors pre-trained network or the human pre-trained network. Namely, creating a large dataset seems infeasible when other options exist that require much less data. Namely, DeepLabCut reports 50-200 for most applications, and LEAP requires only ~500-1,500, which is nearly a factor of 10 less images to match similar performance (I understand that 2K are hand-labeled, while the rest are not, but even that is a 4X increase over the best method, i.e. DeepLabCut). Thus, the amount of training data for the performance is very high, given current alternatives. These limitations should be discussed.

In principle, our Stacked Hourglass network also does not require a large amount of data to perform well. However, like all deep models, it gradually improves in performance and generalizes better with increasing amounts of data. Thus, we used a large training set to increase our network’s robustness and utility. Importantly, our DeepFly3D toolset is designed to simplify and automate annotation such that the large-dataset high-accuracy regimes can be reached relatively easily.

To illustrate these assertions and test the performance of the network in a low data regime, we trained a single 2 stacked network using ground-truth annotations data from 7 cameras. In Figure 2B, we compare the results with an asymptotic prediction error (i.e., the error observed when the network is trained using the full dataset of 40,000 annotated images). We also compare the results to human annotation variability (‘manual annotation error’). In this particular dataset, we observed an asymptotic MAE of 10.5 pixels and a human variability MAE of 9.2 pixels. Thus, the ratio between human variability remains close to 1.12. We observe that, with 800 annotations, our network achieves a similar accuracy to manual annotation error and the asymptotic prediction error. Further annotation only generates diminishing returns.

3) Speed. There is no report of the speed of the network, thus the authors should benchmark the performance on video analysis speed (only network training time is reported).

We agree with the reviewer that this may be an important piece of information for the end-user. Using the desktop configuration described in our original submission, we found that our network can run at 110 Frames-Per-Second (FPS) using the 8-stack variant of the Stacked Hourglass network, and can run at 420FPS when using the smaller 2-stack variant. Thanks to our effective initialization step, we observed that calibration takes only 3-4 s. We also measured the time required for error checking and error correction steps. Error checking can be done at 100 FPS and error correction can be done in 10 FPS. Since error correction is scarcely performed in response to a large reprojection error, it does not create a bottleneck in the overall speed of the pipeline.

We now include this information in the revised manuscript as follows: “Using this desktop configuration, our network can run at 100 Frames-Per-Second (FPS) using the 8-stack variant of the Hourglass network, and can run at 420 FPS using the smaller 2-stack version. Thanks to an effective initialization step, calibration takes 3-4 s. Error checking and error correction can be performed at 100 FPS and 10 FPS, respectively. Error correction is only performed in response to large reprojection errors, and does not create a bottleneck in the overall speed of the pipeline.”

4) Since the authors needed a lot of labeled data, making this a most useful as a specialized network for tethered flies, i.e. much like OpenPose has specific networks for hands (which they could clarify). Therefore, if they provide the network weights they should show the network is useable for other labs data.

As illustrated in our earlier response to point #2, our Stacked Hourglass network does not require 37K images to achieve an accuracy comparable with that reported in Mathis et al., 2018. We have publicly published our dataset (https://dataverse.harvard.edu/dataverse/DeepFly3D), but we do not have access to video data from a synchronized, multi-camera system and thus could not test our network on another laboratory’s data. We would also like to emphasize that DeepFly3D is specialized for tethered flies but that the computational tools behind our data analysis pipeline are general, as demonstrated by our adaptation of DeepFly3D to the human H3.6m dataset (Figure 11).

5) 3D pose in the literature. The authors do not discuss Nath et al., which is a 3D extension of DeepLabCut (bioRxiv Nov 2018, and Nature Protocols, 2019) and given it's a small field, the authors should not claim theirs is the first to do 3D pose. Beyond deep learning multiple 3D pose estimation papers exist in Neuroscience (ie. Mimica et al., 2018 – Efficient cortical coding of 3D posture in freely behaving rats).

