K E Y WORDS. X-ray microanalysis, cryosectioning, X-ray imaging. S U M M A R YApplication of quantitative X-ray imaging to frozen hydrated tissue sections has presented a number of major problems including lack of a suitable algorithm which could deal effectively with mass loss due to radiation damage, problems of low characteristic X-ray signal to background ratios, and provide a means of analysis of the same location in both hydrated and dried states. This paper presents details of the application of our algorithm for analysis of frozen hydrated, then dried cryosections applied to quantitative X-ray imaging, which provides relatively high precision quantitative measurement of elemental content (related to both wet and dry weight) and water content of each pixel. This algorithm largely circumvents many of the problems of analysis of frozen hydrated tissue sections. Our algorithm for X-ray imaging obtains reasonably precise quantitative measurements coupled with morphological information by trading speed and image resolution.

A method is reported for preparing, examining and analysing frozen hydrated tissue sections using transmission electron microscopy and X-ray microanalysis. Use of this method permits localization and measurement of water soluble or diffusible elements within the hydrated cell matrix. Since any change in total fresh weight of the specimen will affect the concentration of all components, great care has been taken to demonstrate that the mass neither increases nor decreases and to ensure that the tissue remains frozen-hydrated. Criteria for assessing whether or not the tissue remains frozen-hydrated are reported. After quench freezing, 1-2 mum thick sections of mouse liver were cut at 193 degrees K and picked up on a specially designed annular specimen holder covered with an aluminium coated nylon film. Using a transfer device which prevents contamination of the tissue sections while maintaining them at a low temperature (below 143 degrees K), the sections are transferred either to the vacuum evaporator cold stage or the scanning microscope cold stage. The tissue sections may be coated with an aluminium layer to improve electrical and thermal conductivity. The specimens are examined in the scanning transmission imaging mode and analysed using an energy dispersive X-ray analyser. Concentration of intra-nuclear and intra-cytoplasmic K, P, S and Cl are reported for mouse hepatocytes as ratios of the characteristic radiation to the continuum radiation used as a measure of mass. Ratios for all four elements were higher in the nucleus than the cytoplasm. Examples are given of this method as applied to plant and insect tissue.

The electrolyte and water content of cellular and interstitial compartments in the renal papilla of the rat was determined by x-ray microanalysis of frozen-hydrated tissue sections . Papillae from rats on ad libitum water were rapidly frozen in a slush of Freon 12, and sectioned in a cryomicrotome at -30 to -40°C . Frozen 0.5-,um sections were mounted on carbon-coated nylon film over a Be grid, transferred cold to the scanning microscope, and maintained at -175°C during analysis . The scanning transmission mode was used for imaging. Structural preservation was of good quality and allowed identification of tissue compartments . Tissue mass (solutes + water) was determined by continuum radiation from regions of interest . After drying in the SEM, elemental composition of morphologically defined compartments (solutes) was determined by analysis of specific x-rays, and total dry mass by continuum. Na, K, CI, and H2O contents in collecting-duct cells (CDC), papillary epithelial cells (PEC), and interstitial cells (IC) and space were measured . Cells had lower water content (mean 58 .7%) than interstitium (77.5%) . Intracellular K concentrations (millimoles per kilogram wet weight) were unremarkable (79-156 mm/kg wet weight) ; P was markedly higher in cells than in interstitium . S was the same in all compartments . Intracellular Na levels were extremely high (CDC, 344 ± 127 SD mm/kg wet weight ; PEC, 287 ± 105; IC, 898 ± 194) . Mean interstitial Na was 590 ± 119 mm/Kg wet weight . CI values paralleled those for Na . If this Na is unbound, then these data suggest that renal papillary interstitial cells adapt to their hyperosmotic environment by a Na-uptake process.Cells of the rat renal papilla are exposed to wide changes in the ionic and osmotic composition of their environment . The papillary epithelium of the rat is exposed to urine whose osmolality ranges from <100 to >3,000 mosmol/kg H2O. Tubular and interstitial cells within the papilla live in the hypertonic environment associated with the urine-concentrating mechanism. Because few mammalian cell types are exposed to such environmental conditions, the mechanism by which these cells adapt to the high salt and urea content of their environment is of great interest to cell biologists. and Morgan (20), using in vitro centrifugation or incubation techniques, have reported that the Na content of papillary cells increases with increasing osmolality and reaches more than 400 mM. However, until recently no method existed for the 274 definition of chemical composition of defined cell types within the papilla. The development of techniques for direct x-ray microanalysis of frozen-hydrated tissue sections (19,22,23) now makes such analysis possible. MATERIALS AND METHODSSix male Long-Evans rats (bred in our colony), weighing 125-200 g, were housed individually in metabolic cages for 2 dor more before the experiment . They were given Purina Rat Chow (0.29% Na, 0.46% K; Ralston Purina Co., St. Louis, Mo.) and water ad libitum. An overnight urine sample was collecte...

