This study was supported by NASA Space Biology grant # NNX15AB41G and LLU Department of Basic Sciences. Sungshin Choi, Dennis Leveson and Rebecca Klotz contributed significantly to the success of our spaceflight study and we greatly appreciate their support. The authors would also like to thank the entire NASA Biospecimen Sharing Program group for their great assistance.
The authors would also like to thank the Center for Dental Research for Micro-CT service.

The study followed the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and was approved by both the Institutional Animal Care and Use Committee (IACUC) of Loma Linda University (LLU) and The National Aeronautics and Space Administration (NASA). More detailed information regarding this flight experiment can be found elsewhere9,15.
1. Flight and control conditions
NOTE: A 12th Commercial Resupply Service (CRS-12) payload was launched By SpaceX at the Kennedy Space Center (KSC) on a 35-day mission in August 2017 which included onboard 10-week-old male C57BL/6 mice (n = 20) for NASA&#8217;s ninth Rodent Research experiment (RR-9).
<ol>
	<li>Prior to returning to Earth via SpaceX&#8217;s Dragon capsule, have the mice live in NASA&#8217;s Rodent Habitats (RH) aboard ISS for 35 days at an ambient temperature of 26&#8211;28 &#176;C with a 12-hour light/dark cycle throughout the flight.</li>
	<li>Place Ground Control (GC) mice into the same housing hardware used in flight and match environmental parameters such as temperature and carbon dioxide (CO2) levels as closely as possible based on telemetry data.</li>
	<li>Feed GC mice the same NASA food bar diet as their space-based counterparts. Provide both the spaceflight and GC mice with same ad libitum access to water and food.</li>
</ol>
2. Post-flight evaluation of the mice
<ol>
	<li>Within 28 hours of splashdown on Earth, transport the mice to Loma Linda University (LLU). Once there, remove the mice from the animal enclosure hardware and assess for survival and health. 
	NOTE: Upon observation, inspecting personnel reported that all the mice had survived the 35-day space mission and were in good condition, i.e. no noticeable deficiencies/abnormalities.</li>
</ol>
3. Dissecting and preservation of mouse eyes after spaceflight
<ol>
	<li>Within 38(&#177;4) hours of splashdown (n=20/group), euthanize the mice in 100% CO2 and collect their eyes.</li>
	<li>Dissect the right eye retinas and place individually in sterile cryovials, snap-freeze in liquid nitrogen, and keep at &#8722;80 &#176;C prior to use.</li>
	<li>Fix the whole left eyes in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h and then rinse with phosphate-buffered saline (PBS) for micro-CT assays.</li>
</ol>
4. Sample preparation for micro-CT scanning
<ol>
	<li>After fixation, dehydrate the mice&#39;s eyes in ethanol. To forestall any further or abrupt shrinkage of the fixed sample, use a graded series of ethanol solutions: beginning with 50% ethanol for 1 hour and then increasing the concentrations of the ethanol solutions as follows for 1 hour each: 70, 80, 90, 96 and 100%. 
	NOTE: The mice&#39;s eyes must be handled in a hood chamber.</li>
	<li>Phosphomolybdic acid (PMA) staining 
	CAUTION: On account of PMA being corrosive, a carcinogenic, and toxic to organs, appropriate protective personal equipment is necessary, including the use of a fume hood.
	<ol>
		<li>Prepare the staining solution: 10 mg of PMA in 100 mL of absolute ethanol.</li>
		<li>Stain the mice&#39;s eyes (10 wt. % phosphomolybdic acid - PMA dissolved in absolute ethanol) for 6 days.</li>
		<li>Prior to scanning, first wash the eye samples in absolute ethanol and then place each eye in individual 2 mL plastic containers that are filled with 100% absolute ethanol. Add a cotton pad to stabilized samples during scan.</li>
	</ol>
	</li>
</ol>
5. Micro-CT scanning and analysis
NOTE: The SkyScan 1272 scanner, a desktop X-ray Micro-CT system, was used for evaluation of retinal damage in the mice&#39;s eyes
<ol>
	<li>Mount the soft-tissue sample to an appropriate sample holder. To prevent any movement during the X-ray CT measurements, ensure a tight fit of the sample on its holder (Figure 1).</li>
	<li>Upon meticulous alignment of each sample, individually scan the sample via X-rays.
	<ol>
		<li>After opening the software, center the sample in the frame. In the protocol, use no filter and set the matrix to increase the pixel at 4 &#181;m. Use micro-positioning to keep the sample center on the frame.</li>
		<li>After that, check the parameter to maximize the contrast agent. To perform the calibration, remove the sample and check that the flat-field correction is greater than 80%.</li>
		<li>After calibration, reinsert the sample into the scanning chamber. For scanning, use a rotation step of 0.400, a frame averaging of 4, a random movement of 30, and rotate the samples 180&#176;.</li>
	</ol>
	</li>
	<li>Use a positioning jig for repeated measurements. Due to phase-contrast enhancement performed as described, object details as small as 4 &#181;m can be detected from x-rays generated by a sealed micro-focus X-ray tube (tungsten anode) at 50 keV and 80 mA with an integration time of 90 minutes. 
	NOTE: The acquisition parameters laid out in this section for selection to produce overview CT scans with the highest image quality.</li>
	<li>After scanning, use software (e.g., NRecon) to reconstruct the data.
	<ol>
		<li>Adjust the histogram and use the same range (0 &#8211; 0.24) for all the samples. Reconstruct region of interest was a circle, and no scales or labels were used.</li>
		<li>To reduce artifacts during the scanning, use a beam-hardening correction of 20, a smoothing correction of 1, a ring artifact reduction of 6, and perform no alteration in the misalignment compensation. After reconstruction, it was confirmed that the sample was within the region of interest.</li>
