Difference between revisions of "Microtomography (microCT)"
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Considerations for DNA and damage to specimen tissue | Considerations for DNA and damage to specimen tissue | ||
Iodine stains have been shown to inhibit PCR (Marin, et al., 2000; Auinger, et al., 2008) and the staining and rinsing process (soaking the specimen in stain solution at room temperature for hours/days, then washing post-scan) may leave DNA vulnerable to decay by hydrolysis. | Iodine stains have been shown to inhibit PCR (Marin, et al., 2000; Auinger, et al., 2008) and the staining and rinsing process (soaking the specimen in stain solution at room temperature for hours/days, then washing post-scan) may leave DNA vulnerable to decay by hydrolysis. | ||
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| + | |+ Common Contrast Staining Methods Adapted from Metscher (2009) | ||
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| + | ! Stain !! Stoc Solution !! Staining Procedure | ||
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| + | | PTA || Example || Example | ||
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| + | | IKI || Example || Example | ||
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| + | | I2E, I2M || Example || Example | ||
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| + | | Osmium tetroxide || Example || Example | ||
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1% (w/v) phosphotungstic acid in water | 1% (w/v) phosphotungstic acid in water | ||
Mix 30 ml 1% PTA solution + 70 ml absolute ethanol to make 0.3% PTA in 70% ethanol. Keeps indefinitely. | Mix 30 ml 1% PTA solution + 70 ml absolute ethanol to make 0.3% PTA in 70% ethanol. Keeps indefinitely. | ||
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Change to 70% ethanol. Staining is stable for months. | Change to 70% ethanol. Staining is stable for months. | ||
Scan samples in 70% – 100% ethanol | Scan samples in 70% – 100% ethanol | ||
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1% iodine metal (I2) + 2% potassium iodide (KI) in water | 1% iodine metal (I2) + 2% potassium iodide (KI) in water | ||
Dilute to 10% in water just before use. | Dilute to 10% in water just before use. | ||
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Wash in water. | Wash in water. | ||
Can be scanned in water or dehydrated to alcohol. | Can be scanned in water or dehydrated to alcohol. | ||
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1% iodine metal (I2) dissolved in 100% ethanol (I2E) or methanol (I2M) | 1% iodine metal (I2) dissolved in 100% ethanol (I2E) or methanol (I2M) | ||
Use at full concentration or dilute in absolute alcohol. | Use at full concentration or dilute in absolute alcohol. | ||
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Wash in alcohol. | Wash in alcohol. | ||
Stain does not need to be completely washed out before scanning. | Stain does not need to be completely washed out before scanning. | ||
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standard EM post-fixation | standard EM post-fixation | ||
Same as routine EM processing. | Same as routine EM processing. | ||
Revision as of 17:39, 27 April 2023
Contents
Statement of Purpose
This page will provide an overview of the utility for microtomography in a museum setting, considerations for specimens and considerations for destructive sampling, and resources for further information.
Introduction
Micro-CT is ideal for imaging hard, calcified structures such as bone, but the low X-ray absorption of low density non-mineralised tissues means that samples must be stained if soft tissues are to be imaged. (Hall et. al., 2015)
Methods
Considerations for DNA and damage to specimen tissue Iodine stains have been shown to inhibit PCR (Marin, et al., 2000; Auinger, et al., 2008) and the staining and rinsing process (soaking the specimen in stain solution at room temperature for hours/days, then washing post-scan) may leave DNA vulnerable to decay by hydrolysis.
| Stain | Stoc Solution | Staining Procedure |
|---|---|---|
| PTA | Example | Example |
| IKI | Example | Example |
| I2E, I2M | Example | Example |
| Osmium tetroxide | Example | Example |
1% (w/v) phosphotungstic acid in water
Mix 30 ml 1% PTA solution + 70 ml absolute ethanol to make 0.3% PTA in 70% ethanol. Keeps indefinitely.
Take samples to 70% ethanol.
Stain overnight or longer.
Change to 70% ethanol. Staining is stable for months.
Scan samples in 70% – 100% ethanol
1% iodine metal (I2) + 2% potassium iodide (KI) in water Dilute to 10% in water just before use. Rinse samples in water. Stain overnight. Wash in water. Can be scanned in water or dehydrated to alcohol.
1% iodine metal (I2) dissolved in 100% ethanol (I2E) or methanol (I2M) Use at full concentration or dilute in absolute alcohol. Take samples to 100% alcohol. Stain overnight or longer. Wash in alcohol. Stain does not need to be completely washed out before scanning.
standard EM post-fixation Same as routine EM processing. Osmium-stained samples can be scanned in resin blocks, with some loss of contrast.
Dissemination of results
Museum policy often requires CT scans be deposited in a publically available domain, wherein the raw data files can be downloaded. Morphosource, an online tool to find, view and download 3D data from the world's natural history collections is bridging this gap in deliverable dataset for the museum community.
https://www.morphosource.org/?locale=en
Contributors
Equipment
File Formats
Source Material
Links
References
Auinger, B., M., et al. 2008. Improved methodology for identification of Protists and microalgae from plankton samples preserved in Lugol’s iodine solution: combining microscopic analysis with Single-cell PCR, Applied Environmental Microbiology. 74(8). pp.2505.
Hall, A., Sherlock, E., & Sykes, D. (2015). Does micro-CT scanning damage DNA in museum specimens?.
Marin, I., et al. 2000. Preparation of DNA Suitable for PCR Amplification from Fresh or Fixed Single Dinoflagellate Cells. BioTechniques. 30. pp.88-93
Metscher B. D. (2009). MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC physiology, 9, 11. https://doi.org/10.1186/1472-6793-9-11