X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.

X-ray microanalysis of frozen-hydrated tissue sections permits direct quantitative analysis of diffusible elements in defined cellular compartments . Because the sections are hydrated, elemental concentrations can be defined as wet-weight mass fractions . Use of these techniques should also permit determination of water fraction in cellular compartments . Reliable preparative techniques provide flat, smooth, 0.5 p,m-thick sections with little elemental and morphological disruption . The specimen support and transfer system described permits hydrated sections to be transferred to the scanning electron microscope cold stage for examination and analysis without contamination or water loss and without introduction of extraneous x-ray radiation .X-ray microanalysis of frozen-hydrated tissue sections is potentially an ideal method for analyzing diffusible elements in tissues. This paper describes a practical method of specimen handling for quantitative x-ray analysis of fully frozen-hydrated tissue sections in scanning electron microscopes (SEM) fitted with energy dispersive x-ray detectors. The success of the method is based on advances in two areas. First, reliable preparative techniques were developed to provide flat, smooth, 0 .5-Lm-thick sections with little elemental redistribution and morphologic disruption . Second, a specimen support and transfersystem was developed that allowed sections to be transferred to the SEM cold stage for examination and analysis without contamination or loss of water and without introducing extraneous x-ray radiation sources. The cryochamber (Fig . 1) was constructed of foam polystyrene sheets (2.5 cm thick), and attached to the microtome knife stage so that chamber and knife moved together in the horizontal plane. The chamber was cooled by a continuous flow of dry Ns gas at constant low temperature, which entered the chamber beneath a secondary floor 10 mm above the main chamber floor . The cold Nz gas was produced by boiling Liquid nitrogen (LN2) in a pressurized 25-Liter Dewar flask with an electrical immersion heater . Heat input, and hence gas flow rate, was adjusted by an autotransformer. Gas leaving the Dewar flask was reheated to the desired temperature by passing through an insulated resistor (Ohmite, 100 S2,225 W, #0904) connected to a separate autotransformer. Specimen temperature was estimated by using a thermistor mounted on the chuck assembly . All "cutting temperatures" were measured at this point .Sections were cut with a single-edge razor blade (Plus Platinum; Schick, Inc ., Westport, Conn.), mounted in a brass vise, which clamped the razor blade with 2-3 mm of the edge extended, and which incorporated an antiroll device consisting of a microscope slide held (in a movable mounting) with its edge parallel to the upper surface of the blade (Fig . 2) . The angle and spacing of the antiroll plate was set before cooling the chamber and final adjustment was made just before sectioning. All sections were cut with the microtome advance set for 0.5-lm section thickn...

Elemental (Na, P, S, Cl, K, Ca, Mg) composition and water content of neurons and glial cells of the leech (Macrobdella decora) were determined by x-ray microanalysis of frozen hydrated and dried section techniques. Results are reported as elemental mass fractions (mass/mass) and water content as percent mass. Specific cell compartments and cell types had distinct elemental patterns and water content which suggests that chemical composition of specific cell types is unique and may represent an expression of cell differentiation analogous to morphological specialization. Water content of cells was also cell specific and ranged from 55% (neurons) to 90% (vacuolated zone of glial cells). K and Na were present in concentrations greater than predicted by ion-selective microelectrode measurements, indicating that not all the K and Na were simultaneously accessible to such electrodes.

New specimen handling and analytic techniques for the application of x- ray microanalysis to studies of cell and organ biology have been recently described (Saubermann et al., 1981, J. Cell Biol. 88:257-267). Based on these techniques, absolute quantitative standardization has been established through x-ray analysis of frozen-hydrated and then dried sections of independently measured standard solutions of elements. These experiments demonstrate that the specific techniques employed have a probable error of less than 10%. Artificial electrolyte gradients established in gelatin were subjected to analysis to determine whether there was elemental displacement under non-membrane- limited conditions at the temperatures employed for sectioning (-30 degrees to -40 degrees C). No significant difference was observed between such gradients in serial sections cut at -30 degrees and -80 degrees C. Similarly, no additional ice-crystal-damage artifact was found in sections cut at -30 degrees C when compared with sections cut at -80 degrees C. Thus, in terms of ice-crystal size, gradient maintenance, and compartmental differentiation, cryosectioning at -30 degrees to -40 degrees C was not associated with redistribution incompatible with 1- to 2-micrometers spatial resolution, and absolute measurements of elemental concentration were practical within regions of this size.

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