Frontiers in electron probe microanalysis: Application to cell physiology

Ultramicroscopy 24 (1988) 185-220 North-Holland, Amsterdam

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FRONTIERS IN ELECTRON PROBE MICROANALYSIS: APPLICATION TO CELL PHYSIOLOGY A. LeFURGEY Department of PhysioloD', Medical Center, Duke University, P.O. Box 3709, Durham, North Carolina 27710, USA

M. BOND Cleveland Clinic Foundation Research Institute, Clet,eland, Ohio 44106, USA

and P. INGRAM Research Triangle Institute, Research Triangle Parle, North Carolina 27709, USA Work presented August 1986; manuscript received 22 June 1987

The application of electron probe microanalysis techniques, using X-ray and electron energy loss instruments, to problems in cell physiology is reviewed. The details of the special methodological requirements for the analysis of cry osections a~ high spatial resolution in an analytical electron microscope are discussed together with a comprehensive review of data obtained on major organ systems and cell type's.

Contents 1. Introduction 2. Theoretical and methodological consideratior~s 2.1. Cryopreservation 2.2. Cryosectioning 2.3. Freeze-drying 2.4. Ctyotransfer and the use of a cryostage 2.5. Electron probe X-ray microanalysis 2.5.1. Instrumentation 2.5.1.1. The electron microscope 2.5.1.2. The X-ray spectrometer 2.5.2. Analysis 2.5.3. Quantitation 2.5.3.1. Algorithms 2.5.3.2. Standards 2.5.3.3. Minimum detectable concentration and mass L.. J."t.

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2.6. Electron energy loss spectroscopy 3. Physiological apphcatlons of EPXMA 3.1. Skeletal and smooth muscle 3.1.1. Skeletal muscle 3.1.2. Smooth muscle 3.2. Heart 3.2.1. Papillary muscle

0304-3991/88/$03.50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)

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3.2.2. Cardiac cells 3.2.2.1. Cultured cells 3.2.2.2. Isolated cells 3.3. Liver 3.4. Pancreas 3.5. Kidney 3.5.1. Intact kidney 3.5.1.1. Cortex: proximal and distal nephron 3.5.1.2. Papillae 3.5.2. Isolated proximal tubule suspension 3.6. Other epithelia 3.6.1. Frog skin 3.6.2. Amphibian urinary bladder 3.6.3. Cornea 3.6.4. Intestine 3.6.5. Salt glands 3.6.6. Salivary glands 3.6.7. Thymus 3.7. Brain/cerebral cortea/nerve 3.8. Other cell types 3.8.1. Photoreceptors, retinal rods 3.8.2. Red blood cells, leukocytes, platelets 3.8.3. Spermatozoa, oocytes 3.8.4. Bacteria 4. Summary and conclusions Acknowledgements References

I. Inlroducfion

In a little over the past decade electron probe X-ray microanalysis (EPXMA) has become established as the most useful method whereby quantitative in situ elemental analysis can be carried out at the ultrastructural level in biological samples [1-12]. Microanalysis allows identification as well as quantitation of diffusible, physiologically active elements within whole cells or subcellular compartments such as mitochondria, nuclei, lysosomes, endoplasmic reticulum, and cytoplasm and provides a basis for interpretation of structural responses to changing functional conditions [1-6]. ,,,~ l.,~ii.m y utijctitlVCb ill ulolottt;atltJll Ol electron probe microanalysis to cell physiology are (A) to determine quantitatively the subcellular compartmentation, distribution and transport of elements (ions) as they are found in vivo: in cytoplasm, in cellular organelles, in intracellular and extracellular spaces, and (B) to utilize these measurements to determine the mechanisms linking structural and elemental compartmentation

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and distribution to cell function. The cells and tissues must be maintained in a physiologically defined state until the moment of preservation arid the in vivo distribution and content of elements within the cells must be maintained throughout further preparatory steps. In addition the electron optical and analytical techniques must be as precise as possible. There are other excellent reviews which treat various aspects of these methodologies in detail [6,10,11]. In this review we focus on the use of EPXMA in cell physiology. We shall discuss mainly the use of cryosectioned material rather than whole cells or bulk tissue, for it is in this arena that it has been possible to obtain relatively high spatial resolution analytical data relating to intracellular element compartmentation. Many of these same preparative considerations appl) to the bulk state [13-15]. We shall discuss data obtained from both frozen hydrated and freeze-dried specimens. In many instances it suffices to acquire data from the dry state, for example in monitoring changes from one (patho)physiological condition to another [16].

,4. LeFurgey et al. / Electron probe mwroanah'sis in cell pf[v.iologr

However, the ultimate goal of correlating physiological function with structure and content requires knowledge not only of the element content but also the water content of intracellular, adjacent intercellular and luminal regions [1,3]. One important premise of this correlative approach to cell physiology is that element content, ultrastructure and physiological or biochemical function be determined in parallel for each cell or tissue type under investigation. For example, measurements of cytoplasmic "free" ions (e.g. Ca) with indicator dyes such as fura-2 provide correlative data important to interpretation of EPXMA determinations of total (free and bound) element content within the cytoplasmic compartment [17]. Microelectrode measurements of cytoplasmic Na, K, CI, or Ca yield ionic activities as well as ionic concentrations in the living cell and can verily EPXMA determinations of the cytoplasmic content of these elements in cryopreserved ,.ells [18,19]. EPXMA can uniquely provide measurements of total element content in intracellular compartments such as mitochondria and endoplasmic reticulum, not usually accessible with dyes or microelectrodes. Uitrastructural definition (in conventional te~ms) of any changes brought about by experimental maripulation should form the basis for EPXMA m~asurements for both scientific and methodological reasons. Of primary scientific importance for structure-function correlation is (a) definition of the cell ultrastructure and (b) determination of whether or not a particular drug or maneuver alters that ultrastructure. This determination is facilitated in conventionally fixed, sectioned and stained cells. If one or more organelles has been changed structurally, then EPXMA analy~es of cryosections may or may not be initially focused on these particular organelle~: r_.ra~v~.,-,, counting statistics is esoptin-tization of '-'"'~'""" sential ~ince obtaining quantitative data is time consuming. Of equal methodological importance is the fact that, in cryosections obtained from rapidly frozen cells or tissues, contrast is low because no stains are used and ultrastructural resolution is generally of the order of 100 ,~ [20,21], more than adequate for EPXMA but not sufficient for discrimination

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of the smallest intracellular structures. Recognition of intracellular structures is also made much easier in low contrast cryosections if one is familiar with cell ultrastructure by conventional transmission electron microscopy (TEM), and can recognize gross structural changes. This ability is also important for evaluating the cryosections themselves, since the complex low temperature sectioning regimen and multiple specimen transfers can also induce structural artifacts [22,23]. Thus conventional TEM and EPXMA must be performed in parallel with other physiological and biochemical measurements, not only to document changes in ultrastructure which any maneuver induces, but also to optimize EPXMA data collection from appropriate intracellular regions. This article is an assessment of the current methodology and techniques necessary to acquire meaningful physioloqical EPXMA data, and a review of the results that have beea obtained to date on major organ systems inclucing muscle, liver, kidney and other epithelia, and other animal cell types.

2. Theoretical and methodological considerations To prepare cells or tissues for localization of diffusible elements by EPXMA, one must first insure that they are maintained in a viable, physiologically active state until the moment of cryoprese~,ation. Second, cryopreservation, or quick freezing, is the only method by which both ultrastructure and intracellular element content can be simultaneously preserved: it must occur at rates of > 10,000 to = 100.000 ° C/s, theoretically without the formation of ice cry'stals (vitrification) or practically with the formation of ice cryslals which are negligibly small in relation to the size of the regions to be analyzed. Third, cryosectioning and/or other subsequent handling of tile frozen still-hydrated, specimens must be performed at temperatures ( _ < - 1 4 0 ° C ) which preclude recrystallization, i.e. warming and refreezing, an event(s) associated with increased ice c~stal size and thus structural disruption and element translation. Fourth, if analyses are to be performed on freeze-dried sections, freeze-drying either inside the microscope or in another vacuum device must

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be stringently controlled so that all water is slowly removed from the frozen specimen by the dehydration process without structural disruption or contamination. Care must be taken that no water (atmospheric moisture) is allowed to re-hydrate the material during storage or transfer of the dried cryosection to the microscope. It is our bias that quantitative X-ray microanalysis of diffusible elements is of limited value unless performed on such sections generally in the absence of additional penetrating cryoprotectants, chemical fixatives, or stains. No embedding medium, including the "low temperature" types, or freeze substitution technique, of which the authors are aware, can be utilized because these agents and methodologies all cause some form of chemical change to occur in the tissues. In that which follows, only the basic principles are described. The reader is referred to numerous excellent texts [24-27] for the fine details.

2.1. Co'opreseroation There are four methods of quick freezing which have been utilized most widely prior to cryosectioning and subsequent X-ray analyses [28--30]. These are (1) plunging the cells or tissues into a liquid cryogen ~uch as liquid-nitrogen-cooled freon, ethane or propane [31-35]; (2) spraying a jet(s) of liquid cryogen onto the cells or tissues [36-41]; (3) clamping cells or tissues between two blocks of frozen cryogen or between the copperclad jaws of pliers pre-cooled in a cryogen [42-44]: (4) slamming the tissues against a polished metal block precooled by cryogen [45-46]. The choice of freezing method is dictated by the geometry and mass of the tissue being frozen, together with the efficiency and capacity of the cryogen or cryogeneOCdecl ~ l l r f : a c o { ~ f e w h e a t r e m t a v a l

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standing structural preservation has been obtained with the slamming method in which 'he copper block is cooled to = 4 K with helium [45]. The rapid impact of material on the block does not disrupt ultrastructure, probably because propagation of freezing is far more rapid than propagation of the mechanical shock wave. Satisfactory freezing for EPXMA has also been obtained with each of the other methods. The quality of freezing

is usually judged by examination of freeze-fracture replicas or freeze-substituted tissue taken from a region adjacent to that which is to be cryosectioned. Depending on the size of ice crystals that can be tolerated, adequate structural preservation can be achieved up to as much as 50 ~m from the surface of first contact with cryogen [28].

2.2. Cryosectioning The theories of cryosectioning are as yet not totally understood but major areas of thought exist defining the process as either (a) true cutting (slicing), (b) fracturing or (c) melting-freezing [47--54]. While a number of experiments provide evidence that melting during sectioning does not occur or, if it does, is insignificant [23,50-53], no consensus has been reached as to whether the process is one of cutting or fracturing. Saubermann et al. [47,48] have compared sectioning of frozen specimens with metal machining, suggesting that the only difference is retention of the "chip" in cryosectioning versus removal of the "chip" in metal machining. In any event the "bottom line" is that a section of well preserved material is ultimately obiained. Sevecal reliable instruments for cryosectioning are currently available which allow maintenance of the frozen specimen, the knife and the cryochamber at temperatures which preclude thawing and recr3,stallization ( < - 140 ° C); these are discussed in detail elsewhere [25,26]. Although the majority of investigators maintain knife, specimen and cryochamber at temperatures of - 130 °C or lowel, Saubermann and colleagues [53] and others [54] perform sectioning at warmer temperatures, = - 35 o to - 50 o C. While this is probably adequate where high spatial resolution is not realllrod

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and "cytoplasm", it must be questioned whether or not there is significant disruption, brought about by warming and recrystallization, within the smaller organelles such as mitochondria. Although some authors claim that sectioning is "easier" at these warmer temperat,~res, Dubochet and coworkers point out that when sectioning is performed at temperatures of < - 1 4 0 ° C or lower on well-preserved ice-crystal-free (cryoprotected)

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tissue, it is possible to obtain excellent sections in which organdies can be visualized even in the hydrated state [20,21,55]. Other parameters which affect cryosection quality are analogous to those which affect sections in routine ultramicrotomy and include sectioning speed ( _< 1 m m / s ) , knife angle, knife composition and quality. Glass and diamond knives have been utilized successfully for cutting sections 100rim thick or less, but several authors emphasize that knife quality is of utmost importance in obtaining good cryosections [56-58]. Improved methods of breaking glass knives and techniques for coating the glass knife edge with carbon or tungsten have been described [58]. Diamond knives have the same obvious advantages as in routine ultramicrotomy; the major drawback appears ta be buildup of static charge at the knife edge. Static charge complicates sectioning with either knife type because all sections must be collected "'dry" to avoid leaching or translation of diffusible elements. Sections floated on boat fluids such as DMSO or glycerol are subject to such artifacts. Static can be reduced by placement of small alpha emitter strips at strategic points in the cryochamber or by use of an anti-static gun. Removal of individual sections from the knife edge to the grid is a delicate procedure at temperatures of -- 130 °C and can bt: accomplished with individual eyelashes or dog hairs mounted on dowels, with very fine bamboo fragments or with glass or metal "' needles" of thin dimension. Each i-nplement or tool used must be pre-cooled to avoid warming of the section or specimen block. Many possible artifacts and pitfalls in cryosectioning have been excellently" reviewed by Frederik [22,23,52]. Most investigators mount their samples on thin films of carbon a n d / o r Formvar on high trans• " The " chosen to give as large an opening as can support ,I.'.. g:l-,, ........ ,,.as. Slotted s,~,.,.,, ~-"~ haxc also bccn used. Low background (peak-free) beryllium or nylon grids are generally avoided, since it is much easier to make spectral corrections from. and identify the presence of, discrete peaks rather than t,nknown Bremsstrahlung. Nicholson et al. [59] used aluminum tmlders and grids in order to further isolate the parts of the spectrum to be corrected. IIIIOOIVII

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Grids with support films should be prepared and stored under scrupulously clean conditions: grids from each coating run should be analysed before they are used for sections to determine that they are contaminant-free. Quantitative analytical EM is a trace analysis technique and any contamination from dirty equipment, chemicals and ambient air will be detected - especially from carbon which is a very good scavenger. The detailed precautions necessary to achieve this are well described in the literature. Any laxity in attention to these details, from the moment the material is acquired to the moment the analysis is complete, could subject the results to error.