		<li>Reposition images using a plane parallel to the optic nerve and lens of the eyes.</li>
	</ol>
	</li>
	<li>After scanning, use software (e.g., DataViewer) to visualize the reconstructed images in all three views. 
	NOTE: If necessary, with this software, the images can be re-positioned using a plane parallel to the optic nerve and lens of the eyes to perform a standardized analysis.</li>
	<li>Descriptive analysis
	<ol>
		<li>Measure the structures using a measuring tool in the software (e.g., CTAn). Use the optical nerve to delimit the region of interest for analysis. By calculation, the protocol used the middle slice to perform the measurements. This evaluation was performed by descriptive analysis (Figure 2 and Figure 3).</li>
		<li>Perform measurements of the retina, retinal pigment epithelium (RPE), choroid, and sclera layer in the sagittal (Figure 2) and axial view (Figure 3). Take three measurements of each structure in order to calculate an average.</li>
	</ol>
	</li>
</ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+Exposed+to+Combined+Chronic+Low-Dose+Irradiation+and+Modeled+Microgravity+Develop+Long-Term+Neurological+Sequelae.">Mice Exposed to Combined Chronic Low-Dose Irradiation and Modeled Microgravity Develop Long-Term Neurological Sequelae.</a> International Journal of Molecular Sciences. 20 (17), 4094 (2019).</li><li>Nelson, E. S., Mulugeta, L., Myers, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgravity-induced+fluid+shift+and+ophthalmic+changes.">Microgravity-induced fluid shift and ophthalmic changes.</a> Life. 4, 621-665 (2014).</li><li>Lee, A. G., Mader, T. H., Robert Gibson, C., Brunstetter, T. J., Tarver, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Space+flight-associated+neuro-ocular+syndrome+(SANS).">Space flight-associated neuro-ocular syndrome (SANS).</a> Eye. 32, 1164-1167 (2018).</li><li>Stenger, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evidence Report: Risk of Spaceflight Associated Neuro-ocular Syndrome (SANS). , (2017).</li><li>Garrett-Bakelman, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NASA+Twins+Study:+A+multidimensional+analysis+of+a+year-long+human+spaceflight.">The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight.</a> Science. 364, (2019).</li><li>Mader, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+disc+edema,+globe+flattening,+choroidal+folds,+and+hyperopic+shifts+observed+in+astronauts+after+long+duration+space+flight.">Optic disc edema, globe flattening, choroidal folds, and hyperopic shifts observed in astronauts after long duration space flight.</a> Ophthalmology. 118, 2058-2069 (2011).</li><li>Mao, X. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mouse+ocular+response+to+a+35-day+spaceflight+mission:+Evidence+of+blood-retinal+barrier+disruption+and+ocular+adaptations.">Characterization of mouse ocular response to a 35-day spaceflight mission: Evidence of blood-retinal barrier disruption and ocular adaptations.</a> Science Reports. 9 (1), 8215 (2019).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+developmental+biology:+a+versatile+tool+for+high-contrast+3D+imaging+at+histological+resolutions.">MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions.</a> Developmental Dynamics. , 632-640 (2009).</li><li>Silva, J. M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+non-destructive+soft-tissue+visualization+with+X-ray+staining+micro-tomography.">Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography.</a> Science Reports. 5, 14088 (2015).</li><li>Descamps, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+tissue+discrimination+with+contrast+agents+using+micro-CT+scanning.">Soft tissue discrimination with contrast agents using micro-CT scanning.</a> Belgian Journal of Zoology. , 20-40 (2014).</li><li>Wu, J., Yin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+research+of+nasolabial+muscle+structure+in+fetus+with+cleft+lip:+an+iodine+staining+technique+based+on+microcomputed+tomography.">Anatomy research of nasolabial muscle structure in fetus with cleft lip: an iodine staining technique based on microcomputed tomography.</a> Journal of Craniofacial Surgery. 25 (3), 1056-1061 (2014).</li><li>Roque-Torres, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Application of Micro-CT in Soft Tissue Specimen Imaging. In: Orhan K. (eds) Micro-computed Tomography (micro-CT) in Medicine and Engineering. , 139-170 (2020).</li><li>Overbey, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spaceflight+influences+gene+expression,+photoreceptor+integrity,+and+oxidative+stress-related+damage+in+the+murine+retina.">Spaceflight influences gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina.</a> Science Reports. 9 (1), 13304 (2019).</li><li>Elkhoury, J. E., Shankar, R., Ramakrishnan, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+and+Limitations+of+X-Ray+Micro-CT+with+Applications+to+Sandstones+and+Limestones.">Resolution and Limitations of X-Ray Micro-CT with Applications to Sandstones and Limestones.</a> Transport in Porous Media. 129, 413-425 (2019).</li><li>Sombke, A., Lipke, E., Michalik, P., Uhl, G., Harzsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+and+limitations+of+X-Ray+micro-computed+tomography+in+arthropod+neuroanatomy:+A+methodological+and+comparative+survey.">Potential and limitations of X-Ray micro-computed tomography in arthropod neuroanatomy: A methodological and comparative survey.</a> Journal of Comparative Neurology. 523 (8), 1281-1295 (2015).</li><li>Huang, A. S., Stenger, M. B., Macias, B. 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All authors have nothing to disclose.

