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3D visualization of macromolecule synthesis.

ABSTRACT: Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

SUBMITTER: Duerr TJ

PROVIDER: S-EPMC7669265 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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3D visualization of macromolecule synthesis.

Duerr Timothy J TJ Comellas Ester E Jeon Eun Kyung EK Farkas Johanna E JE Joetzjer Marylou M Garnier Julien J Shefelbine Sandra J SJ Monaghan James R JR

eLife 20201014

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based ...[more]

PMID: 33051003

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Project description:Photoacoustic (PA) imaging (or optoacoustic imaging) is a novel biomedical imaging method in biological and medical research. This modality performs morphological, functional, and molecular imaging with and without labels in both microscopic and deep tissue imaging domains. A variety of innovations have enhanced 3D PA imaging performance and thus has opened new opportunities in preclinical and clinical imaging. However, the 3D visualization tools for PA images remains a challenge. There are several commercially available software packages to visualize the generated 3D PA images. They are generally expensive, and their features are not optimized for 3D visualization of PA images. Here, we demonstrate a specialized 3D visualization software package, namely 3D Photoacoustic Visualization Studio (3D PHOVIS), specifically targeting photoacoustic data, image, and visualization processes. To support the research environment for visualization and fast processing, we incorporated 3D PHOVIS onto the MATLAB with graphical user interface and developed multi-core graphics processing unit modules for fast processing. The 3D PHOVIS includes following modules: (1) a mosaic volume generator, (2) a scan converter for optical scanning photoacoustic microscopy, (3) a skin profile estimator and depth encoder, (4) a multiplanar viewer with a navigation map, and (5) a volume renderer with a movie maker. This paper discusses the algorithms present in the software package and demonstrates their functions. In addition, the applicability of this software to ultrasound imaging and optical coherence tomography is also investigated. User manuals and application files for 3D PHOVIS are available for free on the website (www.boa-lab.com). Core functions of 3D PHOVIS are developed as a result of a summer class at POSTECH, "High-Performance Algorithm in CPU/GPU/DSP, and Computer Architecture." We believe our 3D PHOVIS provides a unique tool to PA imaging researchers, expedites its growth, and attracts broad interests in a wide range of studies.

Project description:BackgroundResolution estimation is the main evaluation criteria for the reconstruction of macromolecular 3D structure in the field of cryoelectron microscopy (cryo-EM). At present, there are many methods to evaluate the 3D resolution for reconstructed macromolecular structures from Single Particle Analysis (SPA) in cryo-EM and subtomogram averaging (SA) in electron cryotomography (cryo-ET). As global methods, they measure the resolution of the structure as a whole, but they are inaccurate in detecting subtle local changes of reconstruction. In order to detect the subtle changes of reconstruction of SPA and SA, a few local resolution methods are proposed. The mainstream local resolution evaluation methods are based on local Fourier shell correlation (FSC), which is computationally intensive. However, the existing resolution evaluation methods are based on multi-threading implementation on a single computer with very poor scalability.ResultsThis paper proposes a new fine-grained 3D array partition method by key-value format in Spark. Our method first converts 3D images to key-value data (K-V). Then the K-V data is used for 3D array partitioning and data exchange in parallel. So Spark-based distributed parallel computing framework can solve the above scalability problem. In this distributed computing framework, all 3D local FSC tasks are simultaneously calculated across multiple nodes in a computer cluster. Through the calculation of experimental data, 3D local resolution evaluation algorithm based on Spark fine-grained 3D array partition has a magnitude change in computing speed compared with the mainstream FSC algorithm under the condition that the accuracy remains unchanged, and has better fault tolerance and scalability.ConclusionsIn this paper, we proposed a K-V format based fine-grained 3D array partition method in Spark to parallel calculating 3D FSC for getting a 3D local resolution density map. 3D local resolution density map evaluates the three-dimensional density maps reconstructed from single particle analysis and subtomogram averaging. Our proposed method can significantly increase the speed of the 3D local resolution evaluation, which is important for the efficient detection of subtle variations among reconstructed macromolecular structures.

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3D visualization of macromolecule synthesis.

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3D visualization of macromolecule synthesis.

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