Quantitative digital X-ray imaging using frozen hydrated and frozen dried tissue sections

Application 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.     

Low temperature techniques for X-ray microanalysis in pathology: Alternatives to cryoultramicrotomy

Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozenhydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation or when data from a large number of samples have to be acquired. Also X-ray microanalysis of cultured or single cells prepared by freeze-drying can be used to rapidly collect information on a large number of cells. Analysis at high resolution has to be carried out on thin sections: Cryosections or sections of freeze-substituted or freeze-dried embedded tissue. For the latter type of specimens, the use of low-temperature embedding methods may have important advantages.     

Study of potassium deficiency in cardiac muscle: quantitative X-ray microanalysis and cryotechniques

An imbalance of potassium in cardiac muscle causes an alteration of heart function. The distribution and concentration of potassium in rat papillary heart muscle was studied using cryofixation and X-ray microanalysis. Freeze-dried cryosections and sections of freeze-dried, embedded tissue were analysed. Bulk frozen specimens were freeze-dried either in a vacuum or by a new technique using liquid propane as a cryodehydration medium. These two methods of freeze-drying were tested for elemental retention in other specimens, with comparable results. A potassium concentration of 120 mmol/l was measured in normal myocytes of cardiac papillary muscle compared to 80 mmol/l in myocytes of animals stressed by a temperature of 45°C for 1 h. The presumed physiological significance of the findings is discussed.     

The measurement of water distribution in frozen specimens

There are three techniques to measure local water fractions in the cryomicroscope. First, water content may be measured by a direct analysis of oxygen in bulk samples using a windowless detector. Secondly, mass thickness may be estimated in frozen-hydrated, then frozen-dried sections. This technique offers unrivalled spatial resolution, especially if the radiation dose in the frozen-hydrated state is kept low by the use of electron scattering techniques instead of an X-ray microanalytical background determination. External water content standards can be used instead of frozen-hydrated sections and the whole analysis can even be performed exclusively on frozen-dried sections at room temperature. Thirdly, local water fractions can be evaluated from X-ray microanalytical measurements of element concentrations per mass in the frozen-hydrated and frozen-dried state. Corrections necessary for the other techniques cancel out. However, the high radiation dose required for a fully quantitative analysis excludes the use of these methods in thin or ultrathin sections.     

The effect of aluminium coating on elemental standards in X-ray microanalysis

The standardisation of frozen hydrated bulk biological specimens using gelatin standards is described. The relationship between corrected elemental X-ray counts and ionic concentration was found to be linear, and minimum detectable limits for each element are stated. Variations in uncorrected standard curves were found to be due to changes in aluminium coating thickness. There was an inverse relationship between coating thickness and elemental X-ray counts. The factors causing this are discussed. To avoid errors arising from inconsistent aluminium thickness, experimental material should only be compared with standards of similar aluminium net counts. This can be achieved most easily by mounting and analysing specimen and standard together.     

Staining plastic sections

Many of the difficulties of staining plastic embedded tissues for light and electron microscopy derive from physical exclusion of hydrophilic staining reagents by hydrophobic embedding media. Structures which stain most intensely with hydrophilic reagents usually contain less hydrophobic plastic than do non-staining structures. Such incomplete infiltration is apparently caused by exclusion of viscous, hydrophobic monomers by physically dense and/or well hydrated tissue elements. In keeping with this, generalized staining of tissues embedded in hydrophobic media does occur when hydrophobic reagents are used. Staining of plastic-free structures with single hydrophilic reagents or with sequences of such reagents, is, however, largely rate-controlled. The surprising similarity of hydrophilic and hydrophobic plastic embedding media is discussed. Limits of this simple model are explored, with a consideration of the roles of fixative and of monomer-tissue reactions.     