The results of this study showed that there were structural changes in the spaceflight mouse eye using the micro-CT technique when compared to GC groups, particularly of the retina, the RPE, and the choroid layers of the eye, as evidenced by their decreased thickness. Micro-CT provides an efficient and non-destructive technique to characterize the changes with no need for manipulation. The use of PMA staining enhanced the quality of the micro-CT images to successfully obtain clear 3D tomographic images after reconstruction, foregoing any need to physically alter the structure of the specimen. An added benefit of these images is that they display the entire region of interest digitally, thereby increasing accessibility as well as reproducibility of the findings. Through the micro-CT images produced during this study, the targeted specimen showed differentiation of the multiple structures like the retina, RPE, choroid, and sclera layer for determination of the thickness of each layer.
A critical step within the protocol is the manipulation of the samples due to their size and texture. The handling of the specimen must be carefully done without putting pressure on the specimen during the preparation. The micro-CT has some limitations: resolution and the lack of standardized values for the parameters. During the scanning, the different micro-CT scanners may have diverse image processing algorithms; yet calibration for a grayscale might be pursued to overcome any problem. After scanning, reconstruction of the images should be based on the tissue and the analysis that will be performed. It can be critical since image quality depends on the tomographic system, the settings, the specimen size as well as the preparation methods16,17.
Due to its successful application in studying several types of normal and pathologic tissues, micro-CT imaging capabilities should be used in future research to compile volumetric data for other analyses. Thus, based on the present study&#8217;s purpose, it was acceptable to use bi-dimensional measurements, but segmentation of the gross 3D structure can also be beneficial to provide a precise outline of the entire specimen. Even with all the advantages of a non-destructive technique, micro-CT will not replace other methods such as immunohistochemistry, but will supplement and allow subsequent histology analyses if desired.
A prolonged spaceflight condition produces a series of structural and functional ocular changes in the astronauts during and after the space mission defined as SANS. The findings include hyperopic shifts, globe flattening, choroidal/retinal folds, and cotton wool spots19. In contrast to astronauts&#8217; optical coherence tomography (OCT) finding of retinal nerve fiber layer thickening, thinning of retina and choroidal layer was documented in this animal micro-CT study. These results were unexpected. This discrepancy may be due to confounding factors. Mice have limited cephalad fluid shift compared to human. This lack of fluid shift may have evoked different responses to gravitational changes. Secondly, mice were dissected within 38 hours after splashdown, and an acute response for re-adaptation may also contribute to morphological changes in the retina and choroid. Confirmation of this possibility requires further measurements during spaceflight and long term after the mission.
The results of this study indicate that space flight conditions, especially gravitational changes, may induce an acute and short-term response in the eye. Further investigation is needed to determine the consequences of the acute changes on ocular on retinal function and mechanism of spaceflight&#8211;induced structure changes.

