Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography

Utilizing micro-X-ray CT (μCT) imaging, we sought to generate an atlas of in vivo and intact/ex vivo lungs from normal murine strains. In vivo imaging allows visualization of parenchymal density and small airways (15–28 μm/voxel). Ex vivo imaging of the intact lung via μCT allows for improved understanding of the three-dimensional lung architecture at the alveolar level with voxel dimensions of 1–2 μm. μCT requires that air spaces remain air-filled to detect alveolar architecture while in vivo structural geometry of the lungs is maintained. To achieve these requirements, a fixation and imaging methodology that permits nondestructive whole lung ex vivo μCT imaging has been implemented and tested. After in vivo imaging, lungs from supine anesthetized C57Bl/6 mice, at 15, 20, and 25 cmH2O airway pressure, were fixed in situ via vascular perfusion using a two-stage flushing system while held at 20 cmH2O airway pressure. Extracted fixed lungs were air-dried. Whole lung volume was acquired at 1, 7, 21, and >70 days after the lungs were dried and served as validation for fixation stability. No significant shrinkage was observed: +8.95% change from in vivo to fixed lung ( P = 0.12), −1.47% change from day 1 to day 7 ( P = 0.07), −2.51% change from day 1 to day 21 ( P = 0.05), and −4.90% change from day 1 to day 70 and thereafter ( P = 0.04). μCT evaluation showed well-fixed alveoli and capillary beds correlating with histological analysis. A fixation and imaging method has been established for μCT imaging of the murine lung that allows for ex vivo morphometric analysis, representative of the in vivo lung.

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Three-Dimensional In Vivo Imaging of the Murine Liver: A Micro-Computed Tomography-Based Anatomical Study

PLoS ONE ◽

10.1371/journal.pone.0031179 ◽

2012 ◽

Vol 7 (2) ◽

pp. e31179 ◽

Cited By ~ 29

Author(s):

Teresa Fiebig ◽

Hanne Boll ◽

Giovanna Figueiredo ◽

Hans Ulrich Kerl ◽

Stefanie Nittka ◽

...

Keyword(s):

Computed Tomography ◽

In Vivo Imaging ◽

Three Dimensional ◽

Anatomical Study ◽

Micro Computed Tomography ◽

Murine Liver

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Dual-energy micro-CT of the rodent lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00359.2011 ◽

2012 ◽

Vol 302 (10) ◽

pp. L1088-L1097 ◽

Cited By ~ 33

Author(s):

C. T. Badea ◽

X. Guo ◽

D. Clark ◽

S. M. Johnston ◽

C. D. Marshall ◽

...

Keyword(s):

Contrast Agent ◽

Vascular Tissue ◽

Ex Vivo ◽

Respiratory Gating ◽

Three Dimensional ◽

Dual Energy ◽

Volume Estimation ◽

Micro Ct ◽

Micro Computed Tomography

The purpose of this work is to investigate the use of dual-energy micro-computed tomography (CT) for the estimation of vascular, tissue, and air fractions in rodent lungs using a postreconstruction three material decomposition method. Using simulations, we have estimated the accuracy limits of the decomposition for realistic micro-CT noise levels. Next, we performed experiments involving ex vivo lung imaging in which intact rat lungs were carefully removed from the thorax, injected with an iodine-based contrast agent, and then inflated with different volumes of air ( n = 2). Finally, we performed in vivo imaging studies in C57BL/6 mice ( n = 5) using fast prospective respiratory gating in end inspiration and end expiration for three different levels of positive end expiratory pressure (PEEP). Before imaging, mice were injected with a liposomal blood pool contrast agent. The three-dimensional air, tissue, and blood fraction maps were computed and analyzed. The results indicate that separation and volume estimation of the three material components of the lungs are possible. The mean accuracy values for air, blood, and tissue were 93, 93, and 90%, respectively. The absolute accuracy in determining all fraction materials was 91.6%. The coefficient of variation was small (2.5%) indicating good repeatability. The minimum difference that we could detect in material fractions was 15%. As expected, an increase in PEEP levels for the living mouse resulted in statistically significant increases in air fractions at end expiration but no significant changes at end inspiration. Our method has applicability in preclinical pulmonary studies where changes in lung structure and gas volume as a result of lung injury, environmental exposures, or drug bioactivity would have important physiological implications.

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High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

Nature Communications ◽

10.1038/s41467-020-19851-1 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Jiang Lan Fan ◽

Jose A. Rivera ◽

Wei Sun ◽

John Peterson ◽

Henry Haeberle ◽

...

Keyword(s):

Blood Flow ◽

High Speed ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Microscopy ◽

Fluorescence Signal ◽

Imaging Method ◽

Spatiotemporal Resolution ◽

Two Photon

AbstractUnderstanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

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In-Vivo Degradation Behavior and Osseointegration of 3D Powder-Printed Calcium Magnesium Phosphate Cement Scaffolds

Materials ◽

10.3390/ma14040946 ◽

2021 ◽

Vol 14 (4) ◽

pp. 946

Author(s):

Katharina Kowalewicz ◽

Elke Vorndran ◽

Franziska Feichtner ◽

Anja-Christina Waselau ◽

Manuel Brueckner ◽

...

Keyword(s):

Three Dimensional ◽

Bone Substitutes ◽

Magnesium Phosphate ◽

Degradation Behavior ◽

Micro Computed Tomography ◽

Calcium Magnesium ◽

Interest In Research ◽

In Vivo Degradation ◽

Volume Decrease

Calcium magnesium phosphate cements (CMPCs) are promising bone substitutes and experience great interest in research. Therefore, in-vivo degradation behavior, osseointegration and biocompatibility of three-dimensional (3D) powder-printed CMPC scaffolds were investigated in the present study. The materials Mg225 (Ca0.75Mg2.25(PO4)2) and Mg225d (Mg225 treated with diammonium hydrogen phosphate (DAHP)) were implanted as cylindrical scaffolds (h = 5 mm, Ø = 3.8 mm) in both lateral femoral condyles in rabbits and compared with tricalcium phosphate (TCP). Treatment with DAHP results in the precipitation of struvite, thus reducing pore size and overall porosity and increasing pressure stability. Over 6 weeks, the scaffolds were evaluated clinically, radiologically, with Micro-Computed Tomography (µCT) and histological examinations. All scaffolds showed excellent biocompatibility. X-ray and in-vivo µCT examinations showed a volume decrease and increasing osseointegration over time. Structure loss and volume decrease were most evident in Mg225. Histologically, all scaffolds degraded centripetally and were completely traversed by new bone, in which the remaining scaffold material was embedded. While after 6 weeks, Mg225d and TCP were still visible as a network, only individual particles of Mg225 were present. Based on these results, Mg225 and Mg225d appear to be promising bone substitutes for various loading situations that should be investigated further.

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Improved in vivo imaging method for individual islets across the mouse pancreas reveals a heterogeneous insulin secretion response to glucose

Scientific Reports ◽

10.1038/s41598-020-79727-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Henriette Frikke-Schmidt ◽

Peter Arvan ◽

Randy J. Seeley ◽

Corentin Cras-Méneur

Keyword(s):

Insulin Secretion ◽

In Vivo Imaging ◽

Motion Blur ◽

Isolated Islets ◽

Imaging Method ◽

Glucose Stimulation ◽

Powerful Approach ◽

Glucose Challenge ◽

The Impact

AbstractWhile numerous techniques can be used to measure and analyze insulin secretion in isolated islets in culture, assessments of insulin secretion in vivo are typically indirect and only semiquantitative. The CpepSfGFP reporter mouse line allows the in vivo imaging of insulin secretion from individual islets after a glucose stimulation, in live, anesthetized mice. Imaging the whole pancreas at high resolution in live mice to track the response of each individual islet over time includes numerous technical challenges and previous reports were only limited in scope and non-quantitative. Elaborating on this previous model—through the development of an improved methodology addressing anesthesia, temperature control and motion blur—we were able to track and quantify longitudinally insulin content throughout a glucose challenge in up to two hundred individual islets simultaneously. Through this approach we demonstrate quantitatively for the first time that while isolated islets respond homogeneously to glucose in culture, their profiles differ significantly in vivo. Independent of size or location, some islets respond sharply to a glucose stimulation while others barely secrete at all. This platform therefore provides a powerful approach to study the impact of disease, diet, surgery or pharmacological treatments on insulin secretion in the intact pancreas in vivo.

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GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca 2+ Dynamics in High-Ca 2+ Organelles

Cell Chemical Biology ◽

10.1016/j.chembiol.2016.05.010 ◽

2016 ◽

Vol 23 (6) ◽

pp. 738-745 ◽

Cited By ~ 22

Author(s):

Paloma Navas-Navarro ◽

Jonathan Rojo-Ruiz ◽

Macarena Rodriguez-Prados ◽

María Dolores Ganfornina ◽

Loren L. Looger ◽

...

Keyword(s):

In Vivo Imaging ◽

Ex Vivo ◽

Protein Sensor

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Heidelberg-mCT-Analyzer: a novel method for standardized microcomputed-tomography-guided evaluation of scaffold properties in bone and tissue research

Royal Society Open Science ◽

10.1098/rsos.150496 ◽

2015 ◽

Vol 2 (11) ◽

pp. 150496 ◽

Cited By ~ 10

Author(s):

Fabian Westhauser ◽

Christian Weis ◽

Melanie Hoellig ◽

Tyler Swing ◽

Gerhard Schmidmaier ◽

...

Keyword(s):

Tissue Engineering ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Three Dimensional ◽

Characteristic Parameter ◽

Micro Computed Tomography ◽

Mineral Density ◽

Independent Analysis ◽

Scaffold Properties

Bone tissue engineering and bone scaffold development represent two challenging fields in orthopaedic research. Micro-computed tomography (mCT) allows non-invasive measurement of these scaffolds’ properties in vivo . However, the lack of standardized mCT analysis protocols and, therefore, the protocols’ user-dependency make interpretation of the reported results difficult. To overcome these issues in scaffold research, we introduce the Heidelberg-mCT-Analyzer. For evaluation of our technique, we built 10 bone-inducing scaffolds, which underwent mCT acquisition before ectopic implantation (T0) in mice, and at explantation eight weeks thereafter (T1). The scaffolds’ three-dimensional reconstructions were automatically segmented using fuzzy clustering with fully automatic level-setting. The scaffold itself and its pores were then evaluated for T0 and T1. Analysing the scaffolds’ characteristic parameter set with our quantification method showed bone formation over time. We were able to demonstrate that our algorithm obtained the same results for basic scaffold parameters (e.g. scaffold volume, pore number and pore volume) as other established analysis methods. Furthermore, our algorithm was able to analyse more complex parameters, such as pore size range, tissue mineral density and scaffold surface. Our imaging and post-processing strategy enables standardized and user-independent analysis of scaffold properties, and therefore is able to improve the quantitative evaluations of scaffold-associated bone tissue-engineering projects.

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