Electron probe X-ray microanalysis of intracellular element concentrations in cryosections in the presence of changes in cell volume

The interpretation of element concentration data for X-ray microanalyses of biological tissues, which are subjected to some experimental treatment, can be complicated by changes in cell volume and total cell dry matter induced by the treatment. We have examined the manner in which such changes would affect the values measured in frozen-dried cryosections of soft tissues, and how they may be taken into account in the interpretation of the results. The element content (mass per unit dry weight) measured by the peak-to-continuum or Hall method is independent of changes in cell volume, but is sensitive to a change in the local dry mass. Conversely, intracellular concentrations in terms of mass per unit volume, as determined by the peripheral or internal standard technique, are dependent on volume changes but independent of dry mass. The estimated dry weight fraction is affected by changes in both volume and dry mass. The results obtained from both quantification methods can therefore provide information on the combination of changes in cellular element levels, volume and total dry mass that may occur following the experimental treatment. In a study of the late effect of the drug cisplatin on electrolyte concentrations in kidney proximal tubules, both quantification methods have been used to obtain wet weight and dry weight concentrations. By applying the above considerations, the analytical results have been interpreted as a combination of changes in element levels and a shrinkage of the tubule cells. Cell shrinkage was confirmed by morphometric analysis of tubular cross-sections.     

Why low temperature embedding for X-ray microanalytical investigations?

Freeze-drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low-temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to ?50°C. Sections of low temperature embedded material can be cut dry at ?60°C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature. The morphological preservation is adequate for the identification of structures such as mitochondria, lysosomes and different types of endoplasmic reticulum in liver cells. Some physical properties of Lowicryl resins, such as mass loss under the electron beam and high contrast, are positive characteristics for the analysis of semi-thick sections. No significant differences in the elemental composition could be detected between tissue which was freeze-dried or freeze-substituted prior to embedding. Freeze-drying is less time consuming. By avoiding contact with organic solvents the risks of ion loss and redistribution are diminished. In contrast to freeze-dried thin cryosections, low temperature embedded material can be sectioned for light microscopy and areas of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogeneous cell populations. The initial preparative step—the cryofixation— determines to a high degree the morphological preservation of freeze-dried and embedded tissue.     

Quantitative X-ray imaging of labelled molecules in tissues and cells

Using cisplatin as a model system, we have been able to demonstrate the feasibility of studying the cellular and subcellular distribution of a labelled molecule containing a single atom of platinum per molecule in bone marrow. An X-ray imaging system consisting of a microcomputer, a 4pi system and a software package was interfaced with an electron microscope enabling the computer to control the beam movements as well as receive signals from the STEM and EDS X-ray detectors. X-ray imaging is useful for both tissue and samples in which the population of cells is not homogeneous. Imaging permits elemental distributions to be measured throughout the sample and not in just randomly selected areas as previously done in X-ray microanalysis. Images are created for not only the element labelling the molecule of interest but also other specified elements present.
Three types of maps for imaging labelled molecules are compared and discussed. When the original (collected) data are mapped, the elements of interest are obscured by the continuum. The maps calculated using an internal standard give a concentration distribution on the basis of volume (mmol L⁻¹ of packed cells). The maps calculated using the continuum normalization method according to Hall produces concentration distribution on the basis of mass (mmol kg⁻¹ dry weight). By recalculating using the 'Peak' or 'Hall' method the continuum problem is removed yielding quantitative images of the intracellular distribution of labelled molecules present in low concentrations.     

Agar standards for quantitative X-ray microanalysis of resin-embedded plant tissues

Calibration standards for quantitative X-ray microanalysis of resin-embedded plant tissue were prepared by adding 6600 mM KC1 to 5% agar. Agar blocks with an edge length of 1–2 mm were rapidly frozen, freeze-dried and embedded in styrene-methacrylate. Dry sections 1 μm thick were mounted on adhesive-coated grids. Apart from fine-scale inhomogeneities caused by ice crystal formation, the KC1 is evenly distributed in the agar blocks. The peak-to-continuum values of K and Cl were highly linearly correlated to the K and Cl contents over the whole concentration range.     

A new sample preparation method for biological soft X-ray microscopy: nitrogen-based contrast and radiation tolerance properties of glycol methacrylate-embedded and sectioned tissue

We describe the preparation of a biological tissue for imaging in a transmission soft X-ray microscope. Sections of exocrine pancreas embedded in glycol methacrylate polymer, an embedding medium widely used in visible light and electron microscopy, were examined. Contrast was based primarily on the nitrogen content of the tissue, and dual-wavelength imaging at the nitrogen K-shell absorption edge was used to map the distribution and provide quantitative densitometry of both the protein and embedding matrix components of the sample. The measurements were calibrated by obtaining the absorption spectrum of protein near the nitrogen edge. The contrast was consistent and reproducible, making possible the first large-scale X-ray microscopic study on sections of plastic-embedded soft tissue. At radiation doses of up to 10⁸ Gray, much more than required for routine imaging, no distortion and little mass loss were observed. This sample preparation method should permit routine imaging of tissues in X-ray microscopes, previously a difficult task, as well as multimodal imaging (using visible light, X-ray, electron, and scanned probe microscopies) on the same sample.     

