DOI: 10.5061/dryad.x0k6djhnf
title: DeepProjection: Specific and robust projection of curved 2D tissue sheets from 3D microscopy using deep learning

description: The efficient extraction of image data from curved tissue sheets embedded
 in volumetric imaging data remains a serious and unsolved problem in
 quantitative studies of embryogenesis. Here we present DeepProjection
 (DP), a trainable projection algorithm based on deep learning. This
 algorithm is trained on user-generated training data to locally classify
 the 3D stack content and rapidly and robustly predict binary masks
 containing the target content, e.g., tissue boundaries, while masking
 highly fluorescent out-of-plane artifacts. A projection of the masked 3D
 stack then yields background-free 2D images with undistorted fluorescence
 intensity values. The binary masks can further be applied to other
 fluorescent channels or to extract the local tissue curvature. DP is
 designed as a first processing step than can be followed, for example, by
 segmentation to track cell fate. We apply DP to follow the dynamic
 movements of 2D-tissue sheets during dorsal closure in Drosophila embryos
 and of the periderm layer in the elongating Danio embryo. DeepProjection
 is available as fully documented Python package.
description: Preparation and imaging of
 <em>Drosophila</em> embryos Cell junctions were labeled with either
 DE-cadherin-GFP or DE-cadherin-mTomato (labeling
 <em>Drosophila</em> E-cadherin, which is concentrated in
 cell-cell junctions), both knock-in lines under control of the endogenous
 promoter (Huang et al.,
 2009). F-actin was labeled
 with the GFP-moesin actin binding domain, expressed under the control of
 the <em>spaghetti squash </em>promoter<em> </em>in
 the sGMCA line (Kiehart et
 al., 2000). All stocks
 were maintained at room temperature or 25°C on standard cornmeal/molasses
 fly food or in embryo collection cages with a grape juice agar plate and
 yeast paste. Embryos were collected either 2–4 hours after egg lays and
 aged overnight at 16°C, or from overnight egg lays at 25°C. To remove the
 chorion, embryos were incubated in a 50% bleach solution for 1.25 min and
 then rinsed extensively with deionized water. Pre-dorsal closure stage
 embryos were selected using a reflected-light dissecting microscope.
 Embryos were prepared for imaging as described previously
 (Kiehart et al.,
 1994). Images were
 acquired using Micro-Manager 2.0 software (Open Imaging, San Francisco,
 CA) to control a Zeiss Axiovert 200M microscope (Carl Zeiss, Thornwood,
 NY) equipped with a Yokogawa CSU-W1 spinning disk confocal head (Solamere
 Technology Group, Salt Lake City, UT), a Hamamatsu Orca Fusion BT camera
 (Hamamatsu, Japan), and a Zeiss 40X LD LCI Plan-Apochromat 1.2NA
 multi-immersion objective (glycerin). Due to the curvature of the embryo,
 we imaged multiple z planes for each embryo at each time point to view the
 dorsal opening. We recorded 8 z-slice stacks with 1 µm step size for
 single color, and 14 z-slice stacks with 0.5 µm step size for dual-color
 movies. Stacks were acquired every 15 s throughout the duration of closure
 with a 100 ms exposure per slice for GFP and a 150 ms exposure per slice
 for mTomato. Zebrafish husbandry and sample preparation for
 live imaging Zebrafish of the Ekkwill strain were maintained
 between 26 and 28.5 °C with a 14:10 hour light:dark cycle. Fish between
 3-6 months were used for experiments. Transgenic
 <em>krt4:lyn-EGFP</em> fish were described previously
 (Lee et al.,
 2014). Male and female
 fish were set up for mating in tanks with dividers. The dividers were
 removed in the morning for timed mating. Embryos were collected in E3
 medium and screened at 1 dpf for expression of GFP in the periderm
 (krt4-lynGFP). The positive embryos were transferred to a dish with E3
 medium and tricaine (Sigma
 E10521-50G)
 at 0.01% concentration. The embryos
 were dechorionated with forceps and mounted in fluorinated ethylene
 propylene (FEP) tubes according to published protocols
 (Weber et al.,
 2014). The FEP tubes were
 coated with 3% methlycellulose and embryos were mounted in 0.1% agarose
 with 0.01% tricaine to immobilize them during imaging. The tube was then
 placed in a 60 mm culture dish with an agarose bed, held in place with 1%
 agarose and immersed in E3 medium. Images were acquired with LASX software
 on a Leica SP8 confocal microscope using an HC Fluotar L 25X/0.95NA W
 VISIR water-immersion objective at 0.75 or 1X zoom. Image stacks with
 40-60 slices were acquired every 15 minutes with a z-step size of 2 µm.
 Work with zebrafish was approved by the Institutional Animal Care and Use
 Committee at Duke University.
description: The DeepProjection algorithm is available on GitHub (https://github.com/danihae/DeepProjection) and PyPI (https://pypi.org/project/deepprojection/), with documentation and a Jupyter notebook with detailed instructions.