Katchalski, Tsvi; Case, Tom; Kim, Keun-young; Ramachandra, Ranjan; Bushong, Eric; Deerinck, Thomas J.; Haberl, Matthias; Mackey, Mason; Peltier, Steven; Castillon, Guillaume; Fujikawa, Nobuko; Lawrence, Albert; Ellisman, Mark (2021). CIL:50354, phantom. In Cell Image Library. UC San Diego Library Digital Collections. Dataset. https://doi.org/10.6075/J0F76B5J

Biological samples are frequently stained with heavy metals in preparation for examining the macro, micro and ultra-structure using X-ray microtomography and electron microscopy. A single X-ray microtomography scan reveals detailed 3D structure based on staining density, yet it lacks both material composition and functional information. Using a commercially available polychromatic X-ray source, energy integrating detectors and a two-scan configuration labelled by their energy- High and Low, we demonstrate how a specific element, here shown with iron, can be detected from a mixture with other heavy metals. With proper selection of scan configuration, achieving strong overlap of source characteristic emission lines and iron K-edge absorption, iron absorption was enhanced enabling K-edge imaging. Specifically, iron images were obtained by scatter plot material analysis, after selecting specific regions within scatter plots generated from the High and Low scans. Using this method, we identified iron rich regions associated with an iron staining reaction that marks the nodes of Ranvier along nerve axons within mouse spinal roots, also stained with osmium metal commonly used for electron microscopy.

60kVp with fe filtration.

• 2021

• Bushong, Eric
• Case, Tom
• Castillon, Guillaume
• Deerinck, Thomas J.
• Ellisman, Mark
• Fujikawa, Nobuko
• Haberl, Matthias
• Katchalski, Tsvi
• Kim, Keun-Young
• Lawrence, Albert
• Mackey, Mason
• Peltier, Steven
• Ramachandra, Ranjan

A whole, intact retina was isolated from an excised mouse eye and dissected away from the retinal pigment epithelium; the retina was flat mounted with the ganglion-cell side up onto a glass chamber and superfused with Ames' medium during ex-vivo patch clamp electrophysiological experiments as previously described (see Estevez et al., 2012, J. Neuroscience). The recording pipette of the cell contained Lucifer yellow dye which was dialyzed into the cell over the course of a 30 minute recording. At the end of the recording, the tissue was fixed with 4% paraformaldehyde in 0.1M phosphate buffered saline (PBS) for 45 minutes at room temperature. The tissue was washed 3 x 15 minutes in PBS, incubated for 2 hours in blocking solution (2% Triton X-100 and 5% donkey serum in PBS at 4℃) at room temperature and incubated with rabbit anti-lucifer yellow (1:500; Life Technologies, Carlsbad, CA) for 2 nights at 4℃. Then the tissue was rinsed 6 x 10 minutes in PBS and counterstained with donkey anti-rabbit alexa 488 (1:200; Life Technologies, Carlsbad, CA 1: 200) for two hours at room temperature. The immunostained retina was washed 3 x 15 minutes in PBS, mounted on a glass slide and coverslipped using Prolong Gold mouting medium (Life Technologies, Carlsbad, CA).

Images were acquired with a Zeiss LSM 510 Meta laser scanning microscope with a plan-apochromat 20x/0.8 objective. The dye was excited with a 488 nm laser at 5% emission was detected through a 505-550 band pass filter and imaged at 12-bits. The image stack consisted of a 450 μm x 450 μm window through 14 stacks at 0.5 μm (total of 7 μm in z-depth) through the ganglion cell layer and ON-sublayer of the inner plexiform layer of the retina.

Item type: recorded image
Imaging mode: X-ray micrograph
Source of contrast: Fe and U aqueous solutions

• Cell Image Library Group ID: 20513

• Phantom

• data
• image

• No linguistic content; Not applicable

Samplenumber: 50354

Doi: https://doi.org/10.6075/J0F76B5J

Source data

• Source Record in the Cell Image Library: https://doi.org/10.7295/W9CIL50354

Reference

• Katchalski, T., Case, T., Kim, Ky. et al. Iron-specific Signal Separation from within Heavy Metal Stained Biological Samples Using X-Ray Microtomography with Polychromatic Source and Energy-Integrating Detectors. Sci Rep 8, 7553 (2018). https://doi.org/10.1038/s41598-018-25099-z

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2022-08-12