Quantitative elemental X-ray imaging of frozen-hydrated biological samples

Quantitative elemental X-ray imaging of frozen-hydratedbiological samples

A. T. MARSHALL & W. XUAnalytical Electron Microscopy Laboratory, School of Zoology, La Trobe University, Bundoora,Melbourne, Victoria 3083, Australia

Key words. Biological, bulk samples, electron probe microanalysis, frozen-hydrated, insect, ions and water, Malpighian tubules, soy bean nodules,transporting epithelia, X-ray imaging, X-ray microanalysis.

Summary

It is shown that quantitative X-ray imaging of planed,frozen-hydrated, biological bulk samples that have not beenetched is possible. X-ray imaging represents a betteralternative to static beam (selected area) analysis offractured frozen-hydrated samples. This procedure avoidsthe undesirable necessity of etching planed frozen-hydratedsamples to provide an interpretable electron image.Qualitative oxygen and carbon X-ray images, which canbe acquired in a short time, can be used for distinguishingmorphological features and remove the requirement forelectron images. In test samples of frozen-hydrated albu-min, containing salts, analyses by X-ray images comparedwell with static beam (selected area) analyses from the samesamples. An example of an analysis of frozen-hydratedinsect Malpighian tubules is given in which the response toouabain treatment was analysed. In this example X-rayimaging showed that ouabain resulted in a significantincrease in cytoplasmic and luminal Na and a significantdecrease in cytoplasmic and luminal K. X-ray imaging alsoshowed that there was a significant increase in cellularwater content. The presence of a potassium gradient insoybean root nodules was also demonstrated. The use ofstandard deviation images for processing low count imagesincreases analytical precision but results in underestimatesof the true concentrations.

Introduction

The X-ray microanalysis of frozen-hydrated samples inbiology has long been recognized as the most desirableprocedure for in situ elemental analysis. It is assumed thatin a fully frozen-hydrated specimen there is little or nopossibility of element redistribution. This is a particularly

important consideration for the analysis of fluids inextracellular space. Where these fluids contain lowconcentrations of proteins then an alternative low-temperature procedure, such as freeze-drying, certainlyresults in elemental redistribution. Such situations may befound in organ lumina and in plant cell vacuoles.Furthermore, quantification of element concentration isobtained directly in terms of mmol kg¹1 wet weight which ismuch more biologically meaningful than concentration interms of dry mass (Gupta, 1993). Ideally, for diffusibleelements, concentration should be expressed in terms ofmmol L¹1 cell water. A considerable amount of effort hasbeen devoted therefore to finding reliable means ofobtaining the water mass fraction in the analysed volume(von Zglinicki, 1991; Echlin, 1992).

Biological samples may be analysed in the frozen-hydrated state as semithin sections (Gupta, 1991) or asbulk samples (Marshall, 1987). Radiation damage pre-cludes, at the present time, the analysis of ultrathin frozen-hydrated sections (von Zglinicki, 1993). Whilst frozen-hydrated semithin sections have many advantages for X-raymicroanalysis they are inherently less stable than frozen-hydrated bulk samples. The latter are easier to prepare andare considerably more resistant to radiation damage.Although the analytical resolution in a bulk sample is ofthe order of 2 mm (depth and lateral resolution), this is stillsufficient to investigate many biological problems. Frozen-hydrated bulk samples have typically been prepared byeither fracturing the frozen sample (Marshall, 1980, 1987;Echlin & Taylor, 1986) or planing the sample andsubsequently etching the surface by raising the temperatureand subliming ice (Potts & Oates, 1983; Canny & Huang,1993). These procedures produce a recognizable morphol-ogy but present some disadvantages for quantitativeanalysis. Obtaining quantitative data from a fracturedsurface necessitates selecting a part of the sample in whichthe local tilt angle is, or is close to, zero degrees (Marshall,

Journal of Microscopy, Vol. 190, Pt 3, June 1998, pp. 305–316.Received 14 May 1997; accepted 11 September 1997

305q 1998 The Royal Microscopical Society

Correspondence to: Dr A. T. Marshall, tel: 66 (0)3 94792279; fax: 66

(0)39479155; E-mail: [email protected]

1981). Alternatively, procedures which normalize the X-raysignal for tilt can be used (Boekstein et al., 1980; Marshall &Condron, 1985a; Echlin & Taylor, 1986). The use of aplaned surface obviates this difficulty but the process ofetching the surface to produce a recognizable morphologyentails a risk of compromising the analysis. The sublimationof ice is not easy to control, particularly when heating isfrom below the sample (Echlin, 1992). Sublimation can behighly variable in rate, even from a homogeneous sample,and it should be recognized that the precision of the analysismay be significantly decreased (Marshall, 1981).