We thank the reviewer for pointing out relevant literature. We now also cite Nath et al., 2019. However, we would like to point out that we do not claim that DeepFly3D is the first approach to generally perform 3D pose estimation. We recognize a rich human pose estimation literature and an increasing number of studies performing pose estimation on large laboratory animals (including rodents). These studies achieve 3D pose by triangulating multiple camera views using, for example a checkerboard pattern, and readily available software. To the best of our knowledge, this checkerboard-based registration approach is what is used by the OpenCV library that is referred to in Nath et al., 2019: “3D kinematics can be reconstructed from one network being trained on multiple views, and the user needs only the camera calibration files to reconstruct the data. This camera calibration and triangulation can be done in many programs, including the free package OpenCV”.

By contrast, by developing DeepFly3D, we tried to be careful to point out that we are performing 3D pose estimation on uniquely challenging fly-sized objects. To make this clear, we stated that “techniques used to translate human 2D pose to 3D pose cannot be easily transferred for the study of small animals like Drosophila” because “precisely registering multiple camera viewpoints using traditional approaches would require the fabrication of a prohibitively small checkerboard pattern”. Therefore, we still contend that DeepFly3D provides a novel contribution to 3D pose estimation because “the unique challenges associated with transforming these 2D measurements into reliable and precise 3D poses have not been addressed for small animals including the fly, Drosophila melanogaster” and “algorithms for reliably estimating 3D pose in such small animals have not yet been developed.” Nevertheless, as described below we modified the language in our revised manuscript to further clarify these points.

Therefore language like this should be modified:

"However, algorithms for reliably estimating 3D pose in such small animals have not yet been developed.…"

We agree that our definition of “small” may have been unclear, and in light of the reviewer’s comments, we have edited this sentence. The manuscript now states: “However, algorithms for reliably estimating 3D pose in such small Drosophila-sized animals have not yet been developed.”

"However, these measurements have been restricted to 2D pose in freely behaving animals"

We agree with the reviewer that it was initially unclear that this sentence refers entirely to work on insects – for which this statement is correct. We have removed a misleading citation to human pose (Mori and Malik, 2006), added a more relevant Drosophila citation (Isakov et al., 2016), and edited two sentences to now read: “Although this approach works well on humans (Moeslund et al.), in smaller, Drosophila-sized animals markers likely hamper movements,…”, and “However, these measurements have been restricted to 2D pose in freely behaving flies.

"We demonstrate more accurate unsupervised behavioral embedding using 3D joint angles rather than commonly used 2D pose data. Thus, DeepFly3D enables the automated acquisition of behavioral measurements at an unprecedented level of resolution for a variety of biological application" (also here "resolution" should be edited; it is not that DeepFly3D is more accurate than other alternatives.)

We agree with the reviewer and have edited this sentence to improve clarity. Here our reference to ‘resolution’ refers to the additional information provided by the 3rd dimension – something that has not otherwise been achieved for Drosophila. We have now edited this sentence in the Abstract to say “Thus, DeepFly3D enables the automated acquisition of Drosophila behavioral measurements at an unprecedented level of detail for a variety of biological applications.”

However, I do think these authors have a nice solution, so I do find it very fair that they state they have an alternative 3D approach, (and can highlight the advantages) solution to multi-camera estimation in body- or head- fixed animals. However, again, sparse bundle adjustment is not a new technique, so the authors should discuss this in light of the literature.

We thank the reviewer for their positive appreciation of our approach. We have now added additional citations to the Discussion of our approach. In the same lines, we now state: “During the calibration process, we also employ sparse bundle adjustment methods, as previously used for human pose estimation.”

6) "Here we present DeepFly3D, a software that infers the 3D pose of tethered, adult Drosophila-or other animals-using multiple camera images" – they don't quantify performance for other animals, so this should be dropped from the Abstract.

We have removed this reference to “-or other animals-” from the Abstract.

7) "DeepLabCut provides a user-friendly interface to DeepCut, a state-of-the-art human pose estimation network" (DeepCut should be DeeperCut, which is different than DeepCut).