In the microgravity environment of spaceflight, increased intracranial pressure (ICP) caused by fluid shift may have contributed to spaceflight-associated neuro-ocular syndrome (SANS)1,2,3,4,5. Indeed, over 40% of astronauts have experienced SANS during and after an International Space Station (ISS) mission6, including the spaceflight subject of the NASA Twins Study7. The current pathophysiology of SANS includes physiological changes such as optic disc edema, globe flattening, choroidal and retinal folds, hyperopic refractive error shifts, and nerve fiber layer infarcts (i.e., cotton wool spots) and are well documented5,8. However, the underlying mechanisms of the changes and factors contributing to the development of damage are unclear. In order to have a better understanding of SANS, animal models are available for characterizing the spaceflight-associated changes in retinal structure and function.
In a previous investigation on the same animals, we reported the impact of 35 days of spaceflight on the mouse retina. The results elucidate that spaceflight induces significant damage in the retina and retinal vasculature, and some proteins/pathways associated with cell death, inflammation and metabolic stress were significantly altered following spaceflight9.
Currently, there are a variety of noninvasive imaging techniques established to monitor disease development and progression, as well as physiological responses to various environmental stressors, which are also widely used in small rodent models. One of these techniques is micro-CT, which evaluates anatomical structures and pathological processes, and has successfully been used on organisms as small as mice10.
Micro-CT can achieve a microsized resolution, and it can provide high contrast for volumetric analysis of soft tissues with the addition of the appropriate contrast agent10,11,12,13,14. Micro-CT technology is advantageous compared to traditional methods such as gross anatomy, light microscopy, and histology examination, as it minimizes physical damage to the geometric profile of the specimens and does not alter the spatial relationship among structures. In addition, three-dimensional (3D) models of structures can be reconstructed from micro-CT images12,14. To date, despite evidence showing vision impairment following exposure to the space environment, few data in animal models are available for a better understanding of the spaceflight-associated changes in retinal structure and function. In the current study, mice were flown on a 35-day mission aboard the ISS to determine the impact of the spaceflight environment on ocular tissue structures by quantifying the microstructure of the retina, the RPE, and the choroid layers using micro-CT.