The reason for etching or subliming ice from the surfaceof a planed sample is that the planed surface is featureless inboth secondary and backscattered electron images. It is, ofcourse, difficult to analyse what cannot be seen. A solutionto this problem is to use X-ray imaging. As increasinglypowerful computers and microscopes with highly stableperformances have become available, digital X-ray imaginghas become more feasible (Somlyo, 1984; Saubermann &Heyman, 1987; Fiori et al., 1988; LeFurgey et al., 1992).We have adopted this approach for the analysis of planed,fully frozen-hydrated bulk samples. This technique isentirely independent of electron imaging of the sample, i.e.there is no requirement to image the morphology of thesample by electrons. Furthermore, planed, unetched, fullyfrozen-hydrated samples can be used for the determinationof water concentration by measuring oxygen concentration(Marshall & Patak, 1993; Marshall, 1994).

In frozen-hydrated bulk samples the quantification of Nais difficult. This is a consequence of the high absorption ofNa X-rays by the sample and by the detector window. Theabsorption of Na X-rays is minimized in an ultrathinwindow. The use of an ultrathin window for Na detection,however, is precluded in the analysis of frozen-hydratedsamples because the large O peak from water produces asum peak in the spectrum exactly at the energy of Na X-rays(Marshall, 1994). Since intracellular Na concentrations areusually low, a typical spectrum from a frozen-hydrated bulksample has a small Na peak, containing few counts, on alarge background. The statistical error for Na in thissituation may be large. It has been suggested by Statham(1988) that, where an elemental image contains very lowcounts, the genuine presence of the element can bedistinguished from statistical noise by setting to zero allthose pixels in the image which have a value less than ntimes the standard deviation of the image, where the valueof n depends on the required probability. This is done byprocessing the original image with a standard deviationimage formed from the square root of peak plus backgroundcounts at each pixel. The original application of this methodwas for the objective determination of the genuine presenceor absence of an element in low count images. We haveinvestigated the use of this method for the quantitativeanalysis of Na.

Two examples of the application of quantitative X-rayimaging are drawn from investigations currently in progressin this laboratory on ion and water transport by insectMalpighian tubules (Xu and Marshall, in preparation) andthe role of ions in relation to the osmotic control of oxygendiffusion pathways in soybean root nodules (Brown andMarshall, in preparation).

Materials and methods

Instrumentation

Analyses were carried out in a JEOL JSM 840A scanningelectron microscope fitted with a cold stage cooled bythermal transfer via multiple strands of fine copper braid toa cold finger, through which liquid nitrogen circulated froma 10-L stainless steel Dewar. The temperature of the stagewas monitored by a fine thermocouple. Two other liquidnitrogen Dewars cooled two large copper plates arrangedparallel to the walls of the sample chamber. The internalwalls of the Dewars and the copper anticontaminator platesresulted in highly effective cryopumping and a chambervacuum, measured with a VG Anavac gas analyser, of<0·5 × 10¹8 Torr (6·7 × 10¹7 Pa). The holding capacity ofthe stage Dewar was < 24 h and that of the antic-ontaminator Dewars was sufficient for overnight operation.Stage temperature was very stable and remained at 101 Kthroughout the overnight acquisition of X-ray images. Nodrift due to stage movement was detected. The airlock of themicroscope was extended and fitted with a separate rotaryvacuum pump, a cold stage, which operated at atemperature of 88 K, and a manipulator for removing thesample protection cap from the sample holder.

The microscope was equipped with a Link eXL X-rayanalyser and a Link LZ4 30-mm2 detector having anultrathin window and a beryllium window. The take-offangle was 408 and the solid angle was 0·011 steradians.Beam current was monitored by means of a Faraday cupinserted into the beam and was stable (6 2%) over periodsof 120 h of continuous operation.

Sample preparation

The samples used for testing the method included Mal-pighian tubules from the black field cricket (Telleogryllusoceanicus) and soybean (Glycine max) root nodules. TheMalpighian tubules were dissected and the tubules fromeach insect were divided into two groups and incubated in asaline medium (Coast, 1988) for 30 min. One group oftubules was incubated in the same medium for a further30 min, whilst the second group was incubated in1 mmol L¹1 ouabain in the saline medium. Prior to freezingthey were dipped in saline containing 20% albumin (w/v).The osmotic concentration of albumin/saline medium was

306 A. T. MARSHALL AND W. XU

q 1998 The Royal Microscopical Society, Journal of Microscopy, 190, 305–316

only slightly higher (373 mosmol kg¹1) than that of thesaline solution alone (350 mosmol kg¹1). Both values werewithin the range of the normal haemolymph osmoticconcentration. Soybeans were removed from pots and theroots rapidly rinsed in distilled water to remove adheringculture medium.