We thank the reviewer for spotting this error. We changed ‘DeepCut’ to ‘DeeperCut’ in the revised manuscript.

8) "Calibration without targets" is misleading. The authors do calibrate; they just use the rigid body of the fly that has set distances per limb segment. To note, this isn't always doable for other animals/videos, again another argument to drop the random "this can be used for anything" language…

We apologize for the confusion. To clarify, our manuscript mentions “Calibration without external targets” and uses the word ‘external’ explicitly to distinguish calibrating using the fly itself versus some external device fabricated separately for this purpose (e.g., a checkerboard pattern). To fix this possible source of confusion, we have now changed the text to read “Calibration without an external calibration pattern".

Notably, our calibration procedure does not depend on a fixed limb segment length or a rigid body, but rather on the assumption that multiple cameras can see the same 3D point. However, a constant limb segment constraint is used during the automatic correction step, after the calibration. If a non-rigid body is present, we suggest discarding the segment length energy and only using the reprojection error and probability maps during the optimization. This can be done by setting the lambda_bone parameter to 0 inside the Config.py file.

Reviewer #2:

The manuscript by Gunel et al. describes a new tool for 3D pose tracking that automatically combines multiple 2D images from different viewing angles while also learning the camera model. There has been a flurry of recent papers that use deep neural networks to track pose in 2D images. This manuscript improves on these by generating 3D positions for each body part that is tracked using a combination of various algorithms that have been developed for human pose tracking. In addition, the authors provide a simple GUI that allows for part labeling and prediction correction. Overall the paper is well written and the most aspects of the system described and justified.

We thank the reviewer for their positive appreciation of our work.

My only major issue is that the experimental setup is not discussed or motivated. In particular, I would like to see more information about the choices/requirements for the number of cameras and other considerations. Why do the authors use 7 cameras? I assume that with fewer cameras you get more error/occlusions/etc. Can you investigate these effects quantitatively? How should a researcher choose the number of cameras (recording from 7 cameras in synch is difficult)?

We agree with the reviewer that this is important information for an end-user. Triangulation requires that any 3D point be seen by at least two cameras. Therefore, to see landmarks on both sides of the animal, one would need to use at least 4 cameras – 2 cameras on each side of the animal. In Author response image 2 we examine the accuracy of 3D pose estimation as a function of number of cameras used for calibration and/or prediction.

Author response image 2

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Our 3D ground truth data are based on human annotated 2D points calibrated using all 7 cameras, and triangulated again using human annotated 2D points. We observe that 3D error doubles as we reduce the number of cameras from 7 to 4. Thus, 3 cameras could be removed from the setup, but this would lead to an approximately 0.05 mm increase in mean error.

Specifically we observe that: (i) Calibration is not a significant source of error. Calibrating with N cameras (blue circles), or 7 cameras (red circles) is not very different, (ii) More cameras lead to improved triangulation. With fewer cameras, even having calibrated using all 7 cameras (red circles), results in larger error. This may be because the camera views are sufficiently different, having largely non-overlapping 2D-detection failure cases. Thus, the redundancy provided by having more cameras mitigates detection errors by finding a 3D pose that is consistent across at least two camera views. (iii) The remaining error (distance between blue and orange circles) can be explained by the 2D network detections having a larger 2D error (13.9 pixels vs. 12.4 pixels RMSE with human annotations).

On the other hand, as the reviewer points out, there are practical tradeoffs for increasing the number of cameras. Ultimately, one could potentially reduce the number of cameras from seven to two (one on each side of the animal) by training a neural network that “lifts” 2D pose into 3D from single camera images (Martinez et al., 2017).

We have now added this additional information in the revised manuscript as follows: “We assessed the optimal number of cameras for DeepFly3D and concluded that increasing the number of cameras increases accuracy by stabilizing triangulation. Specifically, we observed the following. (i) Calibration is not a significant source of error: calibrating with fewer than 7 cameras does not dramatically increase estimation error. (ii) Having more cameras improves triangulation. Reducing the number of cameras down to four, even having calibrated with 7 cameras, results in an increase of ~0.05 mm triangulation error. This may be because the camera views are sufficiently different, having largely non-overlapping 2D-detection failure cases. Thus, the redundancy provided by having more cameras mitigates detection errors by finding a 3D pose that is consistent across at least two camera views.”

In addition, I would suggest that the authors show the distribution of 3D localization errors. Is this isotropic? Is it the same for all body parts (the Discussion subsection “Learning the parameters” suggests not)?

As mentioned, the error is not isotropic. We now plot all of the errors for each joint in Author response image 3. We believe that the tarsus tips exhibit larger error than the other joints due to the increased prevalence of occlusions from the spherical treadmill, and larger positional variance. On the other hand, increasing error for the body-coxa joints can be attributed to the difficulty of human annotation and occlusions from some perspectives.

Author response image 3

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Appendix to Reviewers

1) For a single point, the error can be decomposed into the error in the x and y dimensions separately, e.g. e=ex2+ey2. Assuming the error in the x and y dimensions can be defined by the same distribution, E[ex2+ey2]=E[2ex2]=2ex^=2ey^, where ex^ and ey^ are the means of the error distribution. Scaling the image by sx and sy in the x and y dimensions, respectively, results in the errores=(sxex)2+(syey)2 and therefore E[es]=E[(sxex)2+(syey)2]=(sx)2+(sy)2ex^=(sx)2+(sy)2ey^. Then the final error for the scaled image can be approximated by E[es]=E[e](sx)2+(sy)2/2.

A much simpler argument to understand the effects of image scaling on reported error – assuming that images are scaled by one factor in both dimensions – can be summarized as follows. On the LEAP dataset, using the scale factor of the y dimension to rescale the error would result in a 15 pixel error (3 pixels * 960/192). Similarly, using the scale factor of the x dimension would result in a 7.2 pixel error (3 pixels * 480/192). Our reported error, 13.9 pixels, is between these two values. In the same manner, on the DeepLabCut dataset, using the scale factor of the x dimension to rescale the error would result in a 9.9 pixel error (4.17 pixels * 320/135). Using the scale factor of the y dimension would result in a 10.3 pixel error (4 pixels * 840/340). These results are also similar to our reported error of 13.9 pixels.

Author details

1. Semih Günel

   1. Computer Vision Laboratory, School of Computer and Communication Sciences, EPFL, Lausanne, Switzerland
   2. Neuroengineering Laboratory, Brain Mind Institute & Interfaculty Institute of Bioengineering, School of Life Sciences, EPFL, Lausanne, Switzerland

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   semih.gunel@epfl.ch

   Competing interests

   No competing interests declared

2. Helge Rhodin

   1. Computer Vision Laboratory, School of Computer and Communication Sciences, EPFL, Lausanne, Switzerland
   2. Department of Computer Science, UBC, Vancouver, Canada

   Contribution

   Conceptualization, Software, Formal analysis, Supervision, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2692-0801

3. Daniel Morales

   Neuroengineering Laboratory, Brain Mind Institute & Interfaculty Institute of Bioengineering, School of Life Sciences, EPFL, Lausanne, Switzerland

   Contribution

   Data curation, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7469-0898

4. João Campagnolo

   Neuroengineering Laboratory, Brain Mind Institute & Interfaculty Institute of Bioengineering, School of Life Sciences, EPFL, Lausanne, Switzerland

   Contribution

   Data curation, Software, Writing—review and editing

   Competing interests

   No competing interests declared

5. Pavan Ramdya

   Neuroengineering Laboratory, Brain Mind Institute & Interfaculty Institute of Bioengineering, School of Life Sciences, EPFL, Lausanne, Switzerland

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Contributed equally with

   Pascal Fua

   For correspondence

   pavan.ramdya@epfl.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5425-4610

6. Pascal Fua

   Computer Vision Laboratory, School of Computer and Communication Sciences, EPFL, Lausanne, Switzerland

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing—review and editing

   Contributed equally with

   Pavan Ramdya

   Competing interests

   No competing interests declared

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