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Abstract: Chrysomela populi is a typical leaf beetle feeding on mainly leaves of aspen trees, and a major pest of forestry. Morphological studies of Chrysomela populi, or even of chrysomelid beetles, are surprisingly scarce. This study performed X-ray micro-computed tomography (micro-CT) on Chrysomela populi adults. Before being scanned with a Skyscan 1172 micro-CT (beam strength: 40 keV), the adult samples were dehydrated with ethanol and dried to a critical point. The scanned image sequence (stack) includes 1,554 images with a resolution of 1.04 μm, totaling a data volume of 18.7 GB. The images are clear and there are no annular pseudomonas. The tissue in the head is also clear and intact; muscles, bones, digestive tracts, and even nervous systems are clearly visible. The scanned image sequence (stack) could be used for 3D reconstruction and anatomical study of the head of Chrysomela populi adults. This dataset provides basic data for morphological and anatomical studies of beetle head.
Keywords: micro-CT; 2D image; image stack; 3D reconstruction
|Title||Micro-CT images of the head of Chrysomela populi adults|
|Data authors||Ren Jing, Ge Siqin|
|Data corresponding author||Ge Siqin (firstname.lastname@example.org)|
|Spatial resolution||1.04 µm|
|Data volume||18.7 GB|
|Data service system||<http://www.sciencedb.cn/dataSet/handle/488>|
|Source of funding||National Natural Science Foundation (Nos. 31472028, 31672347)|
|Dataset composition||This dataset consists of 1,554 micro-CT sequence images of the head of Chrysomela populi adults.|
Known as micro-computed tomography (micro-CT), microscopic CT reconstruction is a new, internationally advanced X-ray nondestructive testing technology, which can clearly display the internal microscopic structure of tested objects.1 This technology was first used in industrial, aerospace and medical fields, and gradually spread to zoology and botany in recent years. Since 2002, it has been applied and promoted in the research of insect morphology.2 Microscopic CT can be used to scan the muscle and endoskeleton of insects' thorax, very suitable for the observation of insects' muscle and other internal structure.3–4
In recent years, microscopic CT has become an efficient method to obtain morphological data in insect morphological studies. With the improvement of hardware and software of modern high-resolution desktop scanner, applied researches tend to focus on the anatomy of microstructures. At present, micro-CT produces images with a maximum resolution of about 0.5 µm, while images produced by top scanners can reach a resolution of 0.1 µm or an even more sophisticated level.5
In addition to being nondestructive to testing samples, micro-CT has a biggest advantage – it greatly accelerates the acquisition of anatomical data with no artifacts. This method is far more efficient than tissue slicing techniques, and the images generated therefrom are ordered and intact. After micro-CT scanning, samples can still be used in Scanning Electron Microscope (SEM) observation or tissue slicing. Unlike tissue slicing, micro-CT almost causes no loss of structure in the scanning process, allowing for very detailed morphological records by using a minimum sample size. Since the experimental procedure is nondestructive to specimens, micro-CT can also be used in research on rare insect samples, model specimens, and even fossil insects buried in amber.6–7 Under the excitation of low-energy electron beam, different types of organizations (such as bones, muscles, nervous system) show different specific absorption rates, which enables a high density resolution to better distinguish the structures.8
Chrysomela populi Linnaeus (Chrysomelinae, Chrysomelidae, Coleoptera) is a typical leaf beetle with chewing mouthparts, mainly feeding on poplar leaves, and an important economic pest of forestry. At present, there is very little research on the morphology of leaf beetles, especially of Chrysomela populi. Taking a typical leaf beetle – Chrysomela populi adult – as the research object, this study uses micro-CT to get sequence images of the adult heads, through which to explore the internal and external structural changes in hope of providing statistical sources for morphological and anatomical studies of beetle heads.
Samples of Chrysomela populi adults were collected in Yanqing, Beijing, on May 12, 2012. The samples were stored at the Institute of Zoology, Chinese Academy of Sciences.
2.1 Sample pretreatment
Micro-CT sample preparation follows a simple two-step process. Specimens were first dehydrated with ethanol (alcohol-acetone gradient dehydration) and then dried to a critical point to avoid contractile artifacts before scanning. This method increases the contrast between the tissue and its surrounding medium (air).
Specifically, we prepared the sample used for micro-CT scanning according to the following steps:
Sample selection -> cleaning -> alcohol-acetone gradient dehydration -> critical point drying.
Alcohol-acetone gradient dehydration: alcohol dehydrogenation was performed 15 to 20 minutes apart, and acetone dehydration 30 minutes apart.
75% alcohol (once), 80% alcohol (once), 85% alcohol (once), 90% alcohol (once), 95% alcohol (once), 100% alcohol (three times), acetone (three times).
Finally, a critical point drier (Hitachi hcp-2) was used to dry the samples.
2.2 Resolution calculation
The samples were embedded in a particular support frame or fixed to a small container (such as microcentrifuge tube or pipette). In order to obtain a maximum spatial resolution, the vertical axis of the samples should fit onto the rotation axis of the sample platform. Sample data were collected at Beijing Institute of Technology, on May 23, 2012. Dried samples were placed on the sample stage of the micro-CT imaging system (Skyscan 1172), where scanning parameters were set. The samples were scanned by using 40 kev and 4× camera lens, and the pictures were saved. The scanning and reconstruction parameters are shown in Table 1.