Samples of animal tissue and model test specimens ofdialysed albumin solutions containing known concentra-tions of salts were frozen on aluminium pins for the ReichertFC4E cryoultramicrotome. Plant tissues were first frozenthen glued into a suitable hole in a liquid nitrogen cooledaluminium stub with Tissue Tec (Miles Inc., Elkhart, IN46575, U.S.A.). All samples were frozen by plunging intoliquid propane at 83 K using a Reichert KF80 freezingdevice. The frozen samples on both pins and stubs wereplaned with a diamond trimming knife in a Reichert FC4Ecryoultramicrotome at 163 K. After planing the pins wereinserted into 10-mm aluminium stubs whilst in themicrotome chamber. Two pins were placed in each stuband locked into the tightly fitting holes by grub screws. Thestub also contained a polished copper rod, 1 mm indiameter, which served as a count rate calibration standardfor quantitative analysis. The stubs were each mounted intoa substage fitted with a threaded cap. The substages couldthen be stored in liquid nitrogen indefinitely.

Planed samples in a substage were transferred to acustom built coating device and coated with 20 nm Al at83 K and 1 × 10¹7 Torr (1·3 × 10¹5 Pa). The coating devicewas fitted with an airlock containing a cold stage and amanipulator for removing the substage cap. After coating,the substage was withdrawn into the airlock and recapped.Following withdrawal from the airlock the substage wasstored in liquid nitrogen.

Analytical model

Quantitative data were obtained by applying matrixcorrections, derived from the phi-rho-zed model, to theX-ray intensities from samples and standards (Marshall,1982; Marshall & Condron, 1987a,b). The quantitativemodel used in this investigation was that provided in theLink exL software which is based on the model developed byBastin and colleagues (Bastin et al., 1984; 1986).

The standards used for elements Z>10 were polishedmicroprobe standards (Biorad). These were used for peakfitting (primary standards) and for concentration determi-nation (secondary standards). For C, N and O microprobestandards were used as primary standards for peak fittingbut frozen-hydrated samples of 20% albumin (w/w) wereused as secondary standards to obtain concentrations ofthese elements. Albumin samples were chemically analysedfor C, N and O content. In applying matrix corrections itwas assumed that the biological matrix is sufficiently wellmodelled by a protein solution and that unanalysed

elements could be represented by the concentrationscalculated for a protein solution, i.e. H in the case of UTWanalyses and H, C, N and O in the case of Be windowanalyses. For biological samples the composition of ageneralized protein was used (Engstrom, 1966) and foralbumin samples the composition as determined bychemical analysis was used.

X-ray imaging

All analyses were made using an accelerating voltage of15 kV. The Link X-ray imaging software for quantitativemapping calculates peak integrals, using multiple least-squares fitting of library peaks to the spectrum for eachpixel, and apparent concentrations, i.e. concentrationswhich have not been corrected for matrix effects. Apparentconcentration images were corrected by applying matrixcorrections derived from selected area analyses of the samesamples. Since the corrections for elements Na–Ca varyvery little for either changes in protein concentration orchanges in elemental concentration over the normalbiological range (Marshall, 1982; Marshall & Condron,1987b), this is a reasonable procedure. In the case of Naimages, in which count rates were very low, standarddeviation (SD) images were used to set every pixel in the Naimage below 1·5× SD to zero in order to discriminate thetrue Na counts from noise due to statistical fluctuation(Statham, 1988) prior to the application of matrixcorrections. This procedure produces images of increasedprecision. However, since large regions of the images areaveraged with the inclusion of many pixels of zero intensity,the accuracy of the analysis is decreased. In order to obtainbetter estimates of the true concentrations, Na imageswhich had not been processed by SD images were also usedto provide average concentrations of regions.

Quantitative light element (C,N,O) images can beobtained in a similar manner; however, the phi-rho-zedcorrections for O are strongly dependent upon proteinconcentration. This means that if dry mass changessubstantially within the image field the phi-rho-zed correc-tion will not be accurate. Fortunately, changes in dry mass(protein concentration) within cells are not usually aproblem at the analytical resolution of this technique.Concentration images for water can be obtained byassuming that cell composition is satisfactorily modelledby a protein water solution (Marshall, 1982). The equationdescribing the linear relationship between water and Oconcentrations was applied to transform O concentrationimages into water concentration images (Marshall, 1994).