2.3. Freeze-do,ing Freeze-drying of cryosections may be performed either through use of a crvotransfer device (see next section) within the electron microscope or, as is more usual, under carefull- controlled conditions outside the microscope. F,cept for special cases of lipid-rich cells [60?, tor dynamic monitoring of the freeze-do, ing process [61]. or for sequential frozen-hydrated/freeze-dried analyses to measure water content [62]. there seems little reason to employ ti:e tnk.'~o.~cope as an cxpcn:dvc freeze-docz. Concerns as to the validity of data obtained on sections freeze-dried outside the microscope seem totally unfounded since in the numerous cases where it has been possible, good agreement has been obtained by several laboratories with parallel chemical and other independent techniques on the same tissues [2.6.18,34.42,46. 63-671. Furthermore. in sections freeze-dried outside the microscope, "'microregions'" e.g. of high Ca in the sarcoplasmic reticuh,m or of Ca in at;ial granUI~,S

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elemental redistribution even at stab~micron resoItJ,ion to3]. ~",,,,.,, dl\ilt~,, ,i,.-tl,od also maintains sharp gradients across the cell membrane of the more diffusible elements (Na. K, CI). However. care must be exercised in effecting the drying process, the physics of which has been described by MacKenzie [68] and others [69--72]. The tissue should be dried in a clean vacuum system, prelerably using oil-free pt, mps: a simple crvosorb pump

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in a clean chamber will suffice [73]. The rate of drying s!tculd be controlled, if possible, by incremental :ncreases in temperature for fixed duration or at least a maximum warming rate of no more than = l ° C / m i n . There is a need to strike a balance between (a) the evaporation of water from any non-crystalline or vitreous water and various sizes of ice crystals and (b) the recrystallization of water/ice that may be present. Since the eutectic formed on freezing can be different within different cell types or within different organelles due to the different water contents and protein compositions, the so-called "critical temperature" points also can be quite different. In addition it has been shown that freeze-drying involves a "'front" or "' fronts" of drying passing through regions during the dehydration process [68]. Clearly if this frontal progression proceeds too fast disruption will resuit. The effects of inhomogeneities can sometimes be seen in the appearance of a well-preserved cell adjacent to one with severe ice crystal damage. In summary, it appears that adequate freeze-drying can be obtained in this phase of specimen preparation by transferring the section to a heavy metal block (e.g. copper or brass) pre-cooled ~.o liquid nitrogen temperature; the block then is allowed to gradually warm to room temperature in a clean vacuum environment. Warming the specimen to slightly above room temperature will prevent water condensation before removal and/or coating with a light carl,on fihn in the same or nearby clean vacuum chamber. Carbon coating has the effect of preventing charging in the electron beam and of minimizing possible ~ehydration on exposure to ambient air. Some workers report freeze-drying sections in the cryoultramicrotome cooling chamber, taking advantage of the low vapor pressure of ice in a nitrogen atmosphere. While such drying in some cases can give reasonable structural preservation, the conditions are unstable and not uniformly reproducible since there are always convection currents within the cryochamber; therefore freeze-drying in the cryomicrotome is not recommended when accurate quantitation at high resolution is required. Freeze-dried sections should be stored in a vacuum dessicator without the use of powdered dessicants such as CaSO4 since they can contaminate the -

specimen. P205 should be used with care and changed regularly to prevent overaccumulation of moisture. No measurable elemental redistribution occurs even after prolonged (months) storage in a dessicator at room temperature, although anomalous increases in sulfur and chlorine have been observed in some instances (authors' experience). Many laboratories maintain the ambient air at constant temperature and low humidity. It is especially important to keep the relative humidity below = 50% (at 7 0 ° F / 2 3 ° C ) to prevent rehydra.. tion during insertion of the sample into the microscope. 2. 4. Cryotransfer and use of a cryostage

To examine cryosections in their frozen hydrated state - which is one of the few ways of obtaining data on local wet weight concentrations the sections must be transferred after sectioning and collection on grids directly into the electron microscope and maintained at ,~..nperatures < - 1 4 0 ° C , or below the transi,on temperatt, i e from the vitreous state to hexagonal ice [25]. Commercial transfer devices are available which basically consist of a heater-regulated stage to which is attached a liquid-nitrogen-filled dewar. The cryosection is placed in a small grid-sized cup and transferred in a thermally shiclded holder to the stage which has been precooled within a liquidnitrogen-cooled receptacle. The stage has two movable covers on a mechanical rod to shield the section during the final rapid (< 3 s) transfer to the microscope airlock. The airlock can be pumped with a cryosorb pump to minimize contamination but more often it is simply pre-evacuated with the (trapped) rotary fore-pumps of the microscope. Although cryotransfer stages have been in routine use for many years [25], the use of fully hydrated cryosections for high resolution analytical studies is limited fror~ two aspects. Firstly~ the EDX signal is severely reduced due to absorption of X-rays by water (ice); for resolutions smaller than the size of a nucleus, such a large electron dose has to be applied to obtain reasonable signal statistics that melting or radiolysis effects disrupt the section. Secondly, frozen hydrated sections are usually too thick ( >_ 1200 ,~,) to -

A. LeFurgey et al. / Electron probe microanal.vsis in cell physiology

be useful for energy loss absorption edge studies, although recently the zero-loss peak has been used to measure mass thickness in the hydrated state prior to subsequent X-ray analysis on the same freeze-dried sections [62]. Conversely the question arises whether there is benefit in using the cryostage to examine freeze-dried sections. While Echlin has opined (in an Editorial [13]) that "it is astonishing that one should continue operating an electron beam instrument at ambient temperatures", it remains to be seen whether this is a :;light exaggeration. Cantino, Johnson and colleagues [74] have shown that mass loss of most elements of biological interest (except possibly for sulfur) is about the same at - 160 ° C as at + 25 o C at the doses used for EPXMA. This may not be true for mass gain, i.e. the propensity of elements to diffuse toward the beam [75], although if one has a perfectly clean column then the limitation is only the specimen itself. On balance it would seem preferable to work at low temperatures since if there is slight contamination at the specimen from whatever source, which is unavoidable completely, the effects will be minimized. Since the quantitation procedure (see section 2.5.3), depends on an accurate measurement of the continuum portion of the X-ray spectrum, it is crucial to ensure that both the column and stage in the specimen are free from residual contamination from both organics and water. It is therefore advantageous to place an inexpensive gas analyzer near the specimen region on the microscope in order to monitor partial pressures of H 2 0 and organics, and to appropriately bake out the microscope column and stage rod frequently. Some instruments have been equipped with helium cryopumps to reduce water and hydrocarbon contamination [76], although it is sufficient to have a well-trapped conventional silicon-free oil diffusion pumped vacuum system. As a matter of practice one can leave the cryostage inserted into the column even when it is not in use. This can result in a significant improvement in the consistency of data. Parenthetically, it is of tremendous value to be able to continuously monitor the mass loss (or gain) while acquisition of a spectrum is in progress [77]; this will immediately reveal if there is a contamination prob-

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lem. At minimum, if one is not able to monitor the X-ray spectrum directly, the intensity of the transmitted or backscattered electron signal should be monitored [78].

2.5. Electron probe X-ray microanalysis 2.5.1. Instrumentation 2.5.1.1. The electron microscope. X-ray microanalysis can be performed in either a conventional transmission electron microscope (TEM), a scanning electron microscope (SEM) or more usually in a TEM to which has been added scanning and small spot probe capabilities. The probe size can be made smaller in the latter case, providing t ~ pcssibility for high spatial resolution studies. Wl" a field emission ele.tron source is used, as by Somlyo and his colleagues [79], X-ray imaging resolutions of 87 A have been demonstrated on model specimens, and routine quantitation can be carried out in the 100 A, range. Most commercial instruments today support the operation of a lanthanum hexaboride (LaB6) filament which gives a factor of about three more intensity per unit area probed over a standard tungsten source. Increased intensity reduces the time necessary to acquire acceptable statistics. While there appears to be no rigorous rationale for the choice of accelerating voltage used, generally for high resolution studies voltages between 60 and 120 keV are employed, consistent with obtaining an optimum balance between signal-to-noise ratios and contrast within the specimen. However, lower voltages (10-50 keV) are often all that are availat"e in SEMs and since these are well above the K-shell absorption edges of all elements of biological inter,..,t, quantitative data can easily be obtained. The main practical consideration in analytical instruments is that both the colunm producing the beam at the specimen and the energy-dispersive X-ray detector device are well collimated so as to exclude extraneous signals. There is going to be some contribution from extraneous sources in all electron microscopes due primarily to secondary, or tertiary X-ray fluorescence from grid bars, the column, pole pieces, etc.: however, it can be minimized by use of suitable thick condenser aper-

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ture~, careful alignment and use of a non-beam defining (hard X-ray) aperture placed just above the specimen. This subject has been treated at length by Williams [80] and others [75]. Most modern commercial instruments satisfy the requirement that the extraneous instrument-derived discrete signals are eliminated and that Bremsstrahhmg is less than 1% of the specimen signal [75]. As stated previously, X-ray microanalysis in general, and biological EPXMA in particular, is a trace analysis technique. The quantum efficiency of the X-ray excitation process is only about 1% and the concentrations of elements in the sample can be as low as 0.1% by volume. To obtain the maximum detectable signal, the largest possible Si(Li) detector crystal must be placed as close as possible to the sample. In most situations this requires that a 30 mm 2 detector be placed in a side-entry column configuration (usually at 0 o to 20 ° azimuth) in order to subtend a solid angle of about 0.15 sr. Consequently the specimen has to be tilted between 6 ° and 40 ° toward the detector with concomitant parallax effects on the image. A probe current of about 10-gA at the specimen rarely produces a count rate of more than a few hundred counts per second for other than pathological tissues with large intracellular inclusions [81]. Thus rather long counting times (100 to 500 secs) can be required in order to quantitate elements (see section 2.5.1). With a so-called top-entry detector which is placed above the objective pole piece (azimuth angle, = 60 ° ), the count rate is much lower since the solid angle in this case is only = 0.01 sr. Other problems with this configuration have been discussed by Warner et al. [82].

2.5.1.2. The X-ray spectrometer There are several excellent commercial energy-dispersive instruments available. We have not considered the use of wavelength-dispersive spectrometers in this review since its purpose is to focus more on high resolution analytical electron microscopy. Because of the lar!e probe currents required to obtain acceptable counting statistics, the resolution of WDX equipped instruments is limited usually to whole cells (or microdroplets) as far as accurate quantitation is concerned. For an excellent review

of this field the reader is referred to the work of Lechene and Warner [83], Roinel and de Rouffignac [84] and others [85]. Energy-dispersive systems basically consist of a liquid-nitrogen-cooled Si(Li) crystal in which the impinging X-rays from the sample produce charges in the form of pulses. These are collected by application of a high voltage across the crystal, passed through a sensitive charge amplifier, pulse processor, analog-digital converter and multi-channel analyzer to sort them into electronic voltage bins which are then subjected to various computer-based software algorithms, subsequent display and printout.