By adjusting the scanner lens, (e.g., magnification, distance to sample) or the distance between the ray source and the sample, we could get different image visions used to scan samples of different sizes. Different camera binning sizes generated vastly different image resolutions. In order to obtain high-resolution images, camera binning of 1 × 1 was used, which prolonged camera exposure time and scanning time. For quick acquisition of relatively rough scanned images, camera binning of 2 or more (including 2 × 2, 3 × 3, 4 × 4) was used. The greater the camera binning size, the faster the scanning speed, and the lower-quality the scanned image. In general, when the camera binning was at 1, the scanning took more than four hours. Different camera lens, sample locations and exposure times also affected the scanning time. The larger the camera binning size, the shorter the scanning time (usually within two hours or even a few minutes). To meet different research needs, the scanning time could be varied by adjusting relevant scanning parameters, and the image would be of different qualities accordingly.
|Scanning parameters||Reconstruction parameters|
|Scanner = Skyscan1172||Reconstruction Program = NRecon|
|Source Type = Hamamatsu 100/250||Program Version = Version: 18.104.22.168|
|Camera = Hamamatsu C9300 11Mp||Reconstruction Engine = NReconServer|
|Source Voltage (KeV) = 40||Engine Version = Version: 1.6.3|
|Source Current (µA) = 250||Dataset Origin = Skyscan1172|
|Camera Binning = 1x1||Result File Type = BMP|
|Image Pixel Size (µm) = 1.04|
|Object to Source (mm) =41.120|
|Camera to Source (mm) =340.168|
|Vertical Object Position (mm) =20.403|
|Exposure (ms) = 2010|
|Rotation Step (deg) = 0.600|
Figure 1 shows the crosscutting images of the head of Chrysomela populi adults at different layers. It shows clearly the internal microscopic structure of the insect, including muscle, nerve and endoskeleton. As micro-CT could help obtain data of the samples' internal structures without damaging specimens, the samples were not dissected for obtaining more detailed and complete morphological data. In this study, though our target was the head of Chrysomela populi adults, the head was not dissected from the beetle body during processing. Rather, the beetle body was dehydrated and dried as a whole, and was then put onto the sample stage. Only the scanner camera was adjusted to focus on the head part. As a result, the integrity of the scanned samples was retained, and they could still be used for ordinary morphological observations or Scanning Electron Microscope observations. In addition, compared with dissecting specific parts of the body, this method allows more anatomical and morphological data to be recorded, especially in the conjuncture between the head and the thorax of the beetle body.
Scanning could be performed once the parameters were set. In order to fully capture the required data, we typically expanded the scanning range slightly beyond the required part. As this dataset targeted at the head of Chrysomela populi adults, we actually scanned the head and the prothorax. After the sequence images were reconstructed, we selected the required images and deleted the redundant.
After the scanning was completed, the scanned images were reconstructed to obtain the tomography image sequence. The image sequences were named in two parts: one part is for autonomous naming based on sample name, scanning time, scanning parameters or other information, where an underline "_" was used to delimit different information types; the other part is for the system's automatic naming, which is a serial number automatically assigned to each sequence image by the system. For example, in the sequence image "ch_a_recxxxx", "ch_a_" is the autonomous naming part, where "ch" is short for the sample's Latin name Chrysomela populi, "A" means adult, and "_" represents the delimiter. When reconstructing the sequence, the system automatically assigns a serial number "recxxxx", where "rec" means "reconstruction", "XXXX" represents the sequence number starting from "0000". This dataset begins with "ch_a_rec1100" and ends with "ch_a_rec2653", with a total of 1554 sequence images. Images "ch_a_rec0000" to "ch_a_rec1099" are redundant data of Chrysomela populi prothorax. Without diminishing the integrity of the head data, redundant data were deleted, which also reduced the data storage space.
This dataset contains 1554 2D micro-CT images of the head of Chrysomela populi adults, totaling a data volume of 18.3 GB. Image resolution is 1.04 µm, and each image takes up a space of 12.3 MB. Crosscutting images of different layers (Figure 1) show great clearity, no ring artifacts, and clear internal structures of the samples, like muscle, bone, gut, and nervous system. Whether in terms of sample preparation or scanning parameters, the image sequence fully meets the demand of related research, such as 3D reconstruction of the internal and external morphological structures of the head of Chrysomela populi adults. The dataset also provides data basis for morphological and anatomical studies of beetle heads.
Based on the tomography image sequence (such as micro CT), the 3D computer reconstruction technology reconstructs and visualizes a structure of interest by means of automatic or semi-automatic segmentation and reconstruction algorithms. Through image sequence processing, noise elimination, contour extraction, and feature point matching, we can render the display of the 3D digital model of insects, enabling a multi-scale, multi-perspective and multi-level display of their morphological structure, as well as an intuitive and dynamic display of the relationship between different structures of the organism. The high-resolution CT image sequence obtained after accurate proofreading can help us with quick and accurate 3D-reconstruction and anatomic studies of the samples,5 which also has broad application prospects in 3D printing.
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1. Ren J & Ge S. Micro-CT images of the head of Chrysomela populi adults. Science Data Bank. DOI: 10.11922/sciencedb.488