Values for average concentrations from selected regions ofMalpighian tubules were obtained by applying binary masksto elemental images using the Link program Featurescan.The entire cellular or luminal region of each tubule profilewas included in the masks. Images were also transferred to


X-RAY IMAG ING OF FROZEN SAMPLES 307

a PC, using the Link program PC Link, and average valuesfrom selected areas in individual tubules were obtainedusing the program Scion Image PC. The data from analysesof Malpighian tubules were statistically analysed usingpaired t-tests.

The protocol used for preparing and analysing Malpighiantubles is schematically shown in Fig. 1.

Results

Samples of 20% albumin containing various salt concen-trations were analysed for 100 s by a rapidly scanningraster over an area of < 66 × 66 mm at a beam current of1 × 10¹9 A. Each sample was analysed in three differentregions. Similar areas on the same samples were analysed asX-ray images using the same beam current and a matrix of32 × 32 pixels with a dwell time of 6 s. A comparison of theanalyses of K and Cl (Figs. 2 and 3) shows that the twoanalytical methods gave identical results. Although thedwell time per pixel in the X-ray image was only 6 s themean element concentration for each sample was derived

from 1024 pixels which corresponds to a total analysis timeof 6144 s.

A similar analysis was carried out on frozen-hydratedalbumin samples containing Na. Although the albumin wasdialysed, flame emission spectrophotometry showed that a20% solution contained 34 mmol kg¹1 Na. The results from

Fig. 1. Schematic diagram showing preparation and imaging pro-cedures for planed samples of frozen-hydrated Malpighian tubules.

Fig. 2. Comparison of K concentrations in frozen-hydrated samplesof 20% albumin with added K measured by ‘static beam’ (selectedarea) analyses (mean of three analyses per sample 6 SE) and byaveraging pixel values in 32 × 32 pixel images (the mean and SEof each image can be considered as the mean and SE of 1024 indi-vidual analyses). When error bars are not shown they lie withinthe sample. Concentrations are in mmol kg¹1 wet weight.

Fig. 3. Comparison of Cl concentrations in frozen-hydrated samplesof 20% albumin with added Cl measured by ‘static beam’ (selectedarea) analyses and by averaging pixel values in 32 × 32 pixelimages (means and SEs as in Fig. 2). Concentrations are in mmolkg¹1 wet weight.



X-ray imaging are shown in Fig. 4. When measured Na isplotted against total Na (Fig. 4a) the intercept of theregression line with the x-axis suggests that the minimumdetection limit is 11 mmol kg¹1. When measured Na isplotted against added Na (Fig. 4b) a regression line fitted tothe data points fails to go through the origin, as would beexpected if the albumin contained Na prior to the additionof Na. The Na image was then processed by a simulta-neously collected standard deviation image so that all pixelsin the Na image with counts less than 1·5× the standard

deviation were set to zero. When these measured concen-tration values are plotted against the concentrations ofadded Na an excellent agreement is obtained (Fig. 4b). Thedifference between the intercepts on the y-axis of therespective regression lines is 21·5 mmol kg¹1. This suggeststhat the elimination of statistical noise imposes a minimumdetection limit for Na of between 21·5 (from the y-intercept)and 34 mmol kg¹1 (the amount of Na remaining in thealbumin after dialysis and which is less than 1·5× SD).

Oxygen concentrations were measured in six frozen-hydrated samples of 20% albumin by selected area analysesusing a rapidly scanning raster covering an area of15 625 mm2 for 100 s. Three areas in each sample wereanalysed using a beam current of 1 × 10¹10 A. An X-rayimage was obtained from each of the same samples for asimilar area at a resolution of 64 × 64 pixels and with thesame beam current. The comparison of selected area


Fig. 4. Na concentrations in frozen-hydrated samples of 20% albu-min measured by averaging pixel values in 32 × 32 pixel images(means and SEs as in Fig. 2). Concentrations are in mmol kg¹1

wet weight. (a) Na measurements plotted against total Na concen-trations of albumin samples; (b) Na measurements, obtained withand without image processing by standard deviation images,plotted against added Na concentrations.

Fig. 5. Low-magnification O image from a planed sample of frozen-hydrated Malpighian tubules. The image clearly shows manytubule profiles. In the image the light tones represent high O con-centrations and the dark tones represent low O concentrations.

