Iodine vapor staining for atomic number contrast in backscattered electron and X-ray imaging

Abstract

Iodine imparts strong contrast to objects imaged with electrons and X-rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation-deposition state-change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas-tight container along with a small mass of solid I2 . The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin-embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X-ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron- and photon-sample interactions, such as transmission electron microscopy and light microscopy.

Keywords: SEM; attenuation; desublimation; histology; microCT; segmentation; soft tissue.

Fig 1

BSE SEM of iodine vapor stained PMMA-embedded musculoskeletal tissues. Three year old Thoroughbred horse distal third metacarpal bone, normal articular cartilage, PMMA block stained with iodine vapor for 23 days. 100 μm scale bar in meta-data panel. ACC = articular calcified cartilage. HAC = hyaline articular cartilage.

Fig 2

BSE SEM of iodine vapor stained PMMA-embedded musculoskeletal tissues. Neonatal Thoroughbred horse distal third metacarpal bone, mineralizing front of the articular cartilage, PMMA block stained with iodine vapor for 36 days. 20 μm scale bar in meta-data panel.

Fig 3

BSE SEM of iodine vapor stained PMMA-embedded musculoskeletal tissues. Adult human femoral head showing typical changes of osteoarthritis with fibrillation of the HAC. PMMA block stained with iodine vapor for 20 days. 100 μm scale bar in meta-data panel. SCB = subchondral bone.

Fig 4

BSE SEM of iodine vapor stained PMMA-embedded musculoskeletal tissues. Rat distal femur growth plate (distal top). 20 μm scale bar in meta-data panel. Asterix ^*in marrow space in epiphysis. PMMA block stained with iodine vapor for 2 months.

Fig 5

X-ray microtomography of locust head. A: tomogram of Locusta head exposed to iodine vapor for 7d. Note contrast between tissue and air and among tissue types. Box shown magnified in (C). B: 3D rendering of 8× downsampled tomograms from the series shown in (A), in frontal view with a cutaway through the right frons and antenna showing the internal muscular structures of the pharynx. Box shown magnified in (D). C: Detail from box in (A) showing trichoid sensillum as it curves through the tomographic plane (arrow) and the space in the cuticle through which its sensory dendrite passes (arrowhead). D: Detail from box in (B) without downsampling of original tomogram showing trichoid sensilla (arrow) on external surface and internal anatomy. E: Example projection image from which tomograms were calculated. Note attenuation of X-ray beam by stained structures F: Tomogram of unstained Schistocerca head at similar anatomic plane to (A) displaying contrast between tissue and air but little detail within soft tissue. Noise is due to a faster acquisition (1600 ms per projection) and larger angular increment (0.6°) than in (A). Key: ct, cuticle; m, muscle; b, brain; con, connective tissue; as, air sac; an, antenna; loc, left ocellus; moc, median ocellus; eye, compound eye; px, pharynx. Scale bars 1 mm (A, E, F) and 100 μm (C). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig 6

X-ray attenuation and emission spectra. X-ray mass attenuation coefficients of adipose tissue, water, Pyrex glass, bone, and elemental iodine show how iodine has a 5- to 30-fold stronger attenuation than biological materials and glass in the range of photon energies produced by a tungsten target (W emission). Figure generated de novo from data published in Hammersberg et al., Linköping Electron Artic Mech Eng, 1998, 1, 1–13, and Hubbell and Seltzer, 1996.