A simple nuclear contrast staining method for microCT‐based 3D histology using lead(II) acetate

Metscher, Brian

doi:10.1111/joa.13351

Cited by 16 publications

(16 citation statements)

References 21 publications

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“…It is the aim of this work to adapt and extend the existing contrasting methods (16)(17)(18)(19)21,23) for micro-CT based investigation of ophthalmological samples. The preparation of the eye requires some adjustments for a proper penetration of contrast agents and other solutions due to its voluminous shape and solid outer delineation to obtain successful visualization of the complex and fine anatomy inside.…”

Section: Discussionmentioning

confidence: 99%

“…Micro-CT is an excellent method for detailed anatomical investigations of ocular soft tissues considering their 3D-topography based on simple contrasting procedures. Detailed scanning provides high resolution imaging which comes close to light microscopy ( 20 , 21 , 35 ) and facilitates 3D analysis without destructive procedures.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the content of liquid media within the tissues such as water or organic solvents for conservation and storage are responsible for partial X-ray attenuation and contributes to the low contrast during image acquisition. Within the last years, a number of contrasting methods have been established and evaluated for micro-CT which ensure contrast-rich imaging of soft-tissue samples while maintaining tissue integrity [e.g., (16,(18)(19)(20)(21); see also references herein and reviews on contrasting methods: (17,(22)(23)(24)].…”

Section: Introductionmentioning

confidence: 99%

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Micro-CT in ophthalmology: ex vivo preparation and contrasting methods for detailed 3D-visualization of eye anatomy with special emphasis on critical point drying

Runge¹,

Stahnke²,

Guthoff³

et al. 2022

Quant Imaging Med Surg

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Background: Micro-computed tomography (micro-CT) provides detailed 3-dimensional (3D) visualization of anatomical structures and encourages morphological reinvestigation of organs with delicate features. The low radiodensity of soft tissues necessitates preceding sample preparation to conduct X-ray imaging with decent contrast between different tissues. In this study, we demonstrate the preparation with three radiopaque agents in combination with elimination of liquids by critical point drying (CPD) introduced for ocular samples.Methods: Enucleated porcine eyes were prepared with ethanolic iodine (EI), aqueous iodine-potassium iodide, or ethanolic phosphotungstic acid (EPTA). Micro-CT scans of the samples were conducted in a moist environment with an isotropic resolution of 9.2-12.5 µm voxel size. Subsequently, samples were chemically dehydrated and critical point (CP) dried to conduct a second scan in a dry environment with a resolution up to 4.7-5.4 µm in voxel size. The visualization effects were qualitatively and semi-quantitatively evaluated with regard to the generated contrast between different ocular tissues.Results: All three contrast agents accumulated well in most of the investigated ocular tissues and lead to an increased X-ray attenuation which allowed for differentiated visualization of ocular structures. Problematic agent penetration into the lens was obvious for iodine-potassium iodide and EPTA. Artificial damages of the lens and thickness reduction for the cornea and sclera due to CPD were noticed. The effects of the different contrasting treatments are described and compared with regard to the effects of CPD. Exclusively CP dried samples that were not treated with contrast agents could also be visualized excellently with a good distinction of different ocular structures from each other.Conclusions: All ocular structures can be visualized by micro-CT. To contrast moist samples, the best results were achieved with iodine potassium iodide (IPI). CPD improved the scan quality in all cases. Even without pretreatment with contrasting agents, the CP dried samples showed a contrast similar to the IPI treated samples.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Micro-CT in ophthalmology: ex vivo preparation and contrasting methods for detailed 3D-visualization of eye anatomy with special emphasis on critical point drying

Runge¹,

Stahnke²,

Guthoff³

et al. 2022

Quant Imaging Med Surg

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“…Other approaches involve using reagents known from classical histological stainings. Eosin can be used to stain cytoplasm 9 , while hematein (led-III-acetate) functions as a nucleus specific staining agent 10 , 11 . While these approaches seem to take the specificity of microCT based virtual histology to a new level, they are hindered by limited sample sizes as well as by the fact that only a few applicable protocols exist to this day.…”

Section: Introductionmentioning

confidence: 99%

Elastic transformation of histological slices allows precise co-registration with microCT data sets for a refined virtual histology approach

Albers

Svetlove

Alves

et al. 2021

Sci Rep

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Although X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.

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“…In contrast, for the 3D analysis of bones microCT can already be considered the method of choice. To circumvent the problem of low contrast in soft-tissue different heavy metal ion-based staining protocols have been reported similar to the ones used for electron microscopy [88][89][90][91] . Another option to drastically increase the contrast is the use of different microCT imaging techniques, which do not rely solely on x-ray attenuation as the method for contrast generation.…”

Section: X-ray Based Virtual Histology As a Tool For The 3d Analysis ...mentioning

confidence: 99%

Preclinical evaluation of nanoparticle enhanced breast cancer diagnosis and radiation therapy

Jonas¹

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Triple negative breast cancer (TNBC) is an aggressive type of cancer which makes up 15-20% of all newly diagnosed cases, lacking the main target molecules for tumor specific treatment.Surgery or systemic therapy by chemotherapy are frequently used in the clinic and combined with radiation therapy to improve locoregional control in breast cancer patients after surgery.With a poor prognosis, there is a clear need to explore new treatment options for TNBC. The aim of the here presented PhD project was to evaluate the feasibility to enhance the biological effect of radiation therapy and increase tumor contrast for diagnosis by applying an in vivo microCT imaging system in combination with barium nanoparticles (BaNPs) in a pH8N8 WAP-T-NP8 mouse model for TNBC. Characterization of the BaNPs revealed strong x-ray attenuation and no toxic effects in different cancer and normal cell lines. Furthermore, irradiation of cancer cells using low energy x-rays in the keV range by a microCT resulted in a significant reduction on colony formation capability. In vitro, this low energy irradiation effect on clonogenic tumor cell survival was enhanced in the presence of BaNPs. Next, a subcutaneous lung cancer mouse model in immunodeficient mice and an orthotopic syngeneic mouse model for breast cancer was applied for further in vivo evaluation. Once the treatment plan was optimized regarding the applied x-ray doses and the frequency of irradiation, low energy radiation therapy within a classical in vivo microCT significantly reduced tumor growth or even resulted in shrinkage of the tumors without visible side effects and weight loss in comparison to untreated controls. However, the intratumoral application of BaNPs was not able to increase the irradiation effect on tumor growth kinetics. This might be in part due to inhomogeneous distribution of BaNPs within the tumor observed by microCT imaging. K-edge subtraction imaging as well as x-ray fluorescence of explanted tumor samples confirmed these findings. To localize the BaNPs in 3D to specific sites within the tumor environment and to detect morphological alterations within the tumor due to irradiation in proximity to BaNPs an ex-vivo imaging based analytic platform was established, utilizing co-registration of microCT and histology data. This imaging approach co-localized BaNPs with CD68 positive phagocytic cells and revealed a non-uniform distribution of the BaNPs within the tumor, however with no signs of locally enhanced radiation effects. Furthermore, antibody functionalized BaNPs were generated for systemic application. Analysis of biodistribution revealed that EpCAM labeled BaNPs did not reach the tumor after intra-venous administration, but accumulated in liver and spleen, demonstrated by a strong CT contrast within these organs.

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A simple nuclear contrast staining method for microCT‐based 3D histology using lead(II) acetate

Cited by 16 publications

References 21 publications

Micro-CT in ophthalmology: ex vivo preparation and contrasting methods for detailed 3D-visualization of eye anatomy with special emphasis on critical point drying

Micro-CT in ophthalmology: ex vivo preparation and contrasting methods for detailed 3D-visualization of eye anatomy with special emphasis on critical point drying

Elastic transformation of histological slices allows precise co-registration with microCT data sets for a refined virtual histology approach

Preclinical evaluation of nanoparticle enhanced breast cancer diagnosis and radiation therapy

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