Quantification for the X-ray microanalysis of cryosections

Some problems of the quantitative analysis of diffusible elements in cryosections are reviewed. The two prevalent methods for obtaining concentrations from X-ray data, one based on characteristic radiation alone and the other on continuum-normalization, are recapitulated. Both methods seem suitable at cellular level while the latter seems preferable at finer spatial resolution. Recourse to both methods together is desirable in the analysis of frozen-hydrated sections especially when there is no peripheral standard. Selective local contamination is a particular hazard in the analysis of chlorine. In the case of sodium, physical parameters set restrictive limits to the minimum concentration measurable by ‘energy-dispersive’ X-ray spectrometry (about 20 mm kg^?1) and to the spatial resolution attainable by diffractive X-ray spectrometry (～0·2 μm). One obvious danger to meaningful quantitative analysis is inadvertent redistribution of diffusible elements during the moments preceding the freeze-quenching of a tiny piece of tissue. Data are presented to show that concentration changes due to simple evaporation are a real hazard prior to the quenching of sub-millimetre size samples.     

A sample preparation for quantitative determination of magnesium in individual lymphocytes by electron probe X-ray microanalysis

We present a sample preparation method for measuring magnesium in individual whole lymphocytes by electron probe X-ray microanalysis. We use Burkitt's lymphoma cells in culture as the test sample and compare X-ray microanalysis of individual cells with atomic absorption analysis of pooled cell populations. We determine the magnesium peak-to-local continuum X-ray intensity ratio by electron probe X-ray microanalysis and calculate a mean cell magnesium concentration of 39± 19 mmol/kg dry weight from analysis of 100 cells. We determine a mean cell magnesium concentration of 34 ±4 mmol/kg dry weight by atomic absorption analysis of pooled cells in three cell cultures. The mean cell magnesium concentrations determined by the two methods are not significantly different. We find a 10% coefficient of variation for both methods of analysis and a 30% coefficient of variation in magnesium concentration among individual cells by electron probe X-ray microanalysis. We wash cells in ammonium nitrate for microanalysis or in buffered saline glucose for atomic absorption analysis. We find cells washed in either solution have the same cell viability (85%), recovery (75%), cell volume (555 μm³) and cytology. We air dry cells on thin film supports and show by magnesium X-ray mapping that magnesium is within the cells. We conclude that: (a) our microanalysis cell preparation method preserves whole intact lymphocytes; (b) there is no systematic difference in results from the two methods of analysis; (c) electron probe X-ray microanalysis can determine the variation in magnesium concentration among individual cells.     

X-ray microanalysis of ion distribution in frozen salt/dextran droplets after freeze-substitution and embedding in anhydrous conditions

A model system using dextran droplets of different salt solutions either frozen together, or sandwiched together after freezing and then freeze-substituted, embedded and dry sectioned has been investigated by X-ray microanalysis. X-ray maps and spot measur ements taken in transects through the interface of both droplets have shown that the P, K and Ca remain well localized in their original droplet. This validates freeze-substitution as a method for localization of these elements in biological samples. Results with Na were more variable and not always explainable. Success was achieved by the use of super-dry solvents and maintenance of a dry environment at all stages. We emphasize the need to avoid water contamination not only during freeze-substitution but also during sectioning, storage and section transfer to the electron microscope.     

A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy

Using a high-intensity synchrotron X-ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D-periodicity and fibril diameter. The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low-temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast. Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical-point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X-ray pattern. Perhaps the most drastic effect was caused by post-fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D-periodicity which occurred during later stages of processing.     

Standards for the application of X-ray microanalysis to biological specimens

A review on the subject of compounds used as standards for biological X-ray microanalysis is presented. The general approach used for standardization has been to use standards which resemble the specimen closely in composition. Thus, standards based on proteins have been used for analysis of quench-frozen cryosectioned specimens, whereas standards based on embedding resins have been used for resin-embedded material. The properties of, and problems associated with, each type of standard are recognized and have been well documented. The choice and analysis of standard should not be a drawback to fully quantitative analysis of biological material. Attention is drawn to the fact that the problems associated with any quantification procedure need to be kept in mind when analysis of standards is undertaken.     

