Biological cryosection preparation and practical ion yield evaluation for ion microscopic analysis

Proc. Natl. Acad. Sci. U.S.A.

Fullmer

Smith

et al. 1990

The intestinal absorption of calcium includes at least three definable steps: transfer across the microvillar membrane, movement through the cytosolic compartment, and energy-dependent extrusion into the lamina propria. Tracing the movement of calcium through the epithelium has been hampered by lack of suitable techniques and, in this study, advantage was taken of ion microscopy in conjunction with cryosectioning and use of the stable isotope "Ca to visualize calcium in transit during the absorptive process. The effect of vitamin D, required for optimal calcium absorption, was investigated. Twenty millimolar 44Ca was injected into the duodenal lumen in situ of vitamin D-deficient and vitamin D-replete chickens. At 2.5, 5.0, and 20.0 min after injection, duodenal tissue was obtained and processed for ion microscopic imaging. At 2.5 min, 44Ca was seen to be concentrated in the region subjacent to the microvillar membrane in tissue from both groups. At 5.0 and 20.0 min, a similar pattern of localization was evident in D-deficient tissues. In D-replete tissues, the distribution of 44Ca became more homogenous, indicating that vitamin D increased the rate of transfer of Ca2" from the apical to the basolateral membrane, a function previously ascribed to the vitamin D-induced calcium-binding protein (28-kDa calbindin-D). Quantitative aspects of the calcium absorptive process were determined in parallel experiments with the radionuclide 47Ca. Complementary information on the localization of the naturally occurring isotopes of calcium (40Ca) and potassium (39K) is also described.The past 20 years have been marked by a growing recognition of the important role of calcium (Ca) as a second messenger, involved in the mediation of a diverse array of cellular functions (1). As a major component of bones and teeth, large amounts of Ca are required for their formation. The only source of Ca to meet these essential functions is ultimately the diet, and mechanisms have evolved to assure an adequate and proper transfer of dietary Ca across the intestine. Special accommodations must be made by the intestinal epithelial cells to transport the large quantities of Ca and, at the same time, assure that the intracellular free Ca2+ concentrations are maintained at levels that are nontoxic and appropriate for normal cell function.A complete understanding of the Ca absorption process and the role of vitamin D thereon, despite considerable effort, is still lacking. One aspect that requires clarification is the path taken by Ca during its transport across epithelia. In the present study, we have visualized this transport by using the stable isotope "Ca as the absorbed species in combination with the technique of ion microscopy (2-4). The ion microscope, based on secondary ion mass spectrometry, is a direct imaging mass spectrometer. The instrument can discriminate isotopes based on their mass-to-charge ratio and, therefore, allows imaging of isotopic gradients in relation to tissue morphology with a spatial resolution of 0O.5 pum. Beca...

Section: Methodsmentioning

confidence: 99%

Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin D-deficient and vitamin D-replete chickens: a 44Ca stable isotope study.

Proc. Natl. Acad. Sci. U.S.A.

Fullmer

Smith

et al. 1990

“…The frozen tissue was cryosectioned (Reichert's Cryo Cut II microtome, Deerfield, Ill., USA) into 2-µm-thick sections at −35 to −25°C, the sections then pressed onto a liquid nitrogencooled wafer of indium substrate, and lyophilized at −50°C for ion microscopic analysis (Sod et al 1990). With the blood supply intact, the duodenal lumen was briefly rinsed with ice-cold saline, the mucosal surface was then surgically exposed, and a small flap of intestine was immediately frozen by pressing the tissue between liquid nitrogen-cooled copper pliers.…”

Section: Ion Microscopymentioning

confidence: 99%

Ion microscopic imaging of calcium during 1,25-dihydroxyvitamin D-mediated intestinal absorption

Fullmer

Smith

et al. 1996

Histochem Cell Biol

Self Cite

A combination of ion microscopic and conventional radionuclide techniques was employed to investigate the temporal-spatial dynamics of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated intestinal calcium (Ca) absorption. At varying times following the administration of a single intravenous dose of 1,25(OH)2D3 to vitamin D-deficient chicks, transepithelial transport and tissue retention of Ca were quantitated in vivo, using the ligated duodenal loop technique and 47Ca as the tracer. The localization of Ca in the intestinal tissue during absorption was monitored by ion microscopy, using the stable Ca isotope, 44Ca, as the absorbed species. There was little transepithelial absorption of Ca in the vitamin D-deficient animals despite a substantial tissue accumulation of luminally derived Ca, the latter localizing predominantly in the brush border region of the enterocyte, as shown by the 44Ca-ion microscopic images. The early (30 min-1 h) response to 1,25(OH)2D3 was an increased tissue uptake of luminal 47Ca, which also primarily associated with the brush border region, again as shown by ion microscopy. At 2-4 h after the 1,25(OH)2D3 dose, there was a progressive redistribution of Ca from the brush border region throughout the cytoplasm and into the lamina propria. At 8-16 h, 47Ca absorption was maximal and 44Ca was sparsely distributed in the intestinal tissue. 47Ca absorption gradually declined and reached pre-dose levels by 72 h. At this time, tissue 44Ca was again largely limited to the brush border region. These results provide support for the multiple actions of 1,25(OH)2D3 on the intestinal Ca absorption process. The ion microscopic images provided unique information on the specific time-dependent changes in the tissue localization of Ca during the process of its intestinal absorption as affected by 1,25(OH)2D3.

“…This hindered ion microscopic analysis due to severe topography and/or specimen charging arising from poor contact with the conducting substrate. By using indium metal as a substrate, this obstacle has been overcome [49]. Indium (stable isotopes of mass 113 and 115) is available in many forms at very high purity and it does not interfere with physiologically important diffusible elements.…”

Section: Ion Microscopic Analysis Of Diffusible Elements In Animal Timentioning

confidence: 99%

“…Since indium was vapor-deposited over an indium cushion, the small grains of indium can be easily recognized in the SEM image. In a recent study it was observed that in rat liver and small intestine the SIMS matrix effects were not significant in frozen-freezedried cryosections [49]. A very powerful and unique capability of SIMS imaging is its isotopic detection which allows the use of stable isotopes as tracers.…”

Section: Ion Microscopic Analysis Of Diffusible Elements In Animal Timentioning

confidence: 99%

Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy

Morrison

1992

Biology of the Cell

Self Cite

108

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.