Table 1. Comparison of percentage weight of oxygen and watermeasured by static beam (selected area) analysis and X-ray imag-ing in frozen albumin solutions compared to theoretical values.

Theoretical % Selected area % X-ray image %

O 76·6 74·5 6 1 87·2 6 2·7Water 81·5 78·3 6 1·8 97·4 6 4·0

Mean 6 SE, n ¼ 6.


analyses with X-ray images for analysing O in frozensolutions of albumin showed that O concentrations wereoverestimated by X-ray imaging (Table 1). This occurredwhen standards data were used that had been obtained,from standards of similar composition, by selected areaanalyses. A possible explanation of this discrepancy is thatthe considerably lower electron dose during X-ray imagingresults in reduced radiation damage, i.e. mass loss. Insubsequent analyses of biological samples, the data obtainedfrom the albumin samples by X-ray imaging were used asstandards data for O analyses by X-ray imaging. In effectthis procedure replaced the standards values obtained byselected area analyses with values obtained by X-rayimaging, i.e. the conditions of standard analysis matchedthe conditions of sample analysis.

Since no morphological detail was apparent in the planedand coated biological samples, an initial O image was madeat a magnification of 200×, a resolution of 64 × 64 pixelsand a dwell time of 0·3 s. The total time required to producean image was < 30 min. The concentration of oxygen infrozen-hydrated samples is usually very high; therefore, alow beam current (0·5 × 10¹10 A) was sufficient to generatea significant count rate for O X-rays. This image providedsufficient morphological detail (Fig. 5) to facilitate selectionof an area for analysis. Selection was carried out bycomparing a secondary (SE) or backscattered (BE) electronimage of the surface of the sample with the oxygen image.Although the surface of a planed sample is featureless therewere usually a number of very small contaminationparticles thinly dispersed over the surface. These particlesmay be due to frost or to debris from the planing process;however, they served as convenient markers for relatingmorphological structure observed in the O image tolocations in the SE or BE image. The correlation was aidedby use of a computer-generated grid overlain on the imagesand by recording the images on a video printer. Regions forselected area analyses, primarily for obtaining the matrixcorrection factors, were also selected in this manner.

Regions of Malpighian tubule samples selected fordetailed analysis were imaged at a resolution of 64 × 64

pixels with a dwell time of 1 s and a beam current of1 × 10¹10 A for light element analyses, principally O, and adwell time of 6 s and a beam current of 1 × 10¹9 A forhigher atomic number elements. Light element X-rayimages were obtained using a UTW and required < 1–2 h(Fig. 6A,B) whereas X-ray images of other elements wereobtained using a Be window and required < 10–12 h(Fig. 6C–F). Similar conditions were used for obtainingimages of soybean root nodules except that some imageswere obtained at a resolution of 128 × 128 pixels and adwell time of 3 s (Fig. 7).

The initial X-ray images obtained in this way wereapparent concentration images and were processed usingthe matrix corrections obtained from static beam (selectedarea) analyses to obtain corrected concentration images.Concentrations in selected regions were averaged andextracted using the Link program Featurescan. Binarymasks were made for the single cell epithelia of theMalpighian tubules by circumscribing the S-containingregions of the tubules from the S image. In the case of thelumen the O image was used for this purpose. These maskswere then applied to all the element X-ray images to obtainmean concentrations per tubule from the averaged pixelintensities. In the case of Na, images were first processedwith a 1·5× SD image to set all pixels below this value tozero. Oxygen concentration images were converted to waterconcentration images by applying a conversion factorderived from the calculated relationship between oxygenconcentration and protein concentration in solution.Selected areas of individual tubules were also analysed,after transfer of images to a PC, by the NIH program Image.

Although processing images, containing very low X-raycounts, with SD images improves the statistical probabilitythat the remaining counts are genuine X-ray events for aparticular element, inevitably very low genuine X-raycounts must be removed. An Na image that is not processedwith an SD image may be more accurate, but less precise,than one that is processed, if the element in question isknown to be present. A comparison of the two methods isshown in Table 2 for five tubules from one insect.

Fig. 6. X-ray images (64 × 64 pixels) from planed samples of frozen-hydrated Malpighian tubules. (A) Quantitative O image showing a low Oconcentration in the tubule cells and a high O concentration in the tubule lumina. The epithelial cell layer of the tubule profiles, tubulelumina and surrounding saline medium are readily identified. The thermal colour scale represents the O concentration in percentage wetweight. The range of the scale exceeds 100% primarily because a few pixels from surface frost contamination give very high O counts.(B) Qualitative C image indicating the distribution of dry mass. The image is based on a thermal colour scale in which black representslow X-ray intensity and white represents high X-ray intensity. (C) Quantitative K image, showing that K concentration is higher in thelumen than in the tubule cell cytoplasm and the surrounding saline medium. The thermal colour scale represents the K concentration inmmol kg¹1 wet weight. (D) Quantitative Na image, after processing with a standard deviation image, shows high Na concentration in thesaline medium and low cellular and luminal concentrations. The thermal colour scale represents the Na concentration in mmol kg¹1 wetweight. (E) Quantitative Cl image, showing high concentrations in the lumen and saline medium and a low cellular Cl concentration.The thermal colour scale represents the Cl concentration in mmol kg¹1 wet weight. (F) Qualitative Ca image showing the distribution of cel-lular inclusions containing calcium phosphate. The image is based on a thermal colour scale in which black represents low X-ray intensityand white represents high X-ray intensity.



An example of this type of analysis applied to images ofMalpighian tubules is shown in Fig. 8 in which thecytoplasmic and luminal concentrations of Na, Cl and Kin control and ouabain-treated tubules are compared. It is

clear that within both the cytoplasm and the lumen thetreatment with oubain resulted in a significant (P<0·05)increase in Na concentration (5·2–19·4 and 10·5–33·2 mmol kg¹1 wet weight, respectively) and a significant

Fig. 7. X-ray images (128 × 128 pixels) from planed frozen-hydrated soybean root nodule. (A) Qualitative O image showing the inner cortex(IC), infected cells (IFC) and sclereid cells (SC). (B) Qualitative C image showing the high C content of the sclereid cell walls. The O and Cimages are based on a thermal colour scale in which black represents low X-ray intensity and white represents high X-ray intensity. (C) Quan-titative K image showing high K concentrations in the inner cortex cells and in an unidentified cell type associated with the sclereid cells. Thethermal colour scale represents the K concentration in mmol kg¹1 wet weight. (D) Quantitative Ca image. High Ca concentrations appear tobe associated with the uninfected cells surrounding the infected cells. The thermal colour scale represents the Ca concentration in mmol kg¹1

wet weight.



(P <0·05) decrease in K concentration (153·2–80·5 and171·6–113·9 mmol kg¹1, respectively). Oubain treatmentalso resulted in an increase in cytoplasmic O concentrationwhich translated into an increase in water concentration(47–62 wt%) (Fig. 9). The presence of calcium-containinggranules or spherites in the cells (Fig. 6D) was responsiblefor the low average water content. The analysis of selectedregions free of spherites in individual control tubules, usingImage, gave a water concentration of 68 6 0·75% (n ¼ 3).

An analysis of soybean root nodule cells using Imageshows the presence of a K concentration gradient across thenodule (Table 3). Potassium concentration was lowest in theuninfected cells and increased towards the outer cortex. Therange of K concentration was highest in the sclereid cells,varying from 139 to 807 mmol kg¹1 wet weight.

Discussion

It is shown that quantitative X-ray imaging of planedfrozen-hydrated biological samples is possible and representsa better alternative to the static beam analysis of fracturedfrozen-hydrated samples (Marshall, 1980; Echlin & Taylor,1986). Qualitative light element images (O,C) are adequatefor resolving morphological detail in planed frozen-hydratedsamples. The gross morphology of the Malpighian tubulesamples was easily discerned and individual cells, cell wallsand nuclei were resolved in the soybean root nodules. Theseimages were obtained in a reasonably short time andobviate the need for obtaining electron images from etchedsamples. The etching or sublimation of ice from planedsurfaces of frozen-hydrated samples cannot be well con-trolled and compromises the analyses (Marshall, 1981).Although the etching procedure may be acceptable for someinvestigations it should be avoided if possible.


Fig. 8. Comparison of element concentrations in the cells (Cyto)and lumina (Lumen) of control and ouabain-treated Malpighiantubules. Mean 6 SE, N1 (insects) ¼ 3, N2 ¼ 12,13 (tubules), N3

(Cyto pixels) ¼ 4440, 4568, N4 (Lumen pixels) ¼ 1155, 987,*P<0·05. The mean concentrations are derived from averages oftubule concentrations in each insect. The total number of imagepixels represents the number of pixels averaged in regions of tubulecytoplasm and lumina. These can be interpreted as the total num-ber of individual analyses that produce the final means.

Fig. 9. Comparison of water concentration in cell cytoplasm andlumen of control and ouabain-treated Malpighian tubules. Mean6 SE, N as in Fig. 8. *P<0·05.

