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    X-ray Microscopic Imaging of a Chemically Dried Cicada

    Figure 1: Volumetric rendering of a chemically dried cicada imaged using the SkyScan 1275 

    The Importance of Sample Preparation

    With a large emergence of the local cicada population along the East coast predicted this year, we decided to re-visit a past specimen and once again examine a cicada with our image of the month study. In this study, we are highlighting the importance of proper sample preparation, especially regarding biological samples. 

    X-Ray Microscopy Imaging of Cicada

    To evaluate different preparations of the same sample we chose to compare an unstained biological sample with a chemically dried biological sample using the SkyScan 1275 high-speed desktop micro-CT. As can be seen from the view in Figure 2 below, obvious physical differences are visible between the two preparations of the same sample. For this study, the cicada was first imaged from a water hydrated state wrapped in wet gauze to prevent dehydration during imaging. Next, the sample was dehydrated in successive solutions of ethanol before chemically drying using hexamethyldisilazine (HMDS) prior to imaging a second time. 

    A loaf of bread

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    A dead bird on a white surface

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    Figure 2: Cicada rendering hydrated (top) and chemically dried (bottom)

    As shown in Figure 2, we observe an extensive contrast increase in the fine insect anatomy details after chemically drying the specimen. The hydrated sample lacks contrast among most internal features and appears with the same signal intensity as the hydrated gauze it is wrapped in. No internal features are particularly evident except for the high-density region within the abdomen. Conversely, the same sample after chemically drying with HMDS allows us to view the internal structures throughout the specimen. 

    Figure 3: Planar 2D views through the hydrated cicada sample

    In inspecting the hydrated sample more closely, we confirm our previous observations about missing contrast within the sample (Figure 3). Contrast with micro-CT imaging arises due to differential attenuation of X-ray energy passing through the sample. Especially for biological soft tissue samples, X-ray energy does not attenuate differently with various tissue types. This often leads to a lack of contrast within these types of samples, which requires us to intervene with some external contrast enhancement medium.  

    Figure 4: Planar 2D views through the chemically dried cicada sample

    When examining the chemically dried cicada sample, we no longer have water filling the internal volume of the sample, allowing us to instead view the fine structural details present (Figure 4).   

    Figure 5: Rendered 3D view slicing through the top of the chemically dried cicada sample

    When inspecting the chemically dried cicada sample using a top-down view, the vast variety of resolved structures within the sample become even more evident. Fine details are captured from the eyes, thorax, and through the abdomen (Figure 5). 

    Interested in learning more about all the ways micro-CT imaging can help with research related to insects? Bruker will be hosting a global webinar on Thursday, June 17, 2021 with featured keynote speaker Dr. Javier Alba-Tercedor, a leading researcher in zoology focusing on studying insects using his SkyScan micro-CT. Registration details are here: Experiences in the Use of Micro-CT in Insect Research

    Conclusion

    The SkyScan 1275 allowed us to compare the effects of soft tissue imaging both with and without pretreatment steps to improve contrast in the final images. Hopefully this article leaves you with a better understanding of why pretreatment may be required for micro-CT imaging. We hope you found this Image of the Month informative and encourage you to subscribe to our newsletter and social media channels in preparation for the continuation of our image of the month series next month. 

    Scan Specifications

    SampleCicada
    Voltage (kV)50
    Current (µA)50
    Pixel Size (µm)20
    Rotation Step0.3
    Scan Time (HH:MM:SS)00:54:10

    These scans were completed on our desktop SkyScan 1275 system at the Micro Photonics Imaging Laboratory in Allentown, PA. Reconstructions were completed using NRecon and visualization of 2D and 3D results were completed using DataViewer, CTVox, and CTAn. 

    Would you like your work to be featured in our monthly newsletter? If so, please contact us by calling Seth Hogg at 610-366-7103 or e-mailing seth.hogg@microphotonics.com

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