2.5.2. Analysis There are three basic methods by which a sample may be analyzed: (1) a stationary (focussed) probe in a conventional TEM; (2) a more finely focussed not-imaging probe in a S(T)EM; and (3) an analog or digitally rastered probe covering a finite imaged area. (A line scan is considered a one-dimensional raster.) Somlyo et al. [10,79] have very effectively used an astigmated probe in a TEM to analyze entire oval-shaped mitochondria, stacks of tubules of rough endoplasmic reticulum, and other regions within cells. A more common practice involves either (2) or (3). A region to be probed is identified in S(T)EM, possibly using the dark-field mode for low contrast specimens, and the lenses then switched so that the electron beam is fixed to probe only a spot within that region for a predetermined time or statistically significant number of accumulated X-ray counts. The problem with this approach is that on very small structures specimen drift can result in movement out of the beam, although there have been methods devised to compensate for this [86,87]. The alternative is t9 place a small raster of known size at high magnification within the structure to be analyzed; if there is any slight movement of the specimen, the beam can be electrically shifted (manually) to bring the region back to its original position. With practice this can rather easily be done by monitoring both the intensity waveform of the small raster and the image itself. A further indication of specimen shift, of which there is always some possibility when using a cryostage, is to monitor, with time, the counts in each X-ray

A. LeFurgey et ai. / Electron probe microanalysis in cell ph.vsiologv

region of interest: any departure from linearity indicates either contamination (mass loss/gain), change in beam current or specimen shift. Digital control of the electron beam implies that, assuming there are enough counts per unit dwell time, every manipulation that is used to quantitate data can be applied to any image data that might result. This is potentially the most exciting development in X-ray microanalysis of biological specimens because for the first time it offers the possibility of performing true "microchemical microscopy" of physiological events on a sub-cellular scale. While this may sound rather grandiose, the initial results of quantitative digital imaging are most encouraging from a number of laboratories [86-91]. Basically, a form of quantitation has to be performed at each pixel and is therefore limited by (a) the dwell time per pixel, and (b) the speed and number-crunching ability of the computer. (a) The dwell time should provide good enough counting statistics so that an image can be formed in a reasonable amount of time, e.g. a 128 x 128 image will take 55 rain at a dwell time of 0.2 s/pixel and over 9 h at 2 s/pixel. (b) As noted elsewhere if one accepts data from nonfiltered and fitted spectra, usually in the form of counts within selected spectral regions including an assumed background, then reasonable "concentration images" can be formed after collection and storage of the image using straightforward mathematical software on commercial analyzers. Quantitative digital imaging has the advantage of convenience since one can always re-probe an interesting region later to obtain better statistics if necessary. Fiori, Leapman and colleagues argue elsewhere in this volume [91] that given the computer power, even if collection is for short times ( = 0.2 s/pixel) with only relatively few counts, good filtered fits can be made at each pixel and an accurate quantitative elemental image obtained. T!.ey p~:gue further that this is a more efficient approach since one can later identify arrays of pixels corresponding to a specified region of the cell and thus ,.,o,~,."t"~:-better overall statistics than would have been practical with a "mandrel" probe. Again any regions which might be to,~ low in concentration for a particular element :an be re-probed in a spot or small raster mode.

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2.5.3. Quantitation 2.5.3.1. Algorithms.

Hall and co-workers [92-97] have summarized the methods available for quantitation of the composition of biological thin sections. Each of these generally used calculations can have application to the determination of concentrations in cryosectioned tissues or cells. There are many detailed reviews of these approaches [92-97], so they will only be summarized here. The first technique as described by Hall and Gupta [1,92-97] involves measurement of elemental ratios, in which, for thin specimens, the count rates of any two elements are proportional to their relative elemental concentrations. Such ratios can be determined without standards or with only a few standards of known composition. The second technique, measurement of elemental concentrations, uses characteristic radiation along with a peripheral standard. In this calculation the signal intensity for a given element is directly proportional to the local mass of element per unit area; this proportion is compared to that of the known mass and intensity of the standard. Since the standard is in direct contact with the unknown, it is assumed that standard and tissue are of identical thickness, and the mass of the element per unit volume is thus determined. Rick and DiSrge and coworkers have developed this method is conjunction with analyses of diffusible elements in cryosectioned kidney, amphibian bladder and skin [65-67]. Sections thus evaluated must be cut without chipping, fracturing or edge compression to insure uniform standard and tissue thickness and must freeze-dry without shrinkage or with uniform shrinkage only. Some questions have been raised regarding transport of ions between sample and standard and water between standard and sample [98] as well as the validity of all the assumptions [99]. Appropriate control experiments should be performed to establish that there is no effect of the standard on the cells. The third technique is generally referred to as the "continuum normalization" method and is the one most commonly used for high resolution studies. It was developed by Hall and Gupta and coworkers and is based on the premise that the continuum X-ray intensity gives a measure of 1o-

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cal total mass of the analyzed volume [1,92-97]. The continuum is usually measured in a region of the spectrum known to be free of any characteristic peaks, zlthough i~t can be measured directly under the peak. For sections thinner than about 1 /tm, X-ray absorption effects can be neglected [1,92-97]. Since the intensity of peaks characteristic of the energies of the excited atoms is proportional to their mass, concentration is simply the ratio of the elemental peak intensity to the continuum intensity. Ragorous uniformity of section thickness and standards is thus not necessary. This is the method that has been used most extensively for freeze-dried cryosections and for frozen hydrated sections. However, in the latter case and for relatively thick (> 1 #m) freeze-dried sections where high resolution is not the main concern, both the peripheral standard and the continuum normalization methods have been shown to give comparable results [47,48,53,98]. The continuum method has been refined by Shuman et al. [10,11] to take account of instrumental conditions and methods of spectral deconvolution. Digitally filtering for background removal and multiple least squares fitting to standards are the usual methods employed; it is preferable to use an external general purpose computer in order to process data either in real-time or subsequently, since most of the currently available commercial analyzers are simply not configured to operate fast enough to accomplish multiple calculations in a reasonable amount of time. The same situation applies for quantitative digital imaging (see articles by Fiori et al. [91] and Leapman and Ornberg [100] in this volume; also Somlyo and Shuman [79]). With the rapid rate of development of computing this will likely change in the near future. Shuman et al. [10], Roomans [101], and more recently Hagler et al. [102], Wendt-Ga!!ite!li and Wolburg [103], Kitazawa et al. [11], and Roos and Morgan [ie.l] have presented detailed methods for the preparation of standards for quantitation in thin cryosections. A widely used technique involves dissolving salts of the elements of interest in a gelatin matrix, freezing the gelatin, cryosectioning and performing EPXMA on the freeze-dried section [100,101]. The 2.5.3.2. Standards.

procedures of Shuman et al. and Kitazawa et al. [10,11] utilize standards which mimic the homogeneous matrices of biological materials, as does gelatin, but which are made from solutions of proteins, such as human or bovine serum albumin and phosvitin. These contain known stoichiometric amounts of covalently bound elements, and are analyzed in the same way as the cell or tissue samples. Small droplets of solutions of the proteins or mixtures are put on carbon-coated grids and allowed to dry before analysis at - 1 2 0 to 135 ° C. Small quantities of salts of the elements of interest (Na, Mg, P, S, CI, K, Ca) can be added to vary the concentration range provided the resultant matrix remains homogeneous. Aliquots of the standard solutions are then analyzed in parallel by two methods: (1) measurement of absolute concentrations by atomic absorption spectroscopy (Na, Mg, K, Ca) or mJcrochemical analyses (P, S, CI) and (2) determination of the appropriate proportionality constant from measurement of the l:eak-to-continuum ratio in the X-ray analyzer. Standard curves, which can be generated for the two parallel data sets, have shown close agreement between EPXMA and atomic absorption spectroscopy measurements [10,11]. -

2.5.3.3. Minimum detectaqle concen:ra:io~; a~;d mass.

Johnson and Cantino outlined the criteria for minimizing errors in EPXMA and concluded that if those errors were made negligible then the minimum detectable mass (MDM) and concentration (MDC) would only be limited by statistical noise due to the finite number of X-rays in a given peak and in its underlying continuum, in addition to those in the continuum region used for quantitation [6]. Parameters affecting both MDM and MDC include the X-ray counting time per spectrum and/or the total number of spectra collected (total counting time), the beam current and the probe diameter which for a given electron source will also determine the spatial resolution. The best experimental values for MDM and MDC obtained to date are those of the Somlyos and colleagues in which = 10-19 g of Fe in ferritin have been detected using a field emission source [79]. Experimental determinations of MDC for most elements of physiological interest are in the range

A. LeFurgey et al. / Electron probe mwroanal.vsis in cell physioiow

of 1 mmol/kg dry wt [6,10,79]. In the special case of the physiologically important element calcium EPXMA has been extended to its practical limit of sensitivity by Bond, Somlyo et al. who were able to detect 300 #tool Ca/kg dry wt in the cytoplasm of cryosectioned smooth muscle cells [2,105,106]. Measurement of Ca in most biological systems is complicated by the facts that (a) small amounts of Ca occur in conjunction with large amounts of K and (b) in an X-ray energy spectrum the K Ko peak overlaps the Ca K,, peak. Significant errors due to the overlap can occur unless both peak controid position and peak resolution are maintained consistently between reference standards and sample. It can be shown [10,11], for example, that a shift of 5 eV in peak calibration can result in a 30% error in measurement of calcium concentration. After developing a multiple least squares fitting routine which included the first and second derivatives of the K K,,t~ X-ray peaks to correct for changes in calibrations, Kitazawa et al. [11] measured in Ca standards 1 _+0.2 mmol Ca/kg dry wt in the presence of 500 mmol K / k g dry wt. Appropriate precautions to eliminate any source of Ca contamination from the environment or from specimen support films must also be observed. In addition to these steps, extension to the sensitivity of 0.3 mmol (300 #mol) Ca/kg d~, wt with an error of _+0.1 mmo|/kg dry wt required long collection times and a large number of spectra (300-500 s/spectrum, 30 h total counting time) [2,105,106]. As Bond and colleagues point out, to double the sensitivity one must quadruple counting time (or increase beam current0 because the X-ray counting process follows Poisson statistics and thus the square law [105,106]. At higher Ca levels (--- 100 or more mmol C a / k g dry wt), such as is found in the terminal cisternae of skeletal muscle under control conditions, or at lower K levels ( < 100 mmol/kg dry wt), such as is found in heart or kidne', cell cytoplasm and mitochondria after treatment with Na-K ATPase inhibitors. quantitation of Ca requires significantly less counting time. 2.5. 4. Statistics

The basic statistical principles of nested sam-

195

piing and analysis of variance apply to element quanfitation data, in determining numbers of experiments, sections or cells to be analyzed to obtain significant results [107]. Using a complete analysis of variance for nested samples on data from a preliminary or pilot study, one can obte.in an overall standard error written in terms of the estimated components of variance among experiments, blocks from each experiment, grids from each block, and sections from each grid. The F test can then be applied to determine significant differences at the 5% or 1% levels among experiments, among blocks, or among grids, etc. Based on these comparisons, changes can be made in sampling size at one or more levels to improve the standard error at no increase in effort or to obtain the same standard error at less effort. For most comparisons of concentration data, obtained by EPXMA, a multiway analysis of variance which takes into account unequal subclass numbers is utilized [108]. F values for experimental variation and manipulation !ifferences in elemental contents of cytoplasmic, mitochondrial, nuclear or other compartments are compute0 For significant F values obtained by analysis of 'variance, a Duncan's multiple range test is carried oat to determine the group or groups that are different from the others [109]. The more simplistic twotailed Student t-test should be utilized with caution because in most cases inter-animal variability (except perhaps for Ca) is significant. 2.6. Electron energy loss spectroscopy

Electron energy loss spe:'tv':'-?.etry trZrZL~t~..,. .___and imaging have not been applied widely in cell physiology, although the5' have been shown to have the potential for detection of low concentrations [110] of biologically important elements, possibly at high resolution [111] apprcmching that of the microscope itself [1i2,113], and potentially at greater sensitivity than EPXMA. The main problem in tissues or isolated cells is in preparing thin enough (cryo)sections to avoid multiple electron scattering which requires spectral deconvolution and necessitates application of special processing techniques [110,115] to extract the reasonably good signalto-noise ratios from very small peak-to-back-

A. LeFurgey et al. / Electron probe rnicroanalysis in cell physiololo'

196

ground spectra. The use of intermediate (300 keV) high voltage microscopes could improve this situation somewhat. The reader is referred to numerous excellent reviews of the field including Leapman and Ornberg [100] in this volume. The basic instrumentation consists of either a magnetic sector spectrometer [116,117] or prism filter [113,114,118] placed so as to collect a n d / o r image the electrons passing through the specimen. Without discussing the merits of either method, most of the quantitative data to date have been obtained from some form of photoelectric counter placed directly behind a slit through which the spectrum can be scanned (serial collection) in a magnetic sector instrument (parallel detectors using diode arrays are now available, which greatly increase the efficiency of collection). As in EDX instruments, data can be fed into a multichannel analyzer for subsequent processing. In our laboratory [62] as well as others [119], a significant potential use for EELS has been found when direct localized information is required on the hydrated state. This is most important when comparisons are being made with the physiological measurements [18] on living cells and tissues. A direct measurement of the mass-thickness can be obtained using the zero loss peak in the electron loss spectrum [62,119]. Massthickness images can be obtained, areas of which can then be X-ray probed after ireeze-drying in the microscope. However, there are several other ways of doing this, for example by measuring and monitoring any aspect of the normal transmitted image [120] as well as the backscattered electron signal [78].

3. Physiological applications of EPXMA This review primarily covers physiological studies published in the past five to ten years. Empha~,J , ~ q v , A ,it

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which correlative physiological, biochemical, ultrastructural and X-ray microanalytical techniques have been utilized to address fundamental questions about cell function. The reader is also referred to other excellent reviews of the literature [2,3,6,121-123].

3.1. Skeletal and smooth muscle 3.1.1. Skeletal muscle Rana pipiens occupies an important niche in the short history of EPXMA as it has been applied to the measurement of the sub-cellular elemental composition of biological tissues. Somlyo and coworkers chose to use freeze-dried cryosections from fast twitch muscles from the frog as test objects to determine the power and validity of this technique for measuring sub-cellular elemental concentrations [124]. Consistency between previous bulk measurements of fiber K and K measurements by EPXMA was demonstrated; the resolution of the technique was defined by measurements of the elemental composition of the terminal cisternae (TC) using probes 50 nm in diameter [125]. The TC contained low Na and C1 and high K, thus unequivocally identifying it as an intraceUular organdie and excluding the possibility of it being in communication with the extracellmar space. In particular, the absence of a C1 gradient across the sarcoplasmic reticulum (SR) membrane and the absence of C1 redistribution during stimulation [126] indicated that there was no major change in electrical potential across the SR membrane during stimulation. The TC was also found to be a major Ca store, its Ca content accounting for 60-70% of total fiber Ca [127]. Most of this Ca (59% or 69 mmol/kg dry wt) was released during a 1.3 s tetanus; this represented an increase of 3-4 mmol C a / k g dry wt in the cytoplasm, sufficient to saturate the Ca specific sites on troponin and the Ca-Mg sites on parvalbumin. Release of Ca from the TC during electrical stimulation was accompanied by uptake of some K and Mg, which partially neutralizes the charge of the released Ca. Ion movements between cytoplasm and SR were also measured during relaxation from a tetanic K, U I I K , ~ I l t l

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accounted for by the rapid uptake of Ca into the SR, in an amount equal to that bound to the low affinity Ca specific sites on troponin. Return of the released Ca to the TC also included a second slower component with a rate (tl/2 = 1.1 s) comparable to the off-rate of Ca from parvalbumin, thus identifying Ca removal from parvalbumin as

A. LeFurgey et al. / Electron probe microanalysis in cell physiologv

the rate limiting factor for return of Ca to the TC. Although EPXMA is unsurpassed in its spatial resolution, it lacks the ability to distinguish free from bound forms of the elements measured. Maughan and Recchia [128] developed an elegant technique for measuring, with EPXMA, diffusible elemental concentrations in frog skeletal muscle cells. They equilibrated microdroplets with single muscle fibers skinned under oil and measured elemental concentrations by wavelength dispersive EPXMA in the freeze-dried droplets. The ratio of diffusible to total fiber K (0.5) implied significant ion binding. While these measurements were not obtained by energy dispersive EPXMA (the focus of this review), the demonstrated ability to extend EPXMA to measure diffusible elemental concentrations should be of general interest. A number of important questions about the mechanism of excitation-contraction coupling in skeletal muscle have been addressed by EPXMA. The area of muscle pathology now awaits the introduction of a similar rigorous quantitative approach. Preliminary reports already indicate that substantial redistributions in total cell Na and K occur in a number of muscle disease states [129]. The potential for correlating ultrastructural and microchemical alterations with physiological events, such as excitation-contraction coupling (ECC), which proceed at ve~ fast rates (milliseconds) has recently been demonstrated by Sommer and colleagues in single intact frog skeletal muscle fibers [45]. These investigators have developed instrumentation and techniques for quick-freezing single fibers at known time intervals after electrical stimulation (0.9 s or less); segments of each quick frozen fiber can then be investigated in parallel by (1) high resolution electron microscopy following freeze substitution, (2) freezefracture/etching and (3) EPXMA after cryosectioning. These experirr, ep's are the first to demon~,-.,,~ ,~,~, ~VYMA ,"an he performed on tissues frozen with an extraordinarily high time resolution, a resolution fast enough to document changing element conccntration such as may occur during ECC. OLIO,.L~,

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197

in smooth muscle SR could also act as an intraceUular Ca store. This was first shown by the accumulation of strontium, an electron dense Ca substitute, into the SR and into the contiguous perinuclear space [130]; Ca localization in the SR of unfixed cryosections soon followed [131]. While Ca release from the SR has been demonstrated in skeletal muscle [126], the physiological role of the SR in smooth muscle remained, until recently, controversial. In !984, however, release of Ca from the junctional SR of rabbit portal vein by norepinephrine (NE) was measured by EPXMA [105]. This occurred in the absence of extracellular Ca and the Ca released was also sufficient to maximally activate the muscle cells [105]. A further series of experiments demonstrated that, even in the presence of normal extraceUular Ca, the central SR in the main pulmonary artery (MPA) released Ca upon NE stimulation [132]. Thus, this series of experiments supports the view that the SR plays a primary role in excitation-concentration coupling in vascular smooth muscle. The unique capability of EPXMA to measure subceUular elemental composition enabled Somlyo and co-workers to obtain, for the first time, dire,'* in situ measurements of the Ca content of mitochondria in smooth muscle cells. It was found that in undamaged cells (low intracellular Na), mitochondria contained less than 3 mmol Ca/kg dry wt [133], even after a maintained 30 min contracture with K and NE [lC6]. Therefore, smooth muscle mitochondria do not appear to constitute an important intracellular Ca store nor to regulate cytoplasmic Ca levels in vascular smooth muscle. These results are consistent with earlier in vitro kinetic measurements of mitochrondrial Ca uptake in the presence of Mg, which demonstrated that the affinity of mitochondria was too low and the rate of Ca uptake too slow to regulate physiological processes [134,135]. Recent improvement in Ca quantitation ir~ "he presence of high K, in which corrections for centroid shifts and changes in peak resolution were included in the quantitation routine [11]. permitted the measurement of low Ca concentrations in the presence of K with even greater accuracy than had previously been achieved. Followin~ these developments, it was possible to show that

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A. LeFurgey et ai. / Electron probe microanalysis in cell physiology

while no change in mitochondrial Ca occurred during a maintained 30 rnin contracture, total cytoplasmic Ca increased significantly, by 1.0 + 0.2 (SD) mmol Ca/kg dry wt of cytoplasm, from 0.8 + 0.2 to 1.8 + 0.2 mmol Ca/kg dry wt cytoplasm [106]. This increase was greater than could be accounted for by Ca binding to calmodulin, suggesting the presence of other, yet unidentified, cytoplasmic Ca-binding sites. 3.2. Heart The importance of ion transport, element content and compartmentation in understanding the physiology of an excitable tissue such as heart has made the cardiac cell an obvious model for examination by EPXMA. However cardiac muscle has not been studied with high resolution EPXMA in detail comparable to skeletal or smooth muscle. Early investigation centered on intact tissues such as papillary muscle from guinea pig, rat or rabbit heart [63,64,136-144], while other more recent studies have utilized single isolated [145] or cultured cardiac cells from rabbit, embryonic chick [18,146] or rat [147]. 3.2.1. Papilla~ muscle Wendt-GallitelU and colleagues have investigated the contribution of vario~as cellular compartments to the events of electrochemical coupling in guinea pig papillary muscle [137-140]. Previous postulates suggest that the development of tension in cardiac muscle, in contrast to skeletal muscle is modulated not only by calcium movements between different intracellu!ar compartments, e.g. sarcoplasmic reticulum, but also by calcium movement from the extracellular space into the cell. The experiments of Wendt-Gallitelli suggest that different intracellular Ca stores can account for the early and late components of contraction. In cardiac muscle calcium accumulation required to maximally elicit a mechanical response either by paired (or short) interval stimuli was located by EPXMA only in sites corresponding to dense rete at the level of the Z-lines of sarcomeres. High potassium and low chlorine at these points confirmed that these were intracellular sites. Calcium accumulation during the late component of contraction was localized at sites corresponding to

T-tubuli and JSR. After stimulation with noradrenaline to produce a "late" type contraction, high concentrations of calcium were located over sarcomeres, Z-lines and cell membranes but not over mitochrondria. These results provided direct information that the intracelluiar distributions of calcium and other elements in guinea pig papillary muscle change during different states of electromechanical coupling, and confirmed the localization of Ca to sarcoplasmic reticulum but not mitochondrial regions. Basal measurements of element distribution in rabbit papillary muscle by Wheeler-Clark et al. [64] and in rat by Cantino et al. [14] are similar to the results of Wendt-Gallitelli in guinea pig. Under certain conditions of stimulation of N a - C a exchange which lead to elevation of total cell calcium such as incubation in low-Na or Na-free media [64], Na and CI contents in all cardiac cell compartments have been observed to change and Ca shown to increase in sarcolemmal boundary regions, over T-tubule boundaries and in junctional sarcoplasmic reticulum, but not in mitochondria. Thus in papillary muscle mitochondria, endogenous Ca appears to be normally low and becomes elevated only during altered metabolic or transport states, such as inhibition of Na-K ATPase with cardiac glycosides [17]. EPXMA of structures such as the sarcolemma and sarcoplasmic reticulum in cardiac muscle is crucial for understanding the role of these cell compartments in excitation-contraction coupling. However, because these structures in heart cells are an order of magnitude smaller than the analogous structures in skeletal muscle, occurring at sizes near the resolution limit for EPXMA (-- 10 nm), Tormey and Wheeler-Clark [136] have developed and applied a method of spatial deconvolution in which X-rays that arise from the structure of interest can be distinguished from those that arise from adjoining phases. Applying the deconvolution correction did not change the relative outcome of paired experiments in which papillary muscles were bathed in control or low Na solutions (see above). However, sarcolemmal Ca concentratiens were doubled by the correction for X-rays coming from adjacent phases but junctional SR (JSR) Na, Mg, P, S, Cl, K, and Ca

A. LeFurgey et aL / Electron probe microanalysis in cell physioloD'

concentrations were not changed by the correction procedure. The values for JSR elem ~,,t concentrations resembled those of the cytoplasm and were also similar to those reported by Somlyo et al. [124,125] for terminal cisternae of skeletal muscle with the exception of Ca which was lower in the cardiac JSR. 3.2.2. Cardiac cells For EPXMA studies of either normal or altered /unction, cultured or isolated cardiac cells offer important physiological advantages, such as short ~1." AP~P. " u u t U s l o n distances and steady state conditions, together with technical advantages including ease and rapidity of manipulation for quick freezing. The early work of Buja, Hagler and colleagues utilizing an intact cardiac preparation illustrates the interpretive problems associated with tissues in which functional, structural, and compartmentation phenomena cannot be or are not measured simultaneously [142,143]. In these preparations pathologically elevated Na [142] could either be attributed to cell damage prior to quick freezing or, as the authors later suggested [60], to preparative artifacts such as rehydration following freezedrying. In cultured or isolated cell preparations, parallel measurements of total cell ion content or ionic activity can easily be performed; the independent data sets allow evaluation of cell viability as well as definition of problems associated with each measurement technique. 3.2.2.1. Cultured cells. EPXMA and ion selective microelectrodes have been utilized to determine the activity, content and compartmentation of Na, K, and CI in cultured embryonic chick heart cells [18]. The results obtained by the two techniques for cell cytoplasm are in remarkably close agreement, with the K" Na ratio being = 10-15 to 1. Calcium determined by EPXMA is low in mitochondria (2.3 __ 2 mmol/kg dry wt); calcium is higher in cytoplasm, which probably includes some regions of sarcoplasmic reticulum (4.5 _+ 1.9 mmol/kg dry wt). The total cell calcium (Cato t) calculated from EPXMA is = 0.75mmolar, less than that of atomic absorption (AA) measurements of total cell calcium, which range from 1.8-3.7mmolar (10 to 20 mmol/kg dry wt).

199

EPXMA eliminates the contribution from extraneous Ca compartments, e.g. extracellular space, which must be estimated and corrected for in bulk measurements. The cultured myocyte model utilized in conjunction with microscopic and microanalytic techniques (EPXMA) has also provided the capability for assessment of cell injury on a cell-to-cell basis, as well as within cellular compartments. Buja and colleagues [147] recently documented changes in ultrastructure and element composition in cultured neonatal rat cardiac myocytes in which impaired energy metabolism and cell injury were produced by the metabolic inhibitor, iodoacetic acid (IAA). Exposure to IAA for one hour produced a mild, reversible decrease in ATP and minimal ultrastructural changes; EPXMA performed on rapidly frozen, cryosectioned and freeze-dried cells demonstrated that by one hour 10 to 25% of cells had increased Na and Ca content in mitochondria and cytoplasm. At 1.5 to 2 h more than 75% of cells had increased Na and Ca and decreased K and Mg in mitocondria, cytoplasm and nuclei; at these times ATP was irreversibly depressed and severe structural changes had occurred. The results demonstrated the heterogeneity of response which occurs during the progression of cell injury; this type of information cannot be obtained with bulk chemical measurements performed either on cultured heart cells or on intact cardiac tissues. 3.2.2.2 Isolated cells. Single cardiac myocytes obtained by enzymatic digestion of rabbit or rat hearts have been utilized by Chiesi et al. [145] for parallel EPXMA and isotopic tracer analyses of Ca transport to determine the role of the sarcoplasmic reticulum (SR) in phasic contractile activation. EPXMA analyses of freeze-dried cryotll~ ~_..u ,~,tl ~.,ff tvsections revealed Na composition in .t.A ...,,., plasm (Na, 140mM; K, 18.2 or 40.7mM) similar to that found in the bathing media (Na, 158mM: K, 5.9mM or 15.9mM). But, the high K concentration in the cytoplasm was not maintained after dissociation, suggesting the presence of electrochemical shunting across the external membrane. These elemental changes were evident even though myocyte ultrastructure was maintained i,a an apparently

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"normal" state. Ca uptake and phasic contractility, although dependent on mitocondrial respiration, were not affected by the mitochondrial uncoupler (FCCP) in the presence of ATP and phosphocreatine. Ca uptake could be increased by oxalate, w.hich was identified within the SR as calcium oxalate crystals by both electron mi-. croscopy and EPXMA. The lowest Ca concentrations sustaining Ca transport ((1-5) x 10-7M) also induced phasic contractile activity in the myocytes. Increasing Ca concentration up to 10-6M increased transport velocity and rates of phasic contraction. If free Ca in the medium was > 10-6M, cardiac myocytes went into contracture and degenerated, with Ca, Mg, and P accumulated in the mitochondria. When the Ca content of the medium was maintained at a constant level, both transport and contractility were affected by Mg, temperature, cyclic-AMP and methylzanthines. The EPXMA results, showing an absence of Na and K transmembrane gradients, and other morphological data documented that the cells were hyperpermeable and did not support cyclic electrical activity that would account for the beating activity of the myocytes. The authors concluded that the beating, as in skinned fibers, could be attributed to a primary release of Ca from the SR, i.e. that phasic contractile activation occurred independently of sarcolemmal excitation. 3.3. Liver

Ca homeostasis in the liver has been an area of contention for many years. Controversy has centered around the relative roles of endoplasmic reticulum and mitocondria as primary intracellular stores of Ca. In vitro measurements of mitocbondrial Ca content suggest that mitocondria could store up to 70% of cell Ca [148]. However, the Ca content of isolated mitochondria appears to be dependent on the composition of the med,a and the procedure used for isolation EPXMA of cryosections of liver rapidly frozen in situ seemed an ideal approach for assessing the true in vivo Ca content of the rnitochondria, since these measurements could be obtained in intact

cells: Cameron and co-workers [150] had already shown that rat liver could be successfully frozen in situ, with sufficiently good ultrastructural preservation (minimum ice crystal damage) to measure subcellular elemental composition by EPXMA in freeze-dried cryosections from the frozen tissue. EPXMA of unstimulated liver rapidly frozen in situ in anesthestized rats revealed that mi,ocondria normally contained only 0.8 + 0.2 mmol C a / k g dry wt; Ca content of the rough endoplasmic reticulum, however, was 6-7 fold higher [151]. These results are inconsistent with mitochondria playing a role in the regulation of cytoplasmic Ca, while the much higher Ca content of the rough ER implicated this organelle as an intracellular store of Ca that could be released by Ca-mobilizing hormones such as vasopressin. 3.4. Pancreas

Quantitative EPXMA has also been performed on rapidly frozen, hydrated or dried sections of human pancreas by Roos and Barrard [152] and of dog pancreas by Nakagaki and colleagues [153]. Changes in elemental composition were examined under resting conditions and conditions of stimulation of the pancreatic acinar cells which lead to membrane depolarization due to an increase in membrane conductance. These changes were postulated to be due to an increase in surface cell membrane permeability to Na, C1, and K which would be reflected by changes in intracellular content of these ions. The cytoplasm had low Na (4.82 + 1 mmol/kg wet wt) and lfigh K (132 _+ 15) concentrations in comparison to acinar cells of other exocrine glands such as the submandibular gland. Secretory (zymogen) granules contained low Na (6 + 5) and K (60 + 15), measurable Ca (7 +_ 5) and very high S (172 + 25). Endoplasmic reticulure (ER) cisternae contained 2-5 mmol Ca/kg wet wt: no data were given for mitochondrial Ca content. Pilocarpine stimulation increased cytoplasmic Na, C1, and Ca (3.7 + 1.4) and decreased K. The results suggest that the ER and secretory granules can serve as intracellular Ca stores; alternatively Ca influx could be from the extracell'alar space.

A. LeFurgey et aL / Electron probe microanah'sis in cell physiology

3.5. Kidney The nephron is specialized structurally and functionally into segments with varied cell types which transport widely differing amounts of electrolytes and fluid; to analyze the mechanism involved in transport, one must determine element content of individual cells. EPXMA studies of kidney cells were begun as early as 1971 by Kfisz and colleagues [154] and 1976 by Trump and co-workers [155]. The majority of quantitative experiments have been performed by investigators utilizing thick (0.25-1 ~m) cryosections from rapidly frozen intact rat kidney for analysis in lower resolution scanning electron microscopes; another important experimental difference between some of these studies and those higher resolution studies cited for skeletal and smooth muscle, liver, and heart is that quantitation was achieved by a method developed by Rick et al. [6:,-67,256-159] which compares the characteristic radiation obtained from cells with that obtained from a standard albumin layer applied directly to the cells (see section 2.5.3).

3.5.1. Intact kidney 3.5.1.1. Cortex: proximal and distal nephron. Initially Beck, Rick and colleagues [156] determined the concentrations of Na, C1, K, and P in the nucleus and cytoplasm of freeze dried cryosections of superficial proximal and distal tubules of the rat kidney, The proximal tubule nuclear concentrations of Na and CI (20 and 23 mmol/kg wet wt) were significantly higher than those of the distal tubule (11 and 13 mmol/kg wet wt), while those of K and P (144 and 150 mmol/kg wet wt) were similar (143 and 175 mmol/kg wet wt). Measurements performed in the cer.~rally located cytoplasm of both cell types, close to the nucleus, showed Na and K values to be similar to those of the nucleus, whereas C1 and P were considerably higher. K: Na ratios were 7.4 : 1 and 12.5 : 1 for proximal and distal tubules, respectively. Compared to cell electrolyte concentrations of mammalian kidney determined by chemical methods, EPXMA values for Na were lower and for K were higher. Beck and colleagues attribute this discrepancy to the difficulties with chemical measure-

201

ments in estimation of the extracellular space with extracellular markers, as well as to the state of viability of tissue slices from which chemical measurements were obtained. In bo!h proximal and distal cell types C1 in the cytoplasm was = 50% greater than in the nucleus, in contrast to the uniform cellular distribution measured for Na and K. The nuclear membrane thus does not appear to represent an effective barrier to movement of sodium and potassium, so that the renal cellular space can be regarded as a single distributional space with respect to these ions. Subsequent studies by these researchers documented changes which occurred in the superficial proximal and distal nephron segments following ischemia [157] or potassium depletion [158]. In proximal tubule cells after 20 or 60 minutes of ischemia, Na increased = 4- to 5-fold (20 to 93 or 112 mmol/kg wet wt); CI increased = 3-fold (21 to 53 or 66); K decreased by more than 50% (141 to 65 or 42); P decreased by --- 30% (145 to 110 or 95); and the dry wt dropped from 22.6 to 20.3 or 17.5% of wet wt. No change occurred in distal tubule cells prior to 60 min of ischemia, at which point Na increased 7-fold (1! to 77 mmol/kg wet wt) and Cl, 3-fold (15 to 48); K decreased by = 40% (134 to 89) and P, by = 10% (168 to 145): the dry wt dropped from 20.8 to 18.4% of wet wt. The authors concluded that the changes in Na and K were caused primarily by inhibition of the Na-K ATPase pump, while changes in CI, P, and dry wt were attributable to an influx of extraceUular fluid. These content changes increased with the duration of ischemia, were reversible and complete within 60 lrdn but returned in some proximal tubule cells by 18 h of reperfusion. At 60 min of ischemia followed by 60 min of reperfusion, the apparently "normal" intracellular contents of Na, K, C1, and P gave no information as ~o the ultimate fate of the cells and thus no clue as to the persistent functioh .l impairment of the whole kidney.

3.5.1.2. Papillae. Saubermann and colleagues have taken a somewhat different technical approach in preparing and analyzing 0.5 ~m sections from rapidly frozen kidney cortex and papillae using higher cryosectioning temperatures and the

202

A. LeFurgey et al. / Electron probe microanalysis in cell physiology

Hall continuum method for quantitation [47,48, 98,160]. Controversies have arisen because the results, particularly with respect to Na and CI in the papillae, do not agree with those earlier results of Beck, Rick, Thurau and colleagues as discussed in the previous sections. Values for Na and C1 obtained by Saubermann et al. [98,160] are consistently higher than those of Beck et al. [156-159], while values for K and P are in good agreement. Resolution of these d:fferences is important because the data, in each case, support substantially different interpretations of the mechanisms by which cells adapt to the hypertonic environment of the medulla. Saubermann [98,160] attempted to resolve the discrepancies by performing experiments using cortical proximal tubule cells and papillary cells in which sectioning and analyses were conducted in parallel by the protocols of the two laboratories. The obvious major technical differences in the protocols were (a) sectioning temperatures ( - 53 ° C versus < - 80 ° C) and (b) quantitation and standardization procedures (Hall continuum normalization method versus Rick contiguous albumin external standard method). He found that both methods provided comparable results for the proximal tubule cells and that no differences were seen in element or water content on cryosectioning with albumin at - 5 3 ° C and at -80°C. Significant differences were obtained with the two methods when they were applied simultaneously to the cells of the rat papillae. The Hall continuum normalization method of analysis of frozen-hydrated/freeze-dried sections was used on undipped papillae while both the Hall method and the Rick peripheral standard method were applied simultaneously to contralateral papillae dipped in albumin standard. Saubermann et al. concluded that (a) albumin dipping significantly changed elemental and H 2 0 content in collecting duct and epithelial cells and (b) the albumin itself also changed element and HzO content in a direction consistent with movement of Na and CI from tissue to standard and H20 from standard to tissue. 3.5. 2. Isolated proximal tubule suspension More recently LeFurgey, lngram and Mandel applied X-ray analysis techniques to a suspension

of isolated kidney proximal tubules using thin cryosections (< 100 nm) in an analytical transmission electron microscope, the combination of which allowed resolution of intracellular compartments such as mitochondria [34]. In addition analytical parameters and instrumental conditions were optimized for determination of intracellular Ca compartmentation as well as Na, Mg, K, CI, P, and S, utilizing the Hall peak-to-continuum normalization method as modified by Kitazawa et al. [11]. In these studies two cell populations were identified: the first, representing the majority of cells, was termed "viable" as defined by a K / N a concentration ratio of = 4 " 1 . The measured cytoplasmic and mitochondrial Ca contents (4.1 _+ 1.4 (SEM) and 3.1 _+ 1.1 mmol/kg dry wt) yielded an average total cell calcium content of = 3.8 mmol/kg dry wt. The second population was termed "nonviable" as defined by a K / N a ratio of = 1" 1 and the occurrence of dense mitochondrial inclusions. The cytoplasmic and mitochondrial Ca contents (15 + 3 and 685 _+ 139 mmol/kg dry wt) yielded an average total cell calcium content of = 210 mmol/kg dry wt. The presence of 4 to 5% nonviable cells in the suspension would account for the average total cell -.alcium content of = 12.6 mmol/kg dry wt ( = 18 nmol/mg protein) measured in perchloric extracts of isolated proximal tubules by atomic absorption spectrophotometry (AA). Intact whole kidney tissues also display a large variability in total cell calcium content (4.5 to 18 nmol/mg protein or 3.4 to 13.5 mmol/kg dry wt) as measured by AA which could be accounted for by 0 to 4% nonviable cells. These results show that the normal mitochondrial calcium content in kidney is sufficiently low to control dehydrogenase activity (= 1 ~m) and probably too low to control cytoplasmic free calcium concentration. This heterogeneity in calcium distribution presents serious problems for the interpretation of results of calcium transport experiments using populations of cells or intracellular organelles. This result also has important implications regarding the heterogeneity which exists in cell populations normally and that which may be developed during the progression of cell injury leading to cell death. Comparison of elemental data shows these results in the proximal

A. LeFur~,ev et al. / Electron probe rnicroanalvsis m cell ph.vsiolow

tubule suspension to be similar to those of Beck and colleagues [156-158] and Saubermann et al. [47,48,98,160] given the limitations of conversion of dry weight to wet weight measurements.

3.6. Other epithelia As described by Thurau and co-workers [161], extensive research has been performed to understand water and solute transport across epithelial cell layers such as occur in the intestinal tract, gall bladder, urinary bladder of frog skin, cornea, and nephron (see section 3.4 above). A variety of experimental techniques, including measurement of electrical potential, uni- and bidirectional fluxes, ionic activities and transepithelial gradients have discl~sed some of the characteristics of transepithelial transport phenomena. EPXMA is ideally suited for determination of intracellular events occurring during transport, in particular, the route of transport, location of the transport pool, intracellular concentration and distribution of elements and their changes at altered transport rates [65-67,162-167].

3. 6.1. Frog skin One of the first important scientific controversies to be settled by EPXMA involved the origin and content of intracellular Na in frog skin and the implied relative permeabilities of the outerand inner-facing cell membranes [162,163]. As demonstrated by Rick, Thurau and colleagues, the outer epithelial layer of the frog skin and cornified cells of the stratum corneum showed element concentrations identical with those of the bathing solutions; ouabain, amiloride, vasopressin and Na-free solution corially produced no alterations. In all the layers of the frog skin composed of living cells, under control conditions, the intracellular Na content determined by EPXMA was = 9.4 and the K content was = 118 mmol/kg dry wt; Na, K, and C1 content in nucleus and cytoplasm were essentially identical. When the active transport step for Na was inhibited by application of ouabain to the corial (inner) membrane, the intracellular Na of all epithelial cell layers increased by = 100 mmol/kg wet wt, balanced by an almost identical drop in K. The ouabain effect was negated by application of an epithelial (outer)

203

bathing solution either free of Na or containing amiloride. These data demonstrated for the first time that the various living cell layers of the frog skin have almost identical intracellular Na and K concentrations under a variety of experimental conditions at various transport rates. Such similarities suggested that the intracellular space of the living cells represented a single distributional space for Na and K i.e. a syncytial Na transport compartment. Because the bulk of the intracellular Na was shown to exchange easily with the epithelial (outer) bathing medium, the authors also con.. cluded that the outer membrane was considerably more permeable to Na than the inner.

3. 6.2. Amphibian urinao" bladder Even in structurally simple epithelia such as frog skin and toad urinary bladder which actively transport Na, several possible routes exist through which Na can be moved from urine, to body fluids, e.g. via intracellular spaces or via a intracellular pathways. Metabolic and isotopic data have demonstrated that Na most likely moxes via transcellular route; however with diffcrent .:ell types (granular, goblet, mitochondria-rich, basal) present in the mucosal cell layer, it is possible that one or more cell types participate in mmsepithelial Na transport. Rick and colleagues util;zed EPXMA to identify cell types involved in Ne, movement and to quantitate the cell Na transpart pool [64]. Granular, mitochondria-rich and basal cells and the basal portions of goblet cells s lowed a similar composition, being high in K (--: 110 mmol/kg wet wt) and low in Na (= 13 mrnol/kg wet wt). The apical portions of goblet cells were higher in Ca and S and lower in P and K presumably reflecting the composition of the mucus within them. Inhibition of the Na-K ATPase pump reduced intracellular K and increased Na. Replacement of mucosal Na with choline, but not serosal Na, prevented these changes. No differences in Na and K concentrations were observed in nuclear or cytoplasmic reDons. These data provided support for the hspothesis that the Na transport pool is derived from the mucosal (urine) side and that no important recycling of Na occurs from the serosal (blood) side. In addition the most outstanding finding was that intracellular Na concentration

204

A. LeFurgey et al. / Electron probe microanalysis in cell physiology

measured by EPXMA was significantly lower than that measured previously by chemical analysis in whole toad bladder tissue or in scraped epithelial cells. 3. 6. 3. Cornea

Rick and co-workers have provided detailed correlative physiological, structural and microprobe data to describe C1 transport in the frog cornea, an epithelium in which active transport of C1 from the inner, stromal to the outer, tear-side bathing medium has been demonstrated to be electrogenic and dependent on the presence of Na in the inner bath [167]. Values obtained by EPXMA, Na, 8 mmol/kg wet wt; C1, 18.4; K, 117.3; were in good agreement with ionic acti;~ties as previously determined. No significant differences between nuclear and cytoplasmic contents were observed nor were there differences in C1 concentrations among cell layers under control conditions or during varying states of CI secretion. A Na concentration gradient was observed between the inner and outer epithelial layer. These data suggest that for C1 but not for Na the epithelium functions as a syncitium. The behavior of the intracellular Na and CI concentrations after removal of Na, CI, or K from the bathing media support the idea that passive electrodiffusive C1 efflux occurs across the apical membrane and that Na-coupled CI uptake occurs across the basolateral membrane. The results cannot define conclusively the precise mechanism of CI uptake, but suggest the existence of either a variable stoick,iometry of the symporter or the presence of more than one transport system. The latter idea was substantiated by the measurement of a dependence ~ intracellular C1 on HCO 3 and CO2. 3.6.4. Intestine

The absorption of many solmes and water by the intestinal mucosa is linked passively to an active transport of Na to produce an absorbate which ~is isotonic with the capillary plasma. As related by Gupta, Hal! and colleagues [168], who have extensively investigated transport phenomena utilizing EPXMA of frozen hydrated sections, the primary sites of Na-transport and solute-water coupling have been postulated to i~e the lateral

intercellular spaces (LIS) within the epithelium; this proposed localization was based on Curran's cell model of a double membrane system with a hypertonic compartment. EPXMA of hydrated sections is the only technique by which the profiles of ionic concentrations within the LIS and surrounding tissue compartments can be measured directly and was utilized by Hall et al. to determine the Na, K, C1, Ca, S, and P concentration profiles in the mucosal tissue of rabbit ileum [168]. The results established for the first time that the LIS fluid was hypertonic and that concentration gradients existed for Na, K, and CI in the cells and the LIS. Average cytoplasmic Na was 36 _+ 3 mmol/kg wet wt; K, 115 _+ 6; Cl, 50 +_4. Measurements were in good agreement with those determined by other analytical methods although Cl was somewhat lower. Analyses along the length of the LIS indicated that midway in the channel the fluid of the LIS may be as much as 100 mOsM hypertonic to the bathing solution, with Na in the interspaces ranging from 78 (apical cell junction) to 157 (midway down LIS) to 155 mmol/kg wet wt (basolateral side). The authors concluded that the observed concentration profiles were not consistent with the standing gradient hypothesis but resembled those predicted by models based on local osmosis. They also found a narrow zone of peripheral cytoplasm constituting a homogeneous compartment, with osmotic concentration intermediate between the mucosal bath and the LIS. They suggested that this compartment may form the main route for transcellular movement of fluid and may determine the osmolarity of the fluid emerging from the LIS. In the invertebrate midgut c~ 'cum, Dow and colleagues [169] found EPXMA data consistent with a model of fluid absorption in which a passive flux of potassium from lumen to blood drives fluid into the blood. Contrary_ to the vertebrate intestine, active absorption of sodium from the lumen would not contribute to fluid transport in vivo. Other EPXMA studies of vertebrate intestine have focused on the analysis of calcium, particularly on the mechanisms of Ca absorption and transport in adult [170] and neonatal rat small intestine [171]. The study of Warner and Coleman,

A. LeFurgey et al. / Electron probe microanalysis m cell physioio~,

which did not employ cryoplescrvation and so cannot be considered from a quantitative point of view, demonstrated that Ca was localized in goblet cell granules [170]. Subsequent studies by Dinsdale utilizing cryopreservation techniques demonstrated the occurrence of Ca not only in goblet cells but also in apical cell vesicles [171]. Both investigations suggest that Ca is compartmentalized as it is transported through the cells.

3. 6. 5. Salt glands Net NaCI secretion in a number of exocrine tissues can apparently be accounted for by a common mechanism, i.e. active chloride transport through a basolateral membrane-bound Na-CI co-transport system. Andrews and colleagues have invoked a similar mechanism for the secretion of a hypertonic saline solution by the avian salt gland, an epithelium which undergoes extraordinary strt, ctural, developmental and functional responses to chronic osmotic stress [46]. The investigators used EPXMA to determine intracellular elemental concentrations of the major electrolytes in principal cells of duckling salt gland, with the underlying assumption that transcellular ion movement would be reflected in the concentrations of intracellular electrolytes. The X-ray results indicated that the cytoplasmic chloride concentration is 157 mmol/kg dry wt (cell apex: 188. cell base) in the unstressed salt gland, a value which implied there is active chloride accumulation since it is greater than that required for electrochemical equilibrium, estimated at < 65 mmol/kg dry wt or 40% of the actual measured concentration. The notion of active C1 transport is also supported by the fact that cytoplasmic chloride concentration in salt-adapted cells is relatively low (124, cell apex: 155 cell base): these dat~ are consistent with chemical and activity measurements and with the idea t~,a! chloride permeability of the apical membrane is higher in secreting cells. As Andrews e t a i . noted, other parallel data have documented that secretory stimulation is accompanied by a change in membrane C1 permeability" however it does not always follow that intracellular C1 content changes with the permeability change, a situation occurring in frog cornea cells (see section 3.6.3 above). Andrews

205

and colleagues also documented some regional differences in the intracellular distributions of ions and water, particularly in the salt-water-adapted cell. The C1 concentration was judged as high in all cell compartments (> 170 mmol/kg dry. wet).

3. 6. 6. Salivary glands Correlative physiological investigations using EPXMA and ion selective microelectrodes have been performed by Gupta, Hall and colleagues to determine the mechanism of transepithelial movement of fluid in the secretory portion of the salivary glands of adult blowflies (Calliphora erythrocephala) [19]. This gland is a structurally typical, simple tubular epithelium which secretes into the lumen a K-rich fluid that is hypertonic to the bathing medium. It consists of a single cell type, with a highly infolded apical membrane which invaginates to form two large secretory, canaliculi that are proposed to be sites of H zO-solute coupling. These researchers found generally good agreement between microelectrode and microprobe measurements, with microprobe data yielding (in m M / k g wet wt) Na, 20: K, 115: CI, 33 in unstimulated glands and Na, 15; K, 125: CI, 23 after stimulation: H,O was 85% and 77.5% respectively. The highest concentrations were found in canaliculi, fluid in these sites being 6-80 mOsM more concentrated. These data support the hypothesis that canaliculi are sites of solute-H,O coupling by some form of local osmosis, and such sites suggest a transcellular route for at least part of the H20 flux, since the closed end of the canalicu!us is not associated with an intercellular junction. The observation of the presence of soptare junctions opening near the mouth of the canaliculus provides a means so that at least some of the H_,O may be moving via a paracellular r o u to. F PXMA

li~in~O

fre~hl,,,

frc~7en

h,¢,-lrntocl

-~n,,.1

dried thin sections of dog submandibular gland was performed to determine the distribution of elements and H:O in the acinar cells of resting and stimulated states [172]. Secretou' granules contained high concentrations of Ca and S while high concentrations of K and P v, ere present in the cytoplasm a n d / o r nucleus of acinar mucous cells of the gland in the resting state. With pilo-

206

A. LeFurgey et al. / Electron probe microanalysis in cell ph.vsioio~"

carpine stimulation, the concentration of Ca increased in the cytoplasm and decreased in the secretory granules, while there was an increase in the Na and C1 in both the cytoplasm and secretory granules of the cells. The authors concluded that the passive Na and C1 influx and the cytoplasmic Ca flowed in from extracellular spaces and was released from secretory granules, an intracellular Ca store, by secretory stimulation; together these events probably trigger the passive or active Na and CI extrusion and consequently the osmotic H 2° flux from the basal part of acinar cells to the secretory granules and the lumen, as well as the serial exocytosis of the granules in the luminal side of the gland. Extensive EPXMA studies of salivary and other secretory epithelia have been performed, in particular by Roomans and colleagues [173] and Izutsu and co-workers [16], to determine the role of ionic defects in the etiology of the disease cystic fibrosis (CF). However, in animal models of CF, neither Roomans nor Izutsu has found evidence to support a Ca transport defect as a primary factor, although in their studies elevated Ca could be detected. 3.6.7. Thymus

Studies in the intact gland and in isolated thymocytes of the rat have been performed by Warley and colleagues [174-176] to establish a basis for clarification of ionic ever:is occurring in the thymus in both normal and diseased states. Cells prepared in different types of media showed variations in the concentrations of Na, K, and CI. Addition of dextra~ to the medivm also ~-!tered ion content, with e!zvated Na and decreased K. Thymocytes prepared by high speed centrifugation showed values for Na, K, and CI similar to values obtained using other analytical methods. 3. 7. Brain / cerebral cortex,~ nerve

As demonstrated with EPXMA by Somlyo and colleagues [2] in other cell types under normal physiological conditions, cells from rat brain cortex frozen in situ have been shown to have very low mitochondrial Ca content (1.5 mmol/kg dry wt

+ 0.26 SEM) and significantly higher cytoplasmic (extramitochondrial) Ca content (6.4 mmol/kg dry wt +_ 0.7 SEM) [177]. Cytoplasmic Na and K and thus K / N a (= 2" 1) ratios obtained simultaneously by EPXMA were also based on normal cell-to-cell distribution and about 4% extracellular space. Although other investigators have estimated extracellular space at = 20%, Somlyo and colleagues found no evidence of such a large space with EPXMA. This K : N a ratio is significantly lower than that found in other viable cell types, for example, heart, = 10 to 15:1 [18] or kidney = 4 to 7.5:1 [34,157-159]. One might suspect that the cells were injured in some way prior to cryofixation but damaged cells most frequently display elevated Na and decreased K accompanied by mitochondrial calcification; in these studies K was maintained at relatively high levels ( = 426 mmol/kg dry wt mitochondria; =600, cytoplasm), while Na appeared to be elevated (189, mitochondria; 293 mmol/kg dry wt, cytoplasm) and Ca appeared to be very low. Although the role of mitochondrial Ca transport in the central nervous system has not been fully resolved, these data demonstrating low endogenous levels of mitochondrial Ca support the conclusion that mitochondrial Ca regulates mitochondrial metabolism through alterations of free [Ca 2+] in the mitochondrial matrix. The high cytoplasmic Ca content, which in these studies included unidentified cell regions containing endoplasmic reticulum, suggest that ER is a possible organelle/site for regulation of cytoplasmic free [Ca 2+]. Andrews, who has utilized EPXMA to examine the role of Ca-sequestering organelles in cholinergic synaptosomes from squid brain, found no evidence of Ca accumulation in synaptic vesicles [178]. Intact synapses of the cerebellar molecular layer also contained low synaptic vesicle and mitochondrial Ca in both resting and stimulated states. However pre- and post-synaptic ER-like cisternae accumulated ~- 5 times as much calcium in stimulated versus resting states. Thcse data strongly suggest that ER-like structures play an integral role in synaptic transmission, while mitochondria and synaptic ve~icles function minimally in Ca-buffering and storing.

A. LeFurgey et al. / Electron probe microanah'sis m cell ph.vsiolog~'

3. 8 Other cell types 3.8.1. Photoreceptors, retinal rods Illumination of the retina has been shown to elicit a hyperpolariz~tion of the outer segment of retinal rods due to the turning off of an inward Na + current. One proposed mechanism for the light response was thought to be an increase in cytoplasmic Ca due to release of Ca from the lumen of the disks of the rod outer segment [179]. Recent EPXMA measurements of dark-adapted and illuminated rod outer segments from frog and toad confirmed the expected reduction in Na content upon illumination [180] but revealed that the Ca concentration was low in dark-adapted rods (0.4 mmol Ca/kg dry wt or 0.1 Ca/rhodopsin) and did not change significantly upon illumination. These results did not therefore support the Ca hypothesis of ph,;totransduction. 3.8.2. Red blood cells, leul~oc;,tes, platelets The earliest quantitative aI alyses of red blood cells were performed by Kirk and colleagues utilizing wavelength dispersive spectrometry of spray frozen intact single cells [181]; Kirk et al. more recently have implemented correlative EPXMA techniques for measuring cation changes during development of erythropoietic cells using energy dispersive spectroscopy of cryosections [182]. Cryosections were necessary to identify the developmental stages of the erythroid cells by morphological criteria as well as to analyse specific subcellular regions, e.g. nucleus and cytoplasm. EPXMA, which allows both morphological and ionic characterization of individual cells within a population, is ideally suited for such studies in which there is no satisfactory separation procedure or synchronized tissue culture method for obtaining pure populations of cells at various developmental stages. These investigators found the switch from high potassium (HK) stem cells to low potassium (LK) red blood cells, which occurs during or slightly before denucleation of the orthochromatophilic e~throblast, to be correlated with the decrease in K content and with major hemoglobin synthesis. They concluded that the most immature erythroid cells were HK (> 80 mmol/kg wet wt) whereas the majority of later

207

erythroid cells were LK (< 10 mmol/kg wet wt). These facts documented that the switch from H K to LK type occurs mainly in the basophilic erythrocytes. A correlative structural, EPXMA and biochemical approach has also been taken by Lew, Somlyo and colleagues [183,184] to address the mechanism of dehydration of the dense subpopulation of sickle-cell anaemia (SC) red blood cells, including the irreversibly sickled cells (ISCs). Discerning this mechanism is important in developing appropriate treatments/interventions for sickle cell disease, the two main clinical features of which are haemolytic anaemia and microvascular occlusion. The earlier working hypothesis for cell dehydration developed from results which showed that SC red blood cells had an elevated Ca content and accumulated Ca during deoxygenation-induced sickling; thus Ca would activate red cell Ca-sensitive K channels with net loss of KCI and H20. However experimental data did not show measurable activation of K channels or Ca pumps, suggesting that Ca might be compartmentalized within the SC cells. Lcw et al. first documented the occurrence of true intracellular vesicles in normal, SC and SC ghc-st cells by the methods of serial sectioni:tg ~,nd three-dimensional reconstruction. In :,ompz.rison with normal cells, they found vesk:les ia SC cells to be increased in size and number, often being multilobed and containing electron-dense inclusions. The compartmentalization of Ca within these vesicles was then established directly by EPXMA, with Ca content within the vesicles averaging = 35 mmol/kg dry wt. Biochemical techniques were then applied to document that these endocytic inside-out vesicles had ATP-dependem Ca pumps capable of bringing Ca inward, against the Ca-extruding pumps of the outer cell membrane. Application of EPXMA to other clinically related questions involving the blood and circulating systems is increasing. Because Mg in peripheral mononuclear blood cells (MBC) is a good indicator of total body Mg, Hook and colleagues proposed development of techniques for measuring MBC Mg by EPXMA using intact single cells or cells in culture [185]. Results with EPXMA and flame atomic absorption spectroscopy were similar

,4. LeFurgey et aL / Electron probe microanalrsis in cell ph)'siolo~"

208

Table 1 Concentrations of elements in major organ systems b'~' cell type a~

Cell Type Skeletal muscle

Animal

Frog

Quant. Method Cont. Norm.

Instr.

EDX TEM EDX

Cellular Compartment

MS

C y t o p l a s m (L) Term C i s t . I-Band Cyto Mitochondria C y t o p l a s m (S)

38±23 56±3~ 45±44 23±25 40±33

41±12 59±22 54±18 23±13 53±17

273±60 415±82 339±80 394±72 359±96

236±35 214±40 263±52 251±35 268±56

18±1.7 27±1.6 37±1.2

58±1.7 56±1.8 59±0.9

623±26 252±8.8 411±7.8

314±11.1 285±10.3 338±3.8

Smooth muscle (pv)

Guinea pig

Cont. Norm.

TEM EDX

Mitochondria C e n t r a l Cyto Per± Cyto

Anterior mesenteric (pv)

Guinea pig

Cont. Norm.

TEM EDX

C y t o p l a s m (L) C y t o p l a s m (S) Mitochondria

Cardiac papillary muscle

Rabbit

Cont. Norm.

59±1 44±1"

Mitochondria Myofibrils SL TSL JSR

Rat

Na

82±8.6

55±4.6

289±17.8

301±18.8

Mitochondria

Cont. Norm.

Myofibrils JSR A-Band I-Band Cultured myocytes

Isolated myocytes (shunted myolemma)

~hick

Cont. Norm.

TE~!/STEM EDX

Mitochondria Cytoplasm

54±7 92±13

39±3 64±6

461±17 527±34

213±8 200±18

Rat

Cont. Norm.

TEM/STEM EDX

Mitochondria Cytoplasm Nucleus

7.2±23.9 30.~:42.4 25±47.5

8.3±8.4 6.±6.4 6.1±6.6

292±95.2 237±104.3 309±108.9

142±45.4 110±27 106±21.7

Rat/ Rabbit

Cont. Norm.

TE~ EDX

Mitochondria Cytoplasm

579±5.3 795±9

35±2 50±3.3

251±1.6 15Y'2.2

454±1 502±3.0

,4. LeFurgey et al. / Electron probe microanalvsis in cell ph.vsiok,~"

C1

Ca

5~i26 43±19 55i27 12±9 56±20

431±95 554±138 510±112 214±60 526±121

8.5±4.6 117±48 4.5±7.1

132+9.2 286±10.3 383±5.7

544±18.0" 759±15.7 783±10.8

0.7±0.3 0,8±0.2 2.8±0.2

[La]

U/M

#/M

mmol/kg d r y wt ±SD

(49) (229) (229)

1.7±3.Y

(39)

3.0±5.8

(40) 0,5x0.3 1.0±0.5 24±1.6

mmol/kg d r y wt ±SEM

(59) (67) (467)

1.3±0.1 0.8±0.2 2.3±0,4

mmol/kg d r y wt ZSEM

(68) (262) (46)

i

mmol/kg dry wt ±SEM

(16)

Dry wt 8/1008

Commen t s

209

Re f . [1261

K+Rb*

[lO61 Mg (n=50) [64]

165±9.3

534z43.4

I. 0±I. 0 8 10 6

K:Cl=

I0:i

1+2

K:CI=

7:1

4-32 3+4 3±4

127±19 134±26

825±44 1052±87

2.3±1.2 4.5±1.9

mmol/kg d r y wt ±SEM

(25) (26)

[iS]

36±14.6 81±35.4 72.5±34.5

335±1~9.2 395±141.2 457.2±134.9

-2.8Z4.8 -1.7Z4.8 -3.1±4.4

mmol/kg d r y wt ±SD

(91) (90) (95)

[1471

405±1 457±2.7

82±0,9 103Zl.5

4~0.7 7±1.4

mmol/kg dry wt ±SE'4

(24) (20)

mmol/kg d r y wt +SF~

[141]

pCa>6

[145]

A. LeFurgey et a L / Electron probe microanalysis in cell physiolo~k~'

210 Table 1 (=ontinued)

C e l l TTpe

Liver

Animal

Rat

Quant. Nethod

Instr.

Cont. Norm.

TEN EDX

Per±. Std.

Cellular Compartment

Nitochondria RER Nucleus Other Total Cell

Na

36o±7

54±3 80±8 77±6 75±6 53±4

43+1 76±2 53±2 53±2 46±2

920±21 557±16 653±20 564±12

SEN/STEM Nucleus

14.1±0.5

11.8±0.6

132.0±1.8

TEM/STEM EDX

4.8±2.1 6±5

Pancreas acinar cells

Dog

Kidney proximal tubule

Rabbit

Cont. Norm.

TEN/STEN N i t o c h o n d r i a EDX Cytoplasm

95±7 125±12

Rat

Peri. Std.

SEM/STEN N uc l e us EDX (Zcytoplasm)

19.1±0.8

Rat

Cont. Norm.

SEN/STEM Cytoplasm EDX

51.6

Rat

Per±. Std.

Rat

Per±. Std.

Kidney distal tubule

Ng

Cytoplasm Zymogen Granules

Nucleus

10.4±0.7

316±4 224±8 245±8 241±9 230e9

165±36 36±8

19+2.8 172+25

27±3 27~4

349±22 356±18

158±6 124±8

14.3±0.4

152.8±3.1

196.8

14.5±0.8

94.6

193.0±5.0

Kidney Papilla ColJecting duct c e l l s

SEM/STEM Nucleus EDX Cytoplasm Nucleus

24.2±3.6 22.6± 3.0 27,5

300.7±22.4 279.2±22.1 316.1±7.2

Epithelial cells

Nucleus

22.5±2.3

333.0±12.3

Interstitial cells

Nucleus

50.2±3.0

309.5±11.8

437.4±18.8 445.0±18.7

14.9±1.2 12.9±0.9

124±12 227±16

363±2 433±17

28.6±1.1 33.9±1.4

151±11 254±31

403±16 433e41

38.6±2.0 40.1±3.8

Interstitium plasma Collecting duct c e l l s

Epithelial cells

Rat

A. LeFurgey et aL / Electron probe microanalysis in cell physiolow

CI

Ca

[La]

U/M

#IM

Dry wt g/lO0$

Comments

211

Ref

75±3 94±5 117±6 103±5 106±4

314±5 579±17 625±22 465±15 479±20

0.8±0.1 5.0±0.4 0.8±0.4 4.0±0.4 3.4±0.4

mmol/kg d r y wt ¢SEM

(97) (24) (21) (29) (20)

21.3±0.4

140.6±1.3

0.3±0.1

mmol/kg wet wt ±SEN

(126)

24.0±0.3

[1561

14i4.7 31±20

132~15 60±16

mmol/kg wet wt ±SD

(15) (9)

22±3.3 37±7%

[153]

7±5

131±12 141±11

349±22 348±23

3.1±1.1 4.1±1.4

nunol/kg d r y wt ±SEN

(23) (23)

19.9±0.7

150.1±2.6

0.2±0.1

mmol/kg wet wt ±SEM

(73)

51.7

132

mmol/kg wet wt ±SEM

(213)

11.4±0.7

151.6±3.5

mmol/kg wet wt ±SEM

(31)

19.1±0.5

67.3±5.9 76.3¢8.4 76.3±2.5

135.7±7.9 129.0±8.9 135.2±2.5

maol/k8 wet wt ±S~I

(18) (18) (154)

19.1±1 . l e 19.1±1.1" 19.7+0.5 *

79.9±3.6

135.9i4.8

(55)

20.9±0.9*

107.2±4.3

138.9±4.2

(56)

21.0±0.9*

437.5±19.9 458.2±20.8

34.9±2.2 32.1±2.0

(56) (56)

11.8±0.5

154±13 269±17

124.7±4.4 154.1±7

(140) (123)

186+11 278±32

140.5+5.7 132+12

( 88 ) ( 27 )

0.2±0.1

mmol/kg wet wt ±SEM

[1511

[341

21.3±0.4

[156]

[98]

[1571

*Urea-free d r y wt.

[159]

25.0 31.7

Album i n - d ippe d Und i p p e d

[1601

30.4 32.2

Album i n - d ippe d Und i p p e d

e

12.0±0.9"

A. LeFurgey et aL / Electron probe microanalysis in cell physiology

212

O~ant.

C e l l Type

Animal

Method

Instr.

Cellular .Compartment

Na

Mg

P

S

Interstitial cells

391±23 362±28

403±18 412±27

21.6±1.5 22.7±2.0

Interstitium

518±15 654±31

29,8±2.5 39.2±4.9

8,6±1.0 2.1±0.4

138.7±18.5 101.9±22.2

Skin

Frog

Per±. Std.

SEN/STEM Nucleus EDX Cytoplasm

11.0±2.8 11.4±4.5

Granular cells

Toad

Per±.

SEM/STEM EDX

Nucleus Cytoplasm

11.2±3.5

153,2±17.0

Std.

13.8±6

124.5±31.3

Mucosal

Toad

Cont. Norm,

SEMISTEM EDX/WDX

ICF Nucleus Cytoplasm

72±13

62±4 74±15

785±47 883±63 714±11

116±34 95±22 126±44

18±3

139±3

32±3

Goblet cells

78±12

388±79

177±39

Smooth muscle

89±28

454±69

205±36

Urinary Bladder

epithelial cells

Cornem

Salivary gland

S a l t Gland epithelisl cells

Frog

Adult bl0#fly

Duck

Per±. Std.

SEM/STEM Nucleus EDX Cytoplasm

Cont. Norm.

SE~/3TE~ EDX/WDX

TEN/STEM EDX

5.8±2.1 6.7±7.3

7.1±4.0 10.5±6.0

117.2±10.8 92.5±17.6

Basemnt memb. Cytoplasm Canal±cull

76±13 20±3 17±10

Lumen

16±2

C e l l Apex C e l l Nucleus Cell Bas~

83±18 76±23 125±11

668±37

Cell Apex C e l l Nucleus Cell Base

23±2.7 12±3.7 34±3.1

153±$8 120±7.2 160±5.5

572±22 596±20

U / M = units of measurement: = / M = n u m b c r of m e a s u r e m e n t s . Q u a n t i t a t i o n m e t h o d ( Q u a n t . M e t h o d ) is either c o n t i n u u m normalization [1] or peripheral s t a n d a r d [65]. I n s t r u m e n t a t i o n " T E M = t r a r s m i s s i o n e l e c t r o n microscope: S E M = s c a n n i n g

A. LeFurgeyet al. / Electron probe microanalysis in cell physiolo~,

213

Dry wt C1

K

Ca

[La]

U/M

#/M

9/1008

....

Comments

429±24 401±31

131.7±5.7 137±8

(103) (54)

33.7 31.5

Albumin-dipped Undipped

517±15 652±32

40.5±3.3 38±2

(119) (78)

17.6 16.9

Albumin-dipped Undipped

31.2±3.1

121.0±15.5

0.1±0.3

mmollkg

37.1±4,8

118.3±14.1

0.6±0.7

w e t wt ±SD

22.9±7.3

118.5±13.7

0.1±0.3

mmol/kg

40.8±10.1

115.9±15.3

2.2±1.8

wet wt ±SD

155±7 135±19 160±4

543±38 594±57

28±3

(16)

23.3±2.2

[162]

26.6±3.5

(12)

23.8±2.2

[65]

29.1±4.0

Freezedried

491±13

mmol/kg dry wt ±SEM

116±8

mmol/kg

Frozenhydrated

wet wt ±SEM 100±13

269±50

mmol/kg dry wt ±SEM

244±50

640±91

mmollkg dry wt

Ref.

[164]

+SEM 17.5±1.9 16.7±3.6

124.4±5.5 119.0±12.6

134121 33±3 114±22

76±8 115±7 160±16

155±7

139±5

124±11 164±13 155±8

423±14 602±22 469±12

mmollkg dry wt ±SEM

(12) (19) (17)

Salt-Water Adapted

33±2,9 33e2.5 43±2.2

1!0±3,8 116e4.2 128±3.4

~o!/kg wet wt ~SEM

(12) (19) (17)

Salt-Water Adapted

0.4±0.4

mmol/kg

0.6±0.5

wet wt ±SD mmol/kg wet wt ±SEM

(9) (9)

[167]

20.8±1.3

21.1±1.9 [19]

(7) (10) (5)

[46]

electron microscope; S T E M = scanning t r a n s m i s s i o n electron m i c r o s c o p e ; E D X = energy dispersive X-ray; W D X = wavelength dispersive X-ray

214

A. LeFurgey et al. / Electron probe microanalysis in cell physiologv

with Mg = 64.0 + 14.3 and 52.2 + 13.8 mmol/kg dry wt respectively. The reader is referred to text reference [81] for a full discussion of EPXMA usage in clinical pathology and diagnostic settings. 3. 8. 3. Spermatozoa, oocytes

Colonna and Oliphant [186] have investigated the relationship between changes in subcellular element distribution and the capacitation and acrosome reactions of mammalian sperm by applying EPXMA to intact frozen sperm cells. Changes occurred in all elements except Mg; statistically significant differences between the two maturational stages existed only in the midpieceassociated Ca, nuclear Na, and P levels. Earlier studies by Cantino and colleagues had also demonstrated changes in Ca and K associated with the acrosome reaction both in cryosections and in whole mounts of frozen sea urchin sperm [187]. 3. 8. 4. Bacteria

As described by Stewart and colleagues in at least four postulated mechanisms of bacterial spore heat resistance, cations are thought to play critical roles [188,189]. The function of ions such as Ca or Mn in any model of resistance or dormancy is dependent on their location and amount within spores. Thus EPXMA of quick frozen, cryosectioned and freeze dried spores has been the method of choice for determining the role of cations in the remarkable physiological properties which bacteria display, i.e. resistance to heat, radiation, enzymes, disinfectants; lack of detectable metabolism during dormancy; rapid germination capacity. Spores of all species have the same general morphology: they consist of an inner core (protoplast) surrounded by a germ cell wall, cortex, coats, and in some species an exosporium. STEM X-ray maps of Bacillus cereus T and B. megaterium Km spores demonstrated almost all the Ca, Mg, Mn, and P to be located in the core area and an unexpectedly high concentration of Si to be in the cortex/coat layers together with S and P. In contrast, EPXMA studies of another species B. coagulans, a strain with much greater heat resistance than B. cereus or megaterium, showed high concentrations of Ca, Mn and Fc in the coat but no Si. According to Stewart two theories of spore resistance and

dormancy, the contractile cortex theory and the expanded cortex theory, require that a substantial concentration of positively charged counterions occur in association with the negatively charged cortical peptidoglycan polymer. The protective chelate element theory requires high Ca to be chelated to dipicolinic acid in a matrix involving spere ligands, or a major proportion of cations to be located in the protoplast or core. The differing results obtained with different species suggest that multiple mechanisms may exist to explain relative dormancies and resistances.

4. Summary and conclusion Table I summarizes many of the EPXMA findings from studies which address specific physiological questions in skeletal and cardiac muscle, kidney and other epithelia and a wide variety of (animal) cell types. Of particular general physiological importance are (a) the findings of Somlyo and colleagues and other investigators that, in the majority of animal cell types studied, Ca in mitochondria is low enough to regulate mitochondrial dehydrogenase but not high enough to regulate cytoplasmic enzymes; (b) the data of Rick, Thurau and coworkers which demonstrate that epithelia such as frog skin, cornea or bladder may function as a syncitium with respect to all or only to some ions; and (c) the results of Hall, Gupta and colleagues which for the first time documented that concentration gradients exist in both cells and lateral intercellular spaces of epithelia s~ch as intestine. The models are consistent with raodels of solute and fluid transport based on local osmosis rather than the standing gradient hypothesis. In situ EPXMA measurements of Na and/or Ca content in many cell types including frog skin, toad D l ~ l O O e I , ~auney, O1-~1,111, I I V ~ I lli~V¢ b l l U W l l t l l ~ l l l t o be lower in general than contents determined in bulk samples because the extracellular space correction and/or "contamination" from non-viable cells are both eliminated from probe measurements. Much of this quantitative information has been obtained using a mathematical algorithm for relat-

A. LeFurgey et al. / Electron probe microanalysis in cell physioiogy

ing X-ray intensities to element content developed by Professor Ted Hall, in whose honor this paper and all those of the symposium are written. As the scientific news reporter, Thomas Maugh, wrote over 10 years ago [190], EPXMA "...gives the physiologist a new way to examine the composition of cellular subunits without having to try to separate those subunits from the rest of the cell". Although Maugh also suggested that EPXMA allows "examination of elementary cell function with an ease never previously imagined", the enormous efforts which have followed between 1977 and 1987 to perfect preservation and analysis techniques attest to the many difficulties of precise microchemical microscopy. But as Maugh pointed out and as the experimentation catalogued here documents, EPXMA is a tool which has made and will continue to make possible a "better understanding of the simple chemical interactions" that are the basis of cell function. Acknowledgements We thank Professors M. Lieberman, J. Sommer, L. Mandel and J. Shelburne for constructive discussion of the manuscript and especially for their combined efforts in establishing the Analytical Electron Microscope Facility in the Department of Physiology, Duke University. Thanks are also due Drs. D. Kopf and M. Lamvik for their help and advice with regard to energy loss spectroscopy. We are also indebted to Drs. A.V. and A.P. Somlyo and Dr. H. Shuman of the Pennsylvania Muscle Institute for their encouragement, support and provision of quantitative computer routines; and to Mr. b. Oavilla for implementing the~ in our facility. Mr. Larry Hawkey, Miss Pamela Schreiner and Miss Gay Blackwell provided excellent technical and editorial skills in preparation of the manuscript. Supported in part by NII-I L/rants l'~os, n L - V t X V ~ , nL-x,u,v, ~ " 27105, DK-38820, DK-37704, RR-05a05 and HL12486.

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2] 5

[3] B.L. Gupta and T.A. Hall, Fed. Proc. 38 (1979) 144. [4] T E. Hutchinson and A.P. Somlyo, Microprobe Analysis of Biological Systems (Academic Press, New York. 198 !i). [5] A.L Morgan, X-Ray Microanalysis in Electron Microscopy for Biologists (Oxford University Press, Oxford,. "1985). [6] D.E. Johnson and M.E. Cantino, High resolution bioiogi~,l X-ray microanalysis of diffusible ions, in: Advanced Techniques in Biological Electron Microscopy Ill, Ed J.K. Koehler (Springer, Berlin, 1986) p. 73. [7] J.J. Hren, J.I. Goldstein, D.C. Joy, Introduction to Analytic;d Electron Microscopy (Plenum, New York, 1979). [8] J.I. Goldstein, D. Newbury