Table 3. Distribution of K across the soy bean root nodule.

K concentrationCell type (mmol kg¹1 wet weight)

Infected cells (5) 200 6 4Uninfected cells (5) 117 6 9Inner cortical cells (7) 346 6 17Sclereid cells (7) 336 6 82Outer cortex cells (5) 390 6 19

Mean 6 SE, (n).

Table 2. Comparison of Na concentrations (mmol kg¹1 wet weight)from control Malpighian tubules after image processing with andwithout SD images.

Lumen Cells

SD 4·5 6 1·2 7·1 6 0·5No SD 22·4 6 2·3 26·4 6 1·5

Mean 6 SE, n ¼ 5.


The advantages and validity of using nonmatchingstandards and phi-rho-zed matrix corrections for theanalysis of frozen-hydrated biological samples have beendiscussed previously (Marshall, 1982). The validity ofsimilar methods using organic standards for the quantita-tive analysis of light elements by static beam analysis inorganic and biological samples has also been established(Marshall & Patak, 1993; Marshall, 1994). The presentwork indicates that these methods can be extended toquantitative X-ray imaging of frozen-hydrated biologicalsamples.

Selected area analyses (rasters) and X-ray images wereobtained from albumin samples containing salts. The dataindicate that averaging concentrations from pixels withsmall numbers of counts provides values comparable tothose obtained from spectra accumulated over a muchlonger time and containing many more counts than thespectra from each pixel. This is consistent with the findingsof Fiori et al. (1988) and LeFurgey et al. (1992) that smallnumbers of counts per pixel give valid quantitative datawhen averaged or summed over regions. An advantage ofX-ray imaging is that the sample experiences considerablyless beam damage. In the case of the albumin samples a32 × 32 pixel image with a dwell time of 6 s is equivalent toa static beam analysis of 102 min but with much reducedradiation damage compared to a 100 s selected areaanalysis because the beam spends far less time on any onelocality. The disadvantage of long acquisition times for X-rayimages could be considerably reduced by using thetechnique of ‘programmed beam acquisition’ described byIngram et al. (1994) where the dwell time in any region ofarbitrary shape can be set according to the statistcs ofacquisition.

The excellent fit for Na added to albumin, after processingNa images with standard deviation images, indicates thatthe amount of Na present in the dialysed albumin beforeaddition of Na, i.e. 34 mmol kg¹1, is close to the concentra-tion at which statistical noise becomes significant. If an SDimage is applied to an Na image then this concentrationlevel becomes the minimum detection limit. The standarddeviation is defined as n times the square root of the peakplus background integral, where the value of n isconservatively set at 3 and in this case relaxed to 1·5.

The precise estimation of Na concentrations is notpossible if an X-ray image contains pixels with Naconcentration values between zero and 34 mmol kg¹1. Inthis range true Na counts cannot be discriminated fromstatistical noise. The application of a standard deviationimage to set pixels of nonzero intensity, which are not morethan 1·5× SD, to zero will inevitably lead to underestimatesof Na concentration in the circumscribed region. Never-theless, accurate relative estimates of Na concentration insamples are still possible and the high precision of theestimates facilitates statistical analysis.

This is demonstrated in the results of Na analysis in theMalpighian tubules in which the control value for Naconcentration was 10·5 mmol kg¹1 and the value for theouabain-treated tubules was 33·2 mmol kg¹1. Both valuesare the result of averaging over a large number of pixels inwhich low values which could not be distinguished fromnoise had been set to zero. They are therefore under-estimates of the true values, and are below the minimumindicated detection limit, but nevertheless show that there isa significant difference between the Na concentrations inthe two groups of tubules. A more accurate, but less precise,estimate of the true Na concentration is obtained by usingimages that are not processed by SD images when regionscontaining low counts are averaged.

The analyses of Na in albumin indicated that the minimumdetection limit obtained from images without SD processing was11 mmol kg¹1 compared with 34 mmol kg¹1 wet weight fromimages after SD processing. Elements of higher atomicnumber than Na such as P, S, Cl and K were accuratelymeasured in albumin models and the values indicate thatthe minimum detection limits for these elements are lessthan 5 mmol kg¹1 wet weight.

The application of phi-rho-zed corrections to X-rayimages of Na and higher atomic number elements is anacceptable procedure. This is because variations in drymass, or protein concentration, and in the concentration ofthe elements themselves (within the usual biological range)make little difference to the individual values of the phi-rho-zed corrections (Marshall, 1982). In the case of O,however, the corrections are strongly dependent on proteinconcentration. For example, a 5% difference in proteinconcentration from an expected value of 20% will result inan error in the calculated O concentration of < 8%. Theanalytical resolution of bulk sample analysis at 15 kV isabout 2 mm for both depth and lateral resolution (Marshall& Condron, 1985b) for static beam analyses and thesedimensions would be included in one pixel in a 64 × 64 pixelimage at a magnification of 400×. It is unlikely that theaverage protein or dry mass fraction included in such alarge analysed volume would change greatly from one partof a cell to another. This is, however, a possible source oferror. A fully corrected analysis at each pixel would over-come this difficulty but this procedure is not supported bythe currently available software. A possible alternative is tocarry out O analyses at low voltage, e.g. 5 kV. At this voltagethe corrections are considerably smaller than at 15 kV;however, the depth resolution for O would no longer besimilar to that of the higher atomic number elements(Marshall, 1982).

The present method was adequate for demonstrating asignificant increase in cytoplasmic water concentration inMalpighian tubules after ouabain treatment. The measuredwater content of the Malpighian tubule cells is lower thanthat normally expected in cells owing to the presence of



large numbers of calcium phosphate inclusions or spherites.Analyses of regions of tubules relatively free of spheritesshowed that the water content (68%) was higher than theaverage water content (47%) in control tubules. Previousestimates of average water content in frozen-hydrated bulksamples of Malpighian tubules (Marshall, 1994) werehigher. This was due to the failure to recognize that thestandards data, obtained from aminoplastic resin, for Oresulted in overestimates of O concentration when used toanalyse matrices with a very high O concentration. Asecondary reason for the higher average water concentra-tions is that the previous estimates included the microvillarregion whilst the present estimates did not do so.

The biological significance of the results obtained here onMalpighian tubules (Xu and Marshall, in preparation) andsoy bean root nodules (Brown and Marshall, in preparation)will be discussed elsewhere. The cytoplasmic values for Na,Cl and K in Teleogryllus Malpighian tubules are consistentwith measurements in Malpighian tubules from Locusta, arelated insect species (Pivovarova et al., 1994), although, aspreviously argued, our Na concentrations are under-estimates of the true values. Changes in intracellular Naand K in response to ouabain treatment are in the directionexpected if inhibition of cellular Naþ Kþ ATPase occurs.Inspection of Fig. 8 shows that there is an apparentimbalance of positive (Naþ þ Kþ) and negative (Cl¹) ionsafter ouabain treatment. This is due to the underestimatedNa concentration resulting from SD image processing. Theconcentrations of Cl and K in the1 luminal fluids areconsistent with microdroplet analyses of secreted fluids(Marshall et al., 1993) although the Na values are againunderestimates of the true values (Table 4). If Naconcentrations are obtained from Na images that have notbeen processed by SD images then the comparison with themicrodroplet analyses is improved (Table 4). This lattercomparison provides good evidence of the validity of thetechnique.

No quantitative X-ray analyses of soybean root noduleshave been previously attempted. There are, however, tworeports of qualitative analyses (Thomas & Minchin, 1992;Minchin et al., 1994). These showed the presence of a Kgradient across the nodule and high levels of Ca inuninfected cells. The quantitative elemental images shownhere confirm these findings but also show that there was amarked variation in K concentration in the cells of both theinner and the outer cortex. There was also a considerablevariation in K concentration in the sclereid cells, whereasthe previous investigations showed that the highest ‘counts’for K were found in these cells. An unidentified cell typeassociated with the sclereid cells had very high concentra-tions of K in the cell wall, cytoplasm and vacuole.

In conclusion, it is demonstrated that quantitative X-rayimaging of planed frozen-hydrated samples is feasible andproduces accurate results. Using this method of analysis it isunnecessary to obtain an electron image of the sample andtherefore it is unneccesary to either fracture the sample orto etch a planed sample. This results in a simpler and moreaccurate analytical procedure.

Acknowledgments

We thank L. Dalton, A. Wright and D. Fernando for skilledassistance with preparation procedures. This work wascarried out with the aid of a grant from the AustralianResearch Council.

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Table 4. Comparison of analyses of Malpighian tubule luminalfluids in control tubules by in situ analysis in frozen-hydratedtubules and by analysis of microdroplets of fluid secreted bytubules. Data for microdroplets are from Marshall et al. (1993). Naimages were processed with (SD) and without (No SD) images.

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Quantitative elemental X-ray imaging of frozen-hydrated biological samples

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