<table>
	<tbody>
		<tr>
			<td>10 wt. % phosphomolybdic</td>
			<td>Sigma</td>
			<td>12026-57-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute by Baker Analyzed</td>
			<td>VWR</td>
			<td>80252500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Merck</td>
			<td>L1825</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray micro-CT system SkyScan 1272 scanner</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of global ocular structure following spaceflight using a micro-computed tomography (micro-ct) imaging method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(&#177;4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

The mean thickness of the retina, RPE, choroid, and sclera layer was recorded using the micro-CT scans after following the protocol above (Figure 1). The technique showed a multiplanar reconstruction of the eyes in three different views. During the analysis, the observer was able to scroll through the whole sample to standardize the analysis right in the middle of the sample.
The micro-CT analysis showed the cross-section areas of the eyes in the sagittal and axial view (Figure 2 and Figure 3) in which were performed the linear measurements. RPE and choroid layer were significantly or trend lower in the spaceflight group when compared to the GC group (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61227/61227fig01v2.jpg" /> 
Figure 1: Micro-CT procedure of soft tissue.&#160;(A) Soft tissue sample (mouse eye). (B) Samples were fixed in 4% formaldehyde in phosphate buffer solution (PBS). After fixation, the mice&#39;s eyes were dehydrated in ethanol. To prevent a further and abrupt shrinkage of the fixed sample, a graded series of ethanolic solutions were used, beginning with 50% ethanol for 1 h and the following ethanol solutions in the concentrations listed, for 1 hour each: 70, 80, 90, 96 and 100%. (C) The mice&#39;s eyes were stained in phosphomolybdic acid (PMA) for 6 days, washed in absolute ethanol and then placed in individual 2 mL plastic containers filled with absolute ethanol. (D) A desktop X-ray micro-CT system scanner was used to evaluate the retinal injury in mice eyes. <a href="https://www.jove.com/files/ftp_upload/61227/61227fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61227/61227fig02.jpg" /> 
Figure 2: Sagittal view of a ground control mouse.&#160;Layers of the eye on the right side of the image are annotated, from top-to-bottom, retina (0.077 mm), retina pigment layer (RPE, 0.038 mm), choroid (0.041 mm), sclera (0.059 mm). This figure has been taken from Overbey et al.15. <a href="https://www.jove.com/files/ftp_upload/61227/61227fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61227/61227fig03.jpg" /> 
Figure 3: Axial view of a ground control mouse.&#160;Layers of the eye on the right side of the image are annotated, from top-to-bottom, retina (0.144 mm), retina pigment layer (RPE, 0.051 mm), choroid (0.041 mm), sclera (0.073 mm). This figure has been taken from Overbey et al.15. <a href="https://www.jove.com/files/ftp_upload/61227/61227fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61227/61227fig04.jpg" /> 
Figure 4: Average thickness of the retinal layer, RPE layer, and the choroid layer measured by micro-CT in the spaceflight and control groups.&#160;Counts were averaged across five retinas per group. Values were represented as mean thickness &#177; standard error (SEM). SEM of the mean is marked with error bars. Significantly lower in cross-section thickness in the spaceflight (FLT) group compared to the ground control (GC) group is denoted &#8216;*&#8217; (p &#60; 0.05). This figure has been taken from Overbey et al.15. <a href="https://www.jove.com/files/ftp_upload/61227/61227fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method at JoVE.com

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage.

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an ...

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5.6K Views.  Loma Linda University. We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage.

Video: Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). 
Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2&#8242;-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs ...