MICROINCINERATION, ELECTRON MICROSCOPY,

AND ELECTRON DIFFRACTION

OF CALCIUM PHOSPHATE-LOADED MITOCHONDRIA

RICHARD S. THOMAS and JOHN W. GREENAWALT

From the Western Regional Research Laboratory, Agricultural Research Service, United StatesDepartment of Agriculture, Albany, California 94710, and the Department of Physiological Chem-istry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT

Isolated rat liver mitochondria were incubated in vitro under conditions supporting themassive accumulation of calcium and phosphate. Samples were embedded, thin sectioned,and examined in the electron microscope. The intramitochondrial distribution of insolubleor structure-bound mineral substances was studied by electron microscopy coupled withrecently developed techniques of high resolution microincineration. As shown previously,the ion-loaded mitochondria acquire large, internal granules which have inherent electronopacity indicative of high mineral content. Study of ash patterns in preselected areas ofsections directly confirmed the high mineral content of the granules, and the appearanceof the residues was consistent with the copresence in the granules of some organic material.Other mitochondrial structures were almost devoid of mineral. Thin sections of unincubatedcontrol mitochondria also were incinerated. They were found to contain appreciableamounts of intrinsic mineral, seemingly associated with membranes. The normal, densematrix granules commonly seen in unaltered mitochondria could be seen in intact sectionsof these control preparations, but after burning no definite correspondence of any ash tothe granules could be demonstrated. The normal granules perhaps do not contain mineral.Heating experiments on ash patterns of all the preparations demonstrated the thermalstability and crystallizability of the ash. The crystallized ash of the in vitro-produced densegranules was tentatively shown by electron diffraction to be 3-tricalcium phosphate (whit-lockite). This, together with evidence from the literature, suggests that the original, non-crystalline mineral may be a colloidal, subcrystalline precursor of calcium-deficient hydroxy-apatite. Experiments were performed on synthetic calcium phosphates for comparison.Other possible applications of the microincineration techniques are briefly discussed.

INTRODUCTION

Isolated mitochondria incubated in vitro in a mitochondria, often in association with the innersuitable ionic medium can utilize either ATP or an membranes (Greenawalt et al., 1964; Brierley andoxidizable substrate to actively accumulate large Slautterback, 1964).amounts of calcium and inorganic phosphate It has seemed likely, a priori, that the granules(Pullman and Schatz, 1967; Lehninger, 1966). must consist largely of some form of precipitatedConcomitantly with the massive accumulation of calcium phosphate, since (a) the calculated inter-ions, large opaque granules are formed inside the nal concentration of calcium and phosphate ions

55

in the mitochondria after maximal uptake greatlyexceeds the solubility product for calcium andphosphate, and (b) the electron-opacity of thegranules does not depend upon heavy metal stain-ing but is intrinsic. Mitochondria heavily loadedwith the granules accumulate calcium and phos-phate with an average molar ratio of about 1.67:1under the conditions initially described by Vasing-ton and Murphy (1962). This has suggested thatthe mineral of granules might be hydroxyapatite(Rossi and Lehninger, 1963 a, b). However, noevidence of crystallinity was detected in the gran-ules in situ (Greenawalt et al., 1964) and so theexact nature of the mineral has not been deter-minable by diffraction analysis.

In the present investigation, we have examinedthe nature of the granules by applying recentlydeveloped techniques of high resolution micro-incineration (Thomas, 1964, 1968) to the granule-loaded mitochondria. These procedures, carriedout on thin sections on electron microscope grids,burn away all organic material in the specimenbut leave in situ the inorganic constituents. Theliberated mineral residues can be visually com-pared with the original, intact structures, and canalso be subjected to further chemical or physicaltreatments. In this way it has been possible to (a)confirm directly the high mineral content of thegranules in contrast to that of other structures inthe mitochondrial sections; and (b) induce themineral residues of the granules to crystallize andtentatively identify the crystals by electron diffrac-tion and comparison with the properties of knownminerals.

Microincineration experiments were also per-formed on control mitochondria not loaded invitro with calcium and phosphate. These studiesrevealed residual deposits of intrinsic, boundmineral, possibly associated with the membranesof the organelles. They also provided new informa-tion on the chemical nature of the dense, matrixgranules commonly seen in normal, unmodifiedmitochondria and regarded by some investigatorsas sites for physiological deposition of minerals invivo (Weiss, 1955; Peachey, 1964; Bruni andPorter, 1965).

Preliminary accounts of the present work werereported earlier (Thomas and Greenawalt, 1964;Greenawalt and Carafoli, 1966). The latter reportdescribes application of the microincinerationtechnique to strontium phosphate-loaded mito-chondria but also gives further details of results

with calcium phosphate-loaded organelles. Sincethese reports appeared, calcium phosphate gran-ules produced in vitro have been isolated frommitochondria and subjected to chemical and physi-cal analysis in bulk (Weinbach and von Brand,1965, 1967). This latter study is in essential agree-ment with our findings on the granules. This is re-assuring for both investigations since the possiblesources of artifact in the two approaches are ratherdifferent.

MATERIALS AND METHODS

Mitochondrial Isolation and

Ion Accumulation

Mitochondria were isolated from the livers ofCarworth Farms (New City, N. Y.) albino rats(Wistar strain) in 0.25 M sucrose by the method ofSchneider (1957) and were then washed three timeswith 0.25 M sucrose.

The washed mitochondria were incubated in themedium described by Vasington and Murphy (1962)for the substrate-supported accumulation of Ca++and Pi. The complete reaction mixture contained80 mM NaCl; 4 mM sodium phosphate, pH 7.0; 10mM Tris maleate (or Tris-chloride), pH 7.0; 10 mMsuccinate; 10 mM MgC12; 4 mM CaC12 (labeled with45Ca); 3 mM ATP; and 3-5 mg of mitochondrialprotein. The total volume was 3.0 ml. Incubationwas for 20 min at 300. Controls consisted of prepara-tions incubated in the complete medium minusCa+ + , in the complete medium minus ATP, or inthe complete medium but removed from incubationat zero time. The incubation was terminated byquickly chilling the media to OOC and centrifugingthe mitochondria at 20,000 g for 4 min. The pelletwas washed once with 0.25 M sucrose.

The accumulation of Ca++ was determined bymeasuring the amount of isotope which disappearedfrom the media by plating and counting aliquotsfollowing sedimentation of the mitochondria. In theexperiments described here, over 90% of the addedCa++ was accumulated. Phosphate uptake was notmeasured but it has been shown that the ratio ofCa++/Pi taken up under these conditions rangesfrom about 1.52 to 1.88 with an average value ofabout 1.67 (Rossi and Lehninger, 1963 a, b).

Preparation of Specimens for Electron

Microscopy and Microincineration

Samples were taken from the pellets of control and"loaded" mitochondria and fixed with 10(% aqueousformaldehyde or with 1% OsO4 in Veronal-acetatebuffer, pH 7.4, for 1 hr at O0C. Samples were takenfrom the top, middle, and bottom regions of each

56 THE JOURNAL OF CELL BIOLOGY VOLUME 39, 1968

pellet; no major differences were observed in themitochondria from these different regions. Mito-chondria were dehydrated by rapid passage througha cold (-10°C) ethanol series and embedded inEpon 812 according to the procedure of Luft (1961).

All of the embedded preparations, both osmium-fixed and formalin-fixed, were routinely thin-sec-tioned and examined in the electron microscope toconfirm that their morphology was similar to thatpreviously reported (Greenawalt et al., 1964). How-ever, for the microincineration study reported here,only one of the controls, the unincubated "zerotime" preparation, was utilized, and most experi-ments were confined to formaldehyde-fixed materialso possible artifacts could be avoided in the ashpatterns which might arise from the introduction ofosmium. In experiments on the "normal" matrixgranules of control mitochondria, it was necessary touse osmium-fixed material, since the granules are notseen in formaldehyde-fixed organelles (see below inResults).

Embedded preparations for microincinerationexperiments were thin-sectioned at about 50-90 m/,or thick-sectioned at 250-500 m, with a diamondknife and a Porter Blum MT-I microtome. Thinsections were necessary to obtain good preservationof structural detail in the ash patterns but thicksections were advantageous for electron-diffractionexperiments since they yielded a greater amount ofash per unit area which could contribute to a diffrac-tion pattern (Thomas, 1968). To avoid the leachingof dense granules from the mitochondria which canoccur on the surface of slightly acidic distilled water,we collected the sections from the knife edge on adilute volatile buffer, 0.001 M ammonium bicarbon-ate, pH 7.5 (Thomas, 1968; Boothroyd, 1964). Thesections were picked up on 200-mesh stainless steelgrids bearing a thin collodion membrane on whichhad been deposited a film of silicon monoxide about200 A thick (Thomas, 1968). The SiO film provideda heat- and oxidation-resistant supporting surface forthe sections. The collodion on the backside impartedadditional mechanical stability to the film duringmanipulation of the grids and was subsequentlyburned away during incineration. It was not necessaryto remove the embedding medium from the sections;this also burned away easily during incineration.

It was directly demonstrated by viewing in theelectron microscope that the total procedures used

for preparation of mitochondria for microincineration

were adequate for the retention of the mineral in thein vitro-acquired dense granules. However, the pro-

cedures almost certainly did not retain all of the

original mineral content of the organelles, and ingeneral, only inherently insoluble minerals or min-

erals bound to insoluble structures were maintained

in place. It was necessary to keep this in mind when

interpreting ash patterns of the mitochondrial speci-mens.

Samples of synthetic calcium phosphates were alsoprepared for electron microscopy for comparisonwith the mitochondrial specimens. The mineralpowder was mixed with 1.5% (w/v) collodion inamyl acetate to form a thick slurry, and a drop of thismixture was allowed to spread on a water surface,forming a film enclosing the dispersed crystals (Brad-ley, 1961). Stainless steel grids (200 mesh) bearingsilicon monoxide-collodion membranes were placedmembrane side down on the upper surface of thecrystal-containing film, and the grids with the filmwere picked up in the usual way and allowed to dry.Alternatively, the mineral powder was mixed withmethyl-butyl methacrylate (inhibitor-free) and cen-trifuged into a pellet, and the pellet was polymerizedovernight under an ultraviolet lamp. Thin sections(50-100 mpu) were cut with a diamond knife ontoammonium bicarbonate buffer and picked up ongrids filmed with silicon monoxide-collodion orcarbon-collodion. The methacrylate was removedfrom the sections by brief immersion of the grids inbenzene.

Microincineration Techniques

The techniques for high resolution microincinera-tion are presented in detail elsewhere (Thomas,1968), and are only briefly described here. Theorganic material of the mounted thin sections wasburned away in one of two ways: (a) by heating thegrids in air to high temperature either in a mufflefurnace or on a special heating stage in a vacuumevaporator (Thomas, 1964); (b) by exposing thegrids to a gaseous plasma of electrically excited oxygenin a modified Tracerlab model LTA 500 low tem-perature asher (Tracerlab Div. of Laboratory forElectronics, Inc., Richmond, Calif.) (Gleit, 1963).The latter technique permits complete combustion ofthe specimen at a temperature below 100°C andminimizes or eliminates the melting and/or volatiliza-tion of minerals and mineral-organic complexeswhich can occur at high temperatures.

For high temperature ashing the grids were heatedto either 500 ° or 600°C for 30 min. Previous experi-ence has shown that this is more than adequate tocompletely combust the specimens. The higher temp-erature was used when it was desired to view thesame area of the specimen in the electron microscopeboth before and after burning. A thin film of con-tamination from the electron beam persisted afterburning at the lower temperature even though theorganic material of the section under the film wasobviously burned away completely.

The low temperature asher, model LTA 500(single sample version) was modified for continuousrather than stepwise adjustment of the power input

RICHARD S. ToMAS AND JOIIN W. (GIEENAWVALT Calcium Phosphate-Loaded Mitochondria 57

Figs. 1-4, thin-sectioned pellet of formaldehyde-fixed, calcium phosphate loaded mitochondria before andafter incineration for 30 min at 600C.

FIGURE 1 Before incineration. The dense granules are easily seen and the mitochondrial profiles whichcontain them are also visible, but with very low contrast. The edge of the section is seen at the top.X 12,000.

FIGURE Same field as Fig. 1, but after incineration Except for a line (presumably contamination)tracing the edge of the section, nothing remains but residues of the dense granules. Lightly shadowed.X 12,000.

FIGURE 3 Before incineration. Area selected from Fig. 1, enlarged to show granules in more detail.X 29,000.

FIGURE 4 Same field as Fig. 3, but after incineration. The residues correspond exactly to the densegranules and only to the granules. The larger residues contain a bubble, however, indicating melting andsome decomposition of the granules. Lightly shadowed. X29,000.

58 THE JOURNAL OF CELL BIOLOGY VOLUME 39, 1968

and also for monitoring of the input (by reading theplate current of the final stage amplifier.) Specimenswere usually incinerated for 30 min at 30 ma.(Highest power input reads 300 ma.) Again, previousexperience has shown this to be more than adequatefor total combustion of the thin electron microscopespecimens. Complete combustion could be confirmed,however, by heating the low temperature-ashedspecimen to 600C and noting no change (see belowin Results). The low temperature asher did notcompletely burn away electron-beam contaminationfilms from previewed specimens, and so its use in con-nection with before-and-after viewing experimentswas not satisfactory.

Specimens were usually shadowed with uraniumimmediately after incineration, unless they weredestined for electron-diffraction examination. Theshadowing angle was nominally tan' ½/, but undula-tions in the support film introduced some uncertaintyin the local shadowing angles. Shadowing appliedbefore exposure of the ash to the moist air of thelaboratory served to monitor the possibility ofhygroscopy in the ash (Thomas, 1968). None of theash patterns in the present experiments showed anyevidence of hygroscopy.

Electron Microscopy, Electron Diffraction,

and X-ray Diffraction

Electron microscopic observations were made witha Hitachi HU-10 with a 35 u objective aperture at50 kv. Micrographs were taken at magnificationsranging from 2700 to 11,000. Higher magnificationswere usually not warranted since very high resolutionwas limited by the relatively thick support films.Special procedures for mapping specimen grids forbefore-and-after-incineration viewing experimentsare described elsewhere (Thomas, 1968). Dark-fieldobservations were made by decentering the objectiveaperture.

Selected area electron diffraction observations(Bigelow, 1960) were made at 50 kv, with a magnifica-

tion of 11,000 and a rectangular (or square) field-limiting aperture which usually included about20 2. The mitochondria tended to occur in clustersin the section and the 20 tz2 area was usually aboutoptimum to include a single cluster but excluderelatively empty support film which contributedunduly to background scattering. The ratio of Braggdiffraction/background scattering was in all cases

Figs. 5-7, effect of incineration temperature on fine structure of dense granule residues. Sections offormaldehyde-fixed, loaded mitochondria. X63,000.

FIGURE 5 Untreated thin section (similar to that of Figs. 1 and 3). The native granules show a some-what diffuse electron density; they apparently are aggregates of very fine mineral grains, 60 A or less indiameter, embedded in some sort of organic matrix.

FIGURE 6 Section, somewhat thicker than in Fig. 5, after incineration for 30 min at 500 0C. The mineralresidues of the granules show a fine porosity, which seems to correspond to the diffuse density of the nativegranules.

FIGURE 7 Section same thickness as for Fig. 6, after incineration for 30 min at 600°C. The granuleresidues are bubble-like. Apparently the granule mass fuses at this higher incineration temperature andbubbles are formed as the organic component vaporizes.

RICHARD S. TIIOMAS AND JOHN W. GREENAWALT Calcium Phosphate-Loaded Mitochondria 59

low, but varying the exposure of the plates caused thecontrast between the two components to be maxi-mized at different radii of the powder pattern. Thus,although no one plate showed all of the diffractionarcs to best advantage, a fairly complete assemblageof arcs could be observed from a collection of plates.The powder patterns were calibrated for determina-tion of dhkl values by reference to the diffractionfrom silicon monoxide membranes bearing vacuum-evaporated gold. Measurements of diffraction ringsand arcs and visual estimations of intensities weremade on the original electron microscope plates. Forpublication, plates were printed through appropriatecontrast-cancellation masks (out-of-focus positives inregister with the negative plates) for suppression ofbackground and enhancement of the visibility of thecrystalline diffraction spots (Gonzales, 1962).

X-ray diffraction powder photographs of calciumphosphate samples were obtained with a cylindricalcamera of a radius 7.184 cm with copper K, radia-tion, X = 1.5418 A. Measurements of line spacingsand visual estimates of intensities were made on thefilms.

RESULTS

Microincineration of Loaded Mitochondria

HIGH TEMPERATURE INCINERATION-EX-

AMINATION OF THE SAME SECTION BEFORE

AND AFTER ASHING: Figs. I and 3 show a

thin section of the formaldehyde-fixed, calciumphosphate-loaded mitochondria, before incinera-tion. In the absence of heavy metal staining, littleof the membrane structure of the organelles can be

seen, but the granules stand out clearly owing totheir intrinsic density. After incineration for 30min at 600C (Figs. 2 and 4) an ash residue re-mains precisely at the site of each granule but allother substance, of both the mitochondria and theembedding, is essentially missing. The association

of ash with the granules directly confirms the highmineral content of the latter, and the absence ofeverything else in the burned section produceswhat is evidently a rather clean preparation of thisparticular ash.

DECOMPOSITION OF THE GRANULES-THE

EFFECT OF TEMPERATURE: Although themineral of the granules is retained in place, manyof the granules, particularly the larger ones, ap-pear somewhat altered by the 600°C treatment;they have a "bubble-like" aspect. Fig. 7, which

shows a thicker section containing more large

granules, demonstrates this strikingly. This evi-

dence of some decomposition is not surprising. As

reported previously (Greenawalt et al., 1964) andas shown in Fig. 5, high resolution examinationof the granules before incineration indicates thatthey are not solid masses of mineral but rather acomplex of very fine dense particles, 60 A or lessin diameter, which seem to be held together insome sort of low density matrix, presumably or-ganic in nature. The presence of the organicmatrix has been confirmed in isolated granules

(Weinbach and von Brand, 1965, 1967). Appar-ently the mineral-organic mass fuses at the high

temperature, and bubbles are formed as the or-ganic material vaporizes.

If microincineration is carried out at 5000Crather than 600C this fusion of the granule mass

does not occur, or is greatly minimized. Fig. 6shows the result of 30 min treatment at this lower

temperature. The mineral residues of the granulesnow have a flocculent, porous appearance which isnot unlike that of the mineral in native granules.Apparently the organic matter is burned out atthis lower temperature without greatly disturbing

the inorganic structure.Except for the improved preservation of granule

fine structure, microincineration at 5000 C gives

results similar to 600C burning. As shown inFig. 8, nothing remains of the mitochondrialsections after 5000 C but the residues of thegranules.

LOW TEMPERATURE (LTA) INCINERA-

TION-NONGRANULE ASH: Incineration of

thin sections of the loaded mitochondria for 30min at 30 ma in the low temperature asher resultsin porous granule residues which are quite similarin appearance to those of the 5000 C treatment(Fig. 6). When one views the incinerated sections

over-all, however, there is a difference. Finestrands or granules of ash other than the densegranule residues are seen scattered here and there.In favorable areas, as shown in Fig. 9, this ashis sufficiently well organized to form a just recog-nizable pattern. It evidently corresponds to mito-

chondrial cross-sections and must represent traceamounts of mineral, other than the dense granules,remaining in the organelles. This mineral is evi-dently volatilized during high temperature ashing(500 ° 600C).

Microincineration of Control Mitochondria

HIGH TEMPERATURE ASHING OF FORMA-

LIN-FIXED AND OSMIUM-FIXED MATERIAL-

THE FATE OF NORMAL DENSE GRANULES:

60 THE JOURNAL OF CELL BIOLOGY VOLUME 9, 1968

Figs. 8 and 9, comparison of 500°C incineration and low-temperature ashing (LTA) on thin sections offormaldehyde-fixed, loaded mitochondria. Preparations shadowed, and micrographs printed with reversecontrast. X 27,000.

FIGURE 8 A section incinerated 30 min at 5000C. Only the mineral residues of the dense granules re-main.

FIGURE 9 A section incinerated in the low temperature asher for 30 min at 30 ma. The granule residuesappear similar to those from 500°C ashing. In addition, a slight ash remains from the rest of the mito-chondrion and delineates its profile in favorable cases.

A typical thin section of formaldehyde-fixed unin-cubated control mitochondria is seen before ashingin Fig. 10. The organdies appear somewhatsmaller and denser on the average than their incu-bated counterparts (see Figs. 1 and 3), which areknown to swell during incubation. As would beexpected, the control mitochondria contain noneof the in vitro-produced dense granules, and in theabsence of heavy metal staining, they display littleevidence of internal structure. The "normal,"dense, matrix granules seen in osmium-fixed mito-chondria (see below in this section) are not ob-served. This agrees with previously reporteddescriptions of aldehyde-fixed mitochondria (Saba-tini et al., 1963; Andre and Marinozzi, 1965).Whether the granules are not present or aresimply invisible in this preparation is not known,

but the former seems most likely (Ashworth et al.,1966).

After 30 min at 5000 or 600°C, sections of thecontrol mitochondria are volatilized completelyand leave no ash residue. They thus behave simi-larly to the loaded mitochondria exclusive of theirdense granules. Their complete volatilization is inagreement with results of classical microincinera-tion studies (Kruszynski, 1966, pages 125 and 129).

So that some sure indication of the fate of thenormal dense matrix granules during incinerationcould be gained, thin sections of control mito-chondria fixed with osmium also were ashed. Be-fore incineration (Fig. 11) the normal granules areclearly visible in this preparation, in contrast toformalin-fixed material. The same preparation,same field of view, after 30 min at 600C is seen in

RIcHARD S. THOMAS AND JOHN W. GREENAWALT Calcium Phosphate-Loaded Mitochondria 61

FIGURE 10 Thin-sectioned pellet of fornmaldehyde-fixed, control mitochondria. The organelles are some-what denser and more distinct in appearance than similarly fixed loaded initochondria. Intracrystatespaces can he recognized but still no definite membrane structure is visible. In vitro-acquired densegranules are absent, of course, and, similarly, there is no evidence of normal dense matrix granules. Sectionsprayed with 0.264 polystyrene latex for focusing aid. X0,000.

Figs. 11 and 12, thin-sectioned pellet of osmium-fixed, control mitochondria before and after incinerationfor 30 min at 600C. Diatom fragments (SiO2) were sprayed on the backside of the grid to serve as perma-nent location markers. X34,000.

FIGURE 11 Before incineration. Membranes are clearly seen in the mitochondrial cross-sections, as arealso the normal dense granules. The latter, although distinct, are of considerably lower density than thein vitro-acquired granules of loaded mitochondria. In the lower right quadrant a granule is seen that issomewhat denser than the others and lies at the margin between two mitochondrial profiles rather thanwithin the matrix of either organelle. It is probably a contaminant particle, either in or on the section.

FIGURE 12 Same field as Fig. 11, but after incineration and shadowing. Nothing remains of the normaldense granules or, in fact, of the mitochondria. The only granule left in the field is the suspected con-taminant particle lying in the lower right quadrant.

62 THE JOURNAl. OF CELL BIOLOGY VOLUME 39, 1968

Fig. 12. Just as with formaldehyde-fixed materialafter 600C, nothing remains of the mitochondria.The osmium is evidently not retained anywhereand the normal dense granules leave no ash fromosmium or anything else.

LOW TEMPERATURE (LTA) INCINERATION---

MINERAL IN THE MEMBRANES (?): In con-

trast to preparations ashed at high temperature,an appreciable mineral residue does remain inLTA-incinerated sections of control mitochon-dria. Fig. 15 shows a thin section of formalde-hyde-fixed control mitochondria after 20 min at 10ma in the low temperature asher. These conditionswere evidently sufficient for complete ashing sinceidentical results were obtained from more strenu-ous treatments, e.g., 30 min at 30 ma. Cross-sections of individual mitochondria are easilyrecognized by their ash patterns. The patternsare somewhat sketchy, owing to fine scale migra-tion of the mineral to form grains (Thomas, 1968),but nevertheless the deposition of the ash definitelysuggests an origin from the membranes of themitochondria.

This interpretation is strengthened by anexamination of sections in various stages of theincineration process. Fig. 13 shows a typical initialpreparation, an unincinerated section which, inthis case, was shadowed. Some indication of in-ternal membrane structure is given by slight sur-face detail, apparently induced by local differencesin cutting properties during sectioning, but thesection surface is nevertheless relatively smooth andcontinuous. Fig. 14 shows a thin section after in-cineration for 5 min at 10 ma. The embeddingEpon and mitochondrial substance are partiallyetched away. The most resistant structures are seenemerging from the etched surface, and their or-ganization indicates fairly convincingly a kinshipboth with the membranes in intact mitochondriaand with the final ash pattern.

Osmium-fixed preparations also were LTA-incinerated. The ash patterns look essentially thesame as from formaldehyde-fixed material, e.g.,Fig. 15. The ash patterns contain no particleswhich are clearly identifiable as residues of thenormal, dense matrix granules. It should also bestated, however, that the general grain size of thepatterns is the same as the granules, and thusresidues of the granules, even if present, might beindistinguishable from the general inorganicresidues.

Heating Experiments on the Ash Patterns

HEATING OF THE LOW-TEMPERATURE

(LTA) ASH PATTERN TO 600 0c: For deter-

mination of the stability of the ash resulting

from low temperature LTA incineration and alsofor verification of the absence of organic material,thin sections of both loaded and control mito-chondria were incinerated in the low temperatureasher for 30 min at 30 ma, and then the resultantresidues were reincinerated in the furnace for 30min at 600C.

After this double treatment the specimens lookno different than after low temperature ashingalone. The residues of loaded mitochondria aresimilar to those shown in Fig. 9 and those of thecontrol mitochondria are as seen in Fig. 15. Fromthis it seems reasonable to conclude that (a) theLTA-ashed specimens are, in fact, free of organicmaterial; (b) the melting and bubble formationseen in dense granules initially incinerated at600C probably results from the presence of or-ganic material, the mineral itself being stable; (c)the absence of nongranule mineral in both loadedand control mitochondria seen after initial in-cineration at high temperature probably resultsfrom a volatile mineral-organic complex, since themineral alone is not volatile.

HEATING OF ASH PATTERNS TO HIGHER

TEMPERATURES-CRYSTALLIZATION OF THE

ASH AND ELECTRON DIFFRACTION: The

ash patterns resulting from both 500 ° and 600°Cburning as well as LTA incineration were routinelyexamined by dark-field electron microscopy andby electron diffraction for detection of crystallinityin the ash if such were present. Usually the ashgave no indication of crystallinity, and in the fewinstances when crystals were detected the presenceof contamination in the specimen was stronglysuspected. In the hope of inducing crystallinity,the ash patterns were heated for a few minutes tohigher temperatures, ranging from 6000 to 1000°Cand then allowed to cool slowly for severalminutes.

The results of these experiments are summarizedin Table I, and the appearance of some of the ashpatterns after heating is shown in Figs. 16-19. Asmay be seen, strong heating melts the ash of all thepreparations and does induce it to crystallize.In most cases, however, the diffraction spots arerelatively weak, and with one exception (groupG) the number of individual spots in the diffrac-tion pattern is insufficient to form recognizable arcs

RICHARD S. THOMAS AND JOHN W. GREENAWALT Calcium Phosphate-Loaded Mitochondria 63

Figs. 13-15, LTA incineration of thin sections of formaldehyde-fixed control mitochondria. Preparationsshadowed, and micrographs printed with reverse contrast.

FIGURE 13 Intact, unincinerated section, sprayed with 0.264 Ag polystyrene latex before shadowing. Themitochondrial profiles and some indications of internal membrane structure are seen by slight surface de-tail (apparently induced by local differences in cutting properties during sectioning), but the surface is rela-tively smooth. X 24,000.

FIGURE 14 A section after incomplete LTA incineration (5 min at 10 ma). The embedding plastic andmitochondrial substance have been partially, and differentially, etched away. The most resistant, emergentmaterial has a configuration strongly suggesting origin from mitochondrial membranes. The edge of thesection is seen at the upper right. X24,000.

which can be used for identification. The spotti-ness is due to an insufficient number of individual

crystals in the selected area field. A contributingfactor here is the tendency of the ash to fuse toform a relatively few, relatively large crystals,rather than to produce a fine dispersion of smallcrystals. This tendency is pronounced with treat-ment at the higher temperatures, and thus thebest diffraction patterns, at least in the case of theloaded mitochondria, are obtained at 700C. Therelative weakness of the spots is partly due torelatively high scattering from the necessarilythick support film, but may also result from a poordegree of crystallinity in most of the particles.

Figs. 18-20 show the bright- and dark-field

images and diffraction pattern of a 6000 C-in-

cinerated section of loaded mitochondria, subse-quently heated to 700C (experiment group G,Table I). The ash pattern consists solely of residuesof dense granules, which although somewhatmelted, are still recognizable. As shown by dark-field, most, if not all, of the residues are detectablycrystalline. Although somewhat weak and grainy,the diffraction pattern shows four fairly easilyrecognized arcs at relatively low angles, corre-sponding to crystal spacings (dhkl) of 5.15, 3.18,

2.86, and 2.57 A. All the arcs recognized in thispattern, as well as those in three other patterns of

64 THE JOURNAL OF CELL BIOLOGY VOLUME 39, 1968

FIGURE 15 A section after complete LTA incineration (20 min at 10 ma). The mitochondrial cross-sections each leave a distinct ash pattern. The patterns are somewhat more sketchy than those of theincompletely incinerated preparations but, still, they suggest origin from the mitochondrial membranes.X30,000.

similar preparations, are listed by dhkl values inTable II. As may be seen, the crystal spacings andthe roughly estimated relative intensities of thearcs agree fairly well with powder X-ray data for,-tricalcium phosphate otherwise known aswhitlockite. Owing to the weak and spotty charac-ter of the patterns, the measurements and particu-larly the judgements of relative intensity weredifficult and should be considered as subject tosome error. Nevertheless, the identification isprobably correct. There is no evidence for thepresence of a major amount of any other crystallinesubstance, and in particular, the prominent, com-plex line of hydroxyapatite at 2.78 A is not seen.However, small amounts of crystalline impuritiescould escape detection.

The diffraction patterns obtained from theheated ash of loaded mitochondria incinerated bythe LTA procedure (group E, Table I) were notof sufficient quality for measurement, but never-theless the patterns appear similar to those of the

high temperature ashed preparations (group G)recorded in Table II. It thus seems likely thatresidues of dense granules ashed by LTA are alsothermally convertible to whitlockite.

PREPARATIONS OF SYNTHETIC CALCIUM

PHOSPHATE-X-RAY DIFFRACTION, ELEC-

TRON MICROSCOPY, AND ELECTRON DIFFRAC-

TION: For comparison with patterns from mito-chondrial dense-granule ash, it seemed desirableto obtain diffraction powder patterns from samplesof genuine whitlockite. Whitlockite is of relativelyrare occurrence in nature (Frondel, 1943) but canbe produced in the laboratory by high temperaturedehydration of calcium-deficient synthetic hy-droxyapatite (Hodge et al., 1938). The calcium/phosphorus molar ratio of the preparation must beabout 1.5 to allow complete conversion to theanhydrous mineral (Hodge et al., 1938; Trautz,1955), and the conversion requires a temperatureof at least 600-800C (Kunin, 1958).

Multimilligram samples of a calcium-deficient

RICHARD S. THOMAS AND JOHN W. GREENAWALT Calcium Phosphate-Loaded Mitochondria 65

Figs. 16 and 17, heated ash patterns of LTA-incinerated thick sections of formaldehyde- fixed, controlmitochondria. (Examples from groups B and C, Table I.) X 17,000.

FIGUtE 16 Ash heated 2 min at 700°C and slowly cooled. The ash pattern still resembles more or less,that of an unheated preparation, but the individual strands of ash have begun to melt and from intodroplets. Except for an occasional small granule or strand, the ash is not crystalline.

FIGURE 17 Adjacent area to that of Fig. 16, after grid was reheated for 2 min at 960°C and slowly cooled.The ash has now grossly melted and formed into distinct droplets. Many of the "droplets" are crystallineand show anomalous high opacity owing to diffraction contrast. (Compare the opacity of various dropletsof similar diameter. The crystallinity of the more opaque droplets was confirmed by dark-field examina-tion, not shown.)

hydroxyapatite (Baker Analyzed calcium phos-

phate, tribasic, powder, J. T. Baker Chemical Co.,North Phillipsburg, N.J.) were heated for 1.5 hrto various temperatures in a platinum crucible ina muffle furnace, and then the X-ray powder pat-tern was determined. The calcium/phosphatemolar ratio of the material was shown analyticallyto be 1.49 0.015. Fig. 21 shows the powderpattern of the unheated material. It is a typicalpattern of hydroxyapatite. Fig. 22 shows the pat-tern of the same material after heating to 1050°C.It is that of whitlockite (see Table II). A similarresult was obtained on material heated to 8250C.If the sample was heated to only 720°, however,it did not dehydrate and the powder patternremained that of hydroxyapatite.

For electron diffraction, various samples of thewhitlockite, prepared as described above, were dis-persed on electron microscope grids either byspreading in a collodion film or by embedding andsectioning. Unfortunately, all of these preparationswere consistently unsuccessful; the crystals, even inthin section preparations, were much too large andunevenly distributed to be suitable for powder

patterns. Evidently the microcrystals of hydroxy-apatite had grossly fused during their recrystalliza-tion to whitlockite.

In the hope of overcoming this problem, samplesof the original hydroxyapatite were dispersed bythe collodion-spreading technique, mounted onsilicon monoxide filmed stainless steel grids, andheated to the same temperatures as before, withthe aim of converting the hydroxyapatite to whit-lockite in situ on the grid. The microcrystals werevery well dispersed. Before heating, they showedthe typical habit of hydroxyapatite (Fig. 23) andgave a typical and quite satisfactory powder pat-tern for hydroxyapatite (Fig. 24). After heating for2.5 hr at 10500C the crystals had a decidedlymelted appearance (Fig. 25) but, curiously, theirpowder pattern, although grainy, remained that ofhydroxyapatite (Fig. 26). Similar results were ob-tained at temperatures down to 700 0C with heat-ing times of a few minutes. Below 7000C, noapparent melting occurred, and the crystal mor-phology and diffraction pattern were completelyidentical with those of unheated specimens.

The in situ recrystallization experiments were

66 THE JOUnNAL OF CEI.L BIOLOGY VOLUME 39, 1968

Figs. 18-20, ash of 600°C-incinerated thick section of formaldehyde-fixed, loaded mitochondria, sub-sequently heated 2 min at 7000 C and slowly cooled. Inspection of the ash in the electron microscope before7000 C heating failed to detect any crystallinity.

FIGURE 18 Bright-field view of a selected area. The ashed granules look more melted than after theirinitial incineration, but they are still recognizable. Although they have no angular, obviously crystallinecontours, they show marked variations in opacity owing to crystalline diffraction contrast. X 17,000.

FIGURE 19 Dark-field view of same area as Fig. 18. Many granules appear bright; this indicates thatthey are crystalline and contribute to that part of the diffraction pattern passed by the off-center aperture.Shifting the aperture to other parts of the diffraction pattern reveals that nearly all of the granules arecrystalline. X 17,000.

FIGURE 20 Diffraction pattern from the selected area shown in Figs. 18 and 19. (The field-limitingaperture, 4 X .5 , included an area slightly greater than shown.) The diffraction spots form only a sketchypowder pattern; there are too few contributing crystals to completely fill out the rings. The four strongestinner rings, which are marked, correspond to crystal spacings of 5.15, 3.18, .86, and 2.57 A.

RICHARD S. THOMAS AND JOHN W. GEENAWALT Calcium Phosphate-Loaded Mitochondria 67

TABLE I

Effect of High Temperatures on Mitochondrial Ash Patterns

Experi-ment Treatment Crystallinity by Quality ofgroup Initial ash pattern* temperature Appearance of ash dark-field exam diffraction pattern

°C

A Control mitochon-dria, LTA incin-erated

BC

D Loaded mitochon-dria, LTA incin-erated

E

F

G Loaded mitochon-dria, 5000 or 600°Cincinerated

H

600 unchanged

700-800 melted900-1000 grossly melted

600 unchanged

700-800 melted

900-1000 grossly melted

700-800 slightly melted

900-1000 grossly melted

absent

weak or absentpresent

absent

present

present

present

present

a few weak spots, noarcs

many weak spots butno clear arcs

a few strong spots,no arcs

many spots, mostlyweak, recognizablearcs

a few strong spots,no arcs

* All of the ash patterns were from formalin-fixed material. The sections before incineration were in mostcases fairly thick (about -/ ), so as to contain a relatively large amount of mineral per unit area. Noneof the initial ash patterns were crystalline.

repeated with another sample of hydroxyapatite,

a semicrystalline colloidal preparation (obtainedfrom Dr. A. Posner) with a calcium/phosphatemolar ratio of 1.51, and a crystallinity index of

30%. (The preparation represented an early stage

in the in vitro formation of hydroxyapatite, and itwas thought that this preparation might moreclosely mimic the successfully converted non-crystalline deposits in mitochondria. See Discus-sion.) Results were similar to those with the Bakerpreparation The colloidal particles fused in thetemperature range, 700-800C, as before (SeeFigs. 27 and 28), but their amorphous-plus-hy-droxyapatite powder pattern could not be con-verted to whitlockite even if the temperaturewas raised above 10000C.

Why the conversion to whitlockite occurred on

the macroscale but not in these microscale experi-ments remains a mystery. A possible explanation inconnection with the mitochondrial results is dis-cussed below (in Discussion).

It is noteworthy that in all of these preparationsthe temperature of apparent fusion of the crystals

was far below their bulk melting point (1 720C fortricalcium phosphate, according to Bale et al.,1945) but in the same temperature range as for themitochondrial dense granule ash.

Although the goal of these experiments, to ob-tain electron-diffraction powder patterns of gen-uine whitlockite, was not achieved, the experi-ments were valuable to illustrate possible pitfallsin applying macroscale data to events in the verysmall world of microincineration.

DISCUSSION

The Nature of the In Vitro-Produced

Dense Granules

ORGANIC CONSTITUENT: The dense gran-ules, when viewed with good resolution, are seen asaggregates of much finer dense grains. If the gran-ules contain organic material serving as a matrix tohold the grains together this observation might beexpected (Greenawalt et al., 1964). That this maybe the case is supported by analytical results onisolated granules, showing a weight loss in incin-

68 THE JOURNAL OF CELL BIOLOGY - VOLUME 39, 1968

TABLE II

Crystal Spacings (dhkl), and Relative Intensities* from Electron-Diffraction Patterns of Dense Granule Ash Heated to700

°or 800°C

Comparison with X-ray data from -tricalcium phosphate (whitlockite).

Dense granule ash; electron diffraction Whitlockite; X-ray diffraction

Plate Plate

8-28-64-19A Plate Plate Plate Frondel C-102A

(Fig. 20) 1-4-66-4A 1-5-66-8A 1-5-66-9A (1943) (Fig. 22)

Relative Relative Relative Relative Relative Relative

dhkl intensity dhkl intensity dhkl intensity dhkl intensity dhkl intensity dlkl intensity

A

3 - -

1 4.03 13 3.43 3

3 3.22 5I - -

5 2.87 51 2.75 3

5 2.59 51 2.53 1

-- 2.27 1-- 2.18 3

-- 2.05 1

-- 1.93 3-- 1.88 3

- 1.80 1

1 1.73 3

- 1.61 1

-- 1.54 3

A A

-- - 8.03 16.36 1 6.55 35.22 5 5.24 2

4.03 3 4.07 23.48 5 3.45 4- - 3.35 1

3.18 5 3.21 5- - 3.02 1

2.87 5 2.88 5- - 2.75 2- - 2.68 1

2.56 5 2.60 5- - 2.52 1- - 2.41 1- - 2.25 1

2.18 3 2.19 1- - 2.16 1- - 2.07 1

2.04 1 2.04 1- - 2.00 1- - 1.93 3

1.88 3 1.88 3- - 1.82 1

- - 1.77 21.72 3 1.72 4- - 1.70 1- - 1.67 1- - 1.63 1

1.60 11.54 1 1.55 3

-- - 1.46 1

-- - 1.43 11.40 1

-- - 1.38 1

* Relative intensities are visual estimates on a scale of 1 to 5, 5 being the most intense. It was impossible

to make five distinctions on the weak and discontinuous arcs of the electron diffraction patterns, so in-tensities are there given as 1, 3, or 5. Both the intensity and the number of spots in the electron-diffractionarcs were considered in assigning the intensity value. The relative intensities taken from the literature

(Frondel, 1943) were converted by us to a scale of 1 to 5.

RICHARD S. THOMAS AND JOHN W. GREFNAWALT Calcisum Phosphate-Loaded Mitochondria 69

A

5.15

3.48

3.183.002.862.74

2.57

2.14

1.891.84

1.72

A

5.22

3.953.48

3.183.002.872.75

2.592.53

1.73

5

3

5151

5

I

31

3

A

8.0 16.50 35.20 34.40 14.05 13.45 3

3.20 43.00 12.90 52.75 1

2.60 42.50 12.40 12.26 12.20 12.16 12.08 12.04 12.00 11.94 31.89 21.83 11.81 11.77 11.73 3

1.68 11.63 11.60 11.55 21.51 11.47 1

1.41 11.39 1

Figs. 21-26, effect of high temperature on a microcrystalline calcium-deficient hydroxyapatite (BakerAnalyzed calcium phosphate, tribasic, powder, calcium phosphate molar ratio 1.49 0.015).

FIGURE 21 X-ray powder pattern of unheated preparation. It is a typical pattern for hydroxyapatite.The strongest arcs which can also be easily recognized in electron-diffraction patterns (Fig. 24) aremarked; they correspond to crystal spacings of 3.44, 2.78 (average of 3 spacings: 2.84, 2.79, and 2.72),1.94, 1.84, and 1.72A.

FIGURE 22 X-ray powder pattern of preparation after heating 1.5 hr at 10500 C. It is a typical pattern ofwhitlockite (see Table II). The strongest arcs which correspond to the four most easily recognized arcs illelectron-diffraction patterns of crystallized dense granule ash (Fig. 20) are marked in the figure.

FIGURE 23 Electron micrograph of an unheated preparation dispersed on a silicon monoxide-collodionsupport film. Nonuniform opacity of some of the crystals is due to diffraction contrast. X 16,000.

FIGURE 24 Electron diffraction pattern of a 4 X 5 selected area including the field shown in Fig. 3.The five easily recognized rings correspond to the marked arcs in the X-ray powder pattern, Fig. 21.

FIGmuE 25 Dispersed preparation similar to that of Fig. 23, but after heating for 2.5 hr at 10500C. All ofthe particles have rounded contours, as though they were molten, but they are all quite crystalline. Darkstriations across many of the crystals are due to diffraction contrast. X 16,000.

FIGURE 26 Electron diffraction pattern of a 4 X 5 selected area including the field shown in Fig. 25.The arcs of the pattern have become quite grainy owing to the increased size and decreased number ofcrystals contributing to them, but nevertheless the pattern is still recognizable as that of hydroxyapatite.

Figs. 7 and 28, electron micrographs showing the effect of high temperature on a dispersed preparation ofsemicrystalline, colloidal calcium phosphate. (The preparation was obtained from Dr. A. Posner. It wasshown to have a calcium/phosphate molar ratio of 1.51, and it gave an X-ray powder pattern of hydroxy-apatite. Detailed analysis of the pattern indicated that the preparation was about 30% crystalline.)XS9,000.

FIGURE 27 Preparation after heating for a few minutes at 600C. The 600°C treatment did not alter thespecimen, as could be shown by comparison with micrographs of the same grid before heating. The ma-terial consists of masses of tiny asymmetric particles with minimum dimension of about 60 A. (The prep-aration both before and after heating gave an excellent, continuous ring, electron-diffraction powder pat-tern of hydroxyapatite.)

FIGURE 28 Preparation, on same grid as for Fig. 27, after reheating for a few minutes at 8000C. Thematerial now consists of a fused reticulum of highly irregular particles with rounded contours. The averageminimum dimension of the particles is about 400 A. Occasional dense spots on the particles are apparentlydue to diffraction contrast. (The preparation still gave a continuous ring, electron-diffraction pattern ofhydroxyapatite, but it was slightly grainy by comparison to that from the 600°C-heated material.)

erated granules of 16-60 % depending on the prep-aration (Weinbach and von Brand, 1967). Thepresent microincineration results are also consist-ent with this picture. The granules after initialrapid burning at 600C are seen to have meltedand formed bubbles during the escape of somevolatilized component. The vaporized materialcould be partly a mineral constituent such as car-bonate (carbonate content of isolated granules is7-10%) or bound water. but probably most vapor-ized material is organic substance.

Granules which have been divested of theirorganic component at lower temperature, either byheating at 500C or by low temperature plasmaashing, retain their inorganic fine structure in theash with little or no evidence of melting and nobubbles. This structured ash shows no evidence ofdecomposition when it is subsequently heated to600C. The ash appears stable until nearly 700°Cis reached, at which temperature it begins to meltwithout bubbles. Carbonate and bound water aregenerally retained after low temperature ashing(Gluskoter, 1965) but they are lost from the gran-

ules at 6000C (Weinbach and von Brand, 1967).Evidently their departure does not much affect thefine structure of the inorganic component of thegranules.

It is noteworthy that the melting range of theintact granules, 5000-600C, is appreciably lowerthan that of their mineral residues, 600-7000 C.This melting point lowering suggests an intimateassociation of organic and inorganic constituentsin the granules.

INORGANIC CONSTITUENT: The tentativefinding in the present study of a 3-tricalcium phos-phate (whitlockite) electron-diffraction pattern forthe 7000C-heated ash of the dense granules agreesquite well with X-ray diffraction results obtainedby Weinbach and von Brand (1967) on bulk prep-arations of isolated granules subjected to prolonged(18 hr) incineration at 600C. In this latter study,four different preparations of isolated granulesyielded X-ray powder patterns variously of hy-droxyapatite alone, hydroxyapatite plus whitlock-ite, or whitlockite alone. However, the two prep-arations which were isolated under chemically mild

RIcHcAs S. THOMAS AND JOHN W. GREENAWALT Calcium Phosphate-Loaded Mitochondria 71

conditions, without the use of strong alkali, yieldedpatterns of only whitlockite. These latter prepara-tions may have been impure in that they con-tained a large proportion of organic material, butthis would only increase their similarity to ourpreparations of unisolated granules in situ. Anymineral associated with the organic impurity prob-ably had little effect on the mineral analyses andX-ray diffraction results after 6000 C-incinerationsince our results show that only the mineral of thedense granules remains after this high temperatureprocessing.

A seeming anomaly in the identification of theashed-granule mineral is the low melting tempera-ture, 600-700°C, of the ash seen under the electronmicroscope both by Weinbach and von Brand(1967) and us. The bulk melting point of trical-cium phosphate is 17200 C (Bale et al., 1945). Indefense of the identification, electron microscopicpreparations of verified calcium phosphates showedsimilar behavior. Actually, the melting seen in mi-croscale had a slight but detectable correlate on themacroscale; high temperature-treated calciumphosphate powders displayed a small change intexture which was evidently due to slight scinter-ing. The apparent melting of electron microscopicspecimens of "heat-stable" minerals is discussedelsewhere (Thomas, 1968).

The finding, in both Weinbach and von Brand'spreparation and ours, of whitlockite in the crys-tallized ash, and also the low crystallizationtemperature, suggests that the original, noncrystal-line, colloidal mineral of the dense granules maybe a subcrystalline precursor of a calcium-deficienthydroxyapatite rather than the stoichiometrichydroxyapatite which had been previously sug-gested (Rossi and Lehninger, 1963 a, b). The cal-cium/phosphate molar ratio of the mineral wouldtheoretically be about 1.50 rather than the 1.67 forstoichiometric hydroxyapatite, Ca10 (PO4)6(OH) 2 -

As has long been known, hydroxyapatite with thestoichiometric ratio, 1.67, maintains its stable,hydrated structure at temperatures up to 1450°C(Bale et al., 1945), but calcium-deficient hydrox-yapatites dehydrate and recrystallize at 600° -

800C (Hodge et al., 1938; Kunin, 1958). If thecalcium/phosphate molar ratio is between 1.67and 1.50, a powder pattern showing both hydroxy-apatite and whitlockite (in relative amount pro-portional to ratio) is obtained after heating. If theratio lies between 1.50 and 1.33 (the minimum ratiofor hydroxyapatites) then a pattern of varyingamounts of whitlockite and pyrophosphate (-

Ca2P 2O 7 ) is obtained. If, however, the ratio is quiteclose to 1.50, only whitlockite is found in thepowder pattern after heating (Hodge et al., 1938;Trautz, 1955). These dehydration results, althoughobtained on preparations which were initially wellcrystallized, are probably applicable to subcrystal-line or noncrystalline preparations as well. Theimportant factor seems to be simply the calcium/phosphate molar ratio of the preparation (Trautzet al., 1954).

So that the interpretation would be reliable fora noncrystalline preparation, it would be necessarythat the entire preparation crystallize; otherwisethe deduced calcium/phosphate ratio might applyto only a biased fraction of the material. WhetherWeinbach and von Brand's mitochondrial densegranule preparation and ours crystallized entirelyis not known.

In the special case of our microincineration ex-periments, confidence in the interpretation re-quires an explanation of why electron microscopicspecimens of calcium-deficient, synthetic hydroxy-apatite did not dehydrate to whitlockite, eventhough the dense granule ash seemingly did. Slightmoisture uptake from the atmosphere in thesespecimens with relatively enormous surface area,and a role of minor mineral components in thedense granule ash might provide an answer here.Even at 1050°C, calcium phosphates can reactwith water vapor (Van Wazer, 1958). Such a reac-tion might have prevented the dehydration of thepure, synthetic hydroxyapatite. In the case of thedense granule ash, 2-4% magnesium (shown to bepresent by Weinbach and von Brand, 1967) mayhave counteracted the effect of moisture uptake andfacilitated the dehydration to whitlockite. Unlikepure calcium whitlockite, calcium-magnesiumwhitlockite can be formed in the presence of water(Trautz et al., 1954).

The actual molar ratio, calcium (plus mag-nesium)/phosphate, shown by Weinbach and vonBrand for their dense granule preparations crystal-lized to whitlockite is 1.51 in one case, in excellentagreement with theory, but in the second case it is1.31. Assuming this is not analytical error, it wouldrepresent anomalous behavior in crystallization.The molar calcium/phosphate ratio for our ownpreparation is unfortunately unknown. In otherexperiments done in the same way as ours (Rossiand Lehninger, 1963 a, b) the molar ratios for up-take of calcium and phosphorus ranged fromabout 1.52-1.88 with an average of 1.67. Thesevalues pertain to whole mitochondria, however,

72 THE JOURNAL OF CELL BIOLOGY VOLUME 39, 1968

and so whether they accurately reflect the ratios inthe dense granules, alone, is not certain.

Quite aside from the crystallographic and ana-lytical results but considering that the original min-eral of the dense granules is colloidal calcium phos-phate, it would be very reasonable to find that thismineral has a calcium/phosphate molar ratio ofabout 1.5. As shown by Watson and Robinson(1953) and more recently by Eanes et al. (1965),hydroxyapatite prepared in the test tube passesthrough a sequence of stages during its formation.First, a colloidal, noncrystalline (or subcrystalline)precipitate appears. This converts to minute,poorly crystalline particles or thin sheets, which,however, continue to grow and mature and finallyform fully developed microcrystals; these give thesharp powder pattern of hydroxyapatite. Thecalcium/phosphate molar ratio of the initial, sub-crystalline deposits is found to be about 1.5 (ac-tually, 1.46 or 1.52 depending, on the method ofpreparation). Only as the preparation becomesdetectably crystalline and the crystals mature doesthe molar ratio rise to the final figure, 1.67, the stoi-chiometric value for Ca10(PO 4)6(OH)2 (Eanes etal.,1965).

Recently, Termine and Posner (1966) have pro-duced evidence and have cited earlier observationsby others to show that also in biological systems,such as bone, the formation of hydroxyapatiteproceeds via initial deposition of a noncrystalline(or subcrystalline) colloid. The colloidal, non-crystalline mineral in the mitochondrial densegranules studied here might similarly represent thisfirst stage of hydroxyapatite formation and onemight expect the material to have a calcium/phos-phate molar ratio of about 1.5.

The success of the present experiments in tenta-tively identifying the chemical nature of mineraldeposits in situ in thin sections of isolated mito-chondria suggests that the microincineration-elec-tron-diffraction technique might be usefully ap-plied to similar noncrystalline, mitochondrial min-eral deposits (Gonzales and Karnovsky, 1961;Caulfield and Schrag, 1964; Heggtveit et al., 1964;Reynolds, 1965; Cameron et al., 1967) and toother mineral bodies seen in thin-sectioned wholecells and tissues. In such whole cell material it isusually impractical to attempt isolation of themineral deposits, and thus an in situ techniqueoffers a unique possibility for identifying the min-eral. Some of the problems of the microincinera-tion-electron-diffraction technique are discussedby Thomas (1968).

Al sh Patterns from Control Mitochondria

" MEMBRANE" ASH: In contrast to the cal-cium phosphate-loaded mitochondria, whichshowed only traces of mineral other than from thedense granules, unincubated control mitochondriashowed an appreciable amount of intrinsic mineralin low temperature ash patterns. The differencebetween these two preparations in this regard cannot be explained with certainty. It could be, how-ever, that the loaded mitochondria lose intrinsicmineral during incubation at the same time thatthey are accumulating vast amounts of calciumand phosphate in the granules. This could resultfrom deterioration in the ultrastructure of themitochondria. In addition, loss of ultrastructuralintegrity during incubation could predispose theorganelles to loss of minerals from specific sitesduring subsequent fixation and embedding. It isknown that, under the incubation conditions usedhere, oxidative phosphorylation in these mitochon-dria is uncoupled and appreciable deterioration ofstructural intactness occurs.