X-ray microanalysis of wet biological specimens in the environmental scanning electron microscope. 1. Reduction of specimen distance under different atmospheric conditions

X-ray microanalysis of non-biological and biological specimens was carried out in the environmental scanning electron microscope (ESEM) under different conditions of specimen distance (the distance travelled by the electron probe within the specimen chamber) and chamber atmosphere. Using both water vapour and argon atmospheres, it was shown that reduction in specimen distance had no effect on atmospheric gas X-ray signal in either case. Unlike water vapour, increased levels of argon (up to 10 torr) caused a marked depression of specimen P/B ratios, with a decrease in both characteristic and background (continuum) counts. These effects in argon were not altered by reduction in specimen distance. Specimen distance was important in relation to beam skirting and elemental analysis. With an extended assembly (short specimen distance), beam skirting in a water-vapour atmosphere was much reduced – leading to enhanced element detectability in a discrete biological specimen (Anabaena cyclindrica).     

Personal-computer-based system for electron beam X-ray microanalysis of biological samples

A system based on a personal computer has been developed which provides a relatively inexpensive way to equip an electron microscopy laboratory for quantitative elemental analyses of cryosectioned biological samples. This system demonstrates the feasibility of making an X-ray analyser from a personal computer, together with commercially available hardware and software components. Hardware and software have been assembled to drive the beam in a scanning electron microscope, collect and analyse X-ray spectra, and save, retrieve, and analyse data. Our software provides a menu-controlled user interface to direct spectra acquisition and analysis. Spot analyses, video images, and quantitative elemental images may be obtained and results transferred in ASCII format to other computers. Wet weight, as well as dry weight, concentrations are calculated, if measurements were made of areas of the hydrated sample before it was freeze-dried. Grey-level copies of video and quantitative elemental images may be made on a laser printer.     

Preparation of cryosections with a modified Sorvall MT2B ultramicrotome and cryoattachment

The Sorvall MT2B ultramicrotome and cryoattachment were modified to extend the duration of cutting and to overcome problems in tissue preparation. Microtome and cryochamber were encased and surrounded by an atmosphere of dry nitrogen gas at room temperature. A specially designed cold block sited within the box close to the microtome enabled tissues to be trimmed at low temperatures in the dry environment. Tissues could be readily visualized both on the trimming block and in the chamber with the existing microscope modified to improve its working distance and lighting system. During transfer from storage in liquid nitrogen, through the trimming procedure, to arrival in the cryochamber, the temperature of tissues as determined using thermocouples embedded in PVP on a stub, never exceeded 140 K. Although there was a considerable standing temperature gradient within the cryochamber, the cutting environment, specimen and edge of the knife were thermally stable. Sections could be routinely cut dry and freeze-dried within the chamber, if desired.     

Properties of frozen sections relevant to quantitative microanalysis

Difficulties in the quantitative X-ray microanalysis of frozen sections may conceivably arise from ice-crystal damage and from electron-beam damage. X-ray peak-to-continuum ratios are commonly taken as a quantitative index of elemental concentrations. But recent reports suggest that in dehydrated frozen sections such ratios vary greatly with the scale of ice-crystal formation existing prior to sublimation. The experiments in these reports are re-interpreted here; it is argued that peak intensities may be affected by ice-crystal scale but that ratios of peak to continuum should not be affected after corrections for exogenous continuum. The accuracy of the peak-to-continuum method is affected by beam-induced loss of mass from microvolumes during analysis. Mass loss can be reduced or slowed by a cold-stage. For example, the radiation sensitivity for loss of chlorine from PVC is reduced by a factor of 1000 or more with reduction of temperature from 300 to 100 K. For sections of soft tissue the effectiveness of cooling is not nearly so striking but at 100 K, analyses of 1 μm frozen-hydrated sections by the continuum method, with spatial resolution of the order of 1 μm, can be completed before substantial mass loss occurs. However, analysis of frozen-hydrated sections by the continuum method at much higher resolution, say 100 nm resolution in 100 nm sections, is precluded by mass loss. Measurements of local mass can be achieved with much lower dose by observation and calibration of the electron transmission or backscattering. But even with these methods, several problems remain in achieving quantitative X-ray analysis at very high resolution.