The low temperature ash patterns of the controlmitochondria suggest that the origin of the ash isfrom the membranes of the organelle. This sugges-tion is strengthened by examination of partiallyashed preparations, but unfortunately the poordefinition in both types of preparations prevents acompletely certain interpretation of this point. Themembranous structure of the mitochondrion un-doubtedly influences in some way the deposition ofthe ash to produce the patterns seen, but whetherthe ash actually derives from native mineral in themembranes or from mineral originally at adjacentsites cannot be answered at this time. It seemslikely that soluble, unbound minerals localizedwithin the matrix would be largely depleted duringfixation and dehydration. Membrane-bound min-erals, on the other hand, could be at least partiallyretained even though the formaldehyde-fixedmembranes might well be stripped of most of theirlipids during acetone dehydration (Ashworth et al.,1966). Rather uniquely, the ultrastructural ap-pearance and macromolecular configuration ofmitochondrial membranes appears to depend little,if at all, upon the presence of the lipid components(Fleischer et al., 1967).

It is noteworthy that ash patterns of osmium-fixed mitochondria looked very similar to those offormaldehyde-fixed preparations. Presumably,membrane lipid was largely retained with osmium(Ashworth et al., 1966). What effect osmium might

RICHARD S. TaHOAS AND JOHN W. GREENAWALT Calcium Phosphate-Loaded Mitochondria 73

have on retention of cations bound to phospho-lipid is, of course, an unanswered question. Thepartially reduced osmium, itself, might well be lostby reoxidation to osmium tetroxide and volatiliza-tion during the low temperature ashing (Gleit,1967).

Further work is in progress to determine theorigin of the membrane ash patterns, and theirconfigurations in other specimens, such as thin-sectioned whole cells. Better retention of mineralsin the specimens presented for microincinerationmay improve not only the significance, but also thedefinition of the ash patterns (Thomas, 1968).

The chemical nature of the membrane ash re-mains unknown, but it is noteworthy that afterinitial formation by low temperature ashing, itproves to be stable at 600°C and it is not hygro-scopic. This suggests that it is either a metallicoxide or a metallic phosphate. Carbonates, chlo-rides, sulfates, and probably other common saltscould be retained by low temperature ashing(Gluskoter, 1965) but these anions would mostlikely be volatilized at 600°C. Loss of the mito-chondrial mineral by initial high temperatureashing at 600C is probably due to organic ma-terial formed into volatile complexes with themineral. Alternatively, the initial state of themineral itself, before low temperature oxidation,might be volatile at high temperature (Gleit,1967). The apparent melting or scintering of theash seen at 700°C is not inconsistent with a metallicphosphate (see previous section of Discussion) oreven with a metallic oxide (see references inThomas, 1968). The ash could, of course, be amixture of several compounds, and this is actuallysuggested by its relatively great reluctance tocrystallize at high temperature.

NORMAL DENSE GRANULES: The small,dense, matrix granules commonly seen in normalmitochondria within cells were not found in iso-lated mitochondria following active uptake of cal-cium and phosphate (Greenawalt et al., 1964)perhaps because of the swollen condition of theincubated mitochondria (Wlodawer et al., 1966).The small granules were found in osmium-fixed,unincubated control preparations, however, andthis provided an opportunity to compare theirbehavior during ashing with that of the in vitro-produced granules. This comparison is of someinterest since it has been suggested that the func-tion of the normal granules in vivo is to sequesterosmotically active, metallic cations (Weiss, 1955;

Peachey, 1964) in a manner analogous to that ofthe larger, in vitro granules.

The high temperature microincineration ex-periments showed unequivocally that the normalgranules are completely volatilized at 600C, afinding which is in sharp contrast to that for invitro-formed dense granules. This perhaps indi-cates that the normal granules contain no mineralsubstances. On the other hand, the membranemineral, demonstrated by low temperature ashing,is also completely volatilized from the same prep-arations during the high temperature ashing; thenormal granules thus might contain mineral whichwas similarly volatilized. Low temperature ashpatterns of the preparations do not display anyparticles clearly recognizable as residues of thenormal granules, but the latter are so small thattheir residues might be overlooked among theubiquitous small grains of the ash patterns. In con-clusion, the present experiments do not rule out thepossibility that the normal granules contain somemineral, but they do show that the granules lackthe stability against high temperature incinerationdisplayed by the in vitro-formed calcium phos-phate granules. The normal granules behave dur-ing ashing like the membranes or other mitochon-drial substances.

The failure to obtain any clear evidence ofmineral in the normal granules is perhaps not sur-prising. The granules are seen only in osmium-fixed or aldehyde-osmium-fixed preparations andnot at all in material fixed by aldehyde alone.Without additional heavy metal staining, theosmium-stained granules do not have an electronopacity which even approaches that of small invitro-produced calcium phosphate granules. Ash-worth et al. (1966) have shown that the aldehyde-fixed normal granules cannot be demonstratedeven by osmium fixation and staining if this followsdehydration with nonpolar solvents and sub-sequent rehydration. This behavior is similar tothat of membranes and other lipid-containingstructures. Those workers conclude that the gran-ules are lipid; the lipid is evidently not fixed byaldehyde but is extracted during dehydration ofaldehyde-fixed specimens. Andr6 and Marinozzi(1965) find that the granules retained by osmiumfixation disappear when the reduced osmium isremoved by hydrogen-peroxide oxidation. Theyalso conclude that the granules have no intrinsicdensity and are probably lipid bodies. Evidencethat the normal granules are lipid bodies does not,

74 THE JOURNAL OF CEILL BIOLOGY ·VOLUME 39, 1968

of course, prove that they do not serve as sites fornative mineral deposition in vivo. However, failureto find any of this native mineral still attached inthe specimens presented for examinations doestend to downgrade the special status of the gran-ules with regard to mineral content and placethem in the same category as other lipid-containingbodies in the cell which can be stained with heavymetals.

The present experience in trying to determinethe nature of the normal dense granules in osmium-fixed mitochondria suggests that microincinerationmay be generally useful in discriminating betweenheavily OsO4-fixed structures and mineralizedstructures seen in osmium-fixed thin sections. Itmay prove generally so, as here, that osmium iscompletely volatilized from high temperature ashpatterns, and even from low temperature ashedspecimens (Gleit, 1967; Thomas, 1968). Thus elec-tron-opaque structures leaving an ash could be un-ambiguously categorized as mineral. Osmium re-moval by microincineration would offer at leastone distinct advantage over other methods such ashydrogen-peroxide treatment (Andre and Mari-nozzi, 1965). The microincineration reagent is

gaseous, rather than aqueous, and thus the possi-bility of leaching out native mineral along with theosmium is eliminated.

Other possible applications of the microincin-eration technique are discussed in Thomas (1968).

The authors thank Drs. C. Rossi and F. D. Vasington(formerly of Johns Hopkins University School ofMedicine) for some of the mitochondrial preparationsused in these experiments. Model preparations ofcolloidal calcium phosphate were gratefully receivedfrom Dr. A. Posner (Cornell University MedicalCollege). The authors are indebted to Mr. A. Potterand Miss R. Young (Western Regional ResearchLaboratory) for chemical and X-ray analyses ofcalcium phosphates.

Reference to a company or product name does notimply approval or recommendation of the product bythe United States Department of Agriculture to theexclusion of others that may be suitable.

This investigation was partially supported by re-search grant No. GM-12125 from the NationalInstitutes of Health, United States Public HealthService.

Received for publication 18 March 1968, and in revisedform 10 June 1968.

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ANDRE, J., and V. MARINOZZI. 1965. Presence dansles mitochondries, de particules ressemblant auxribosomes. J. Microscopie. 4:615.

ASHWORTH, C. T., J. S. LEONARD, E. H. EIGENBRODT,and F. J. WRIGHTSMAN. 1966. Hepatic intracellularosmiophilic droplets. Effect of lipid solvents duringtissue preparation. J. Cell Biol. 31:301.

BALE, W. F., J. F. BONNER, H. C. HODGE, H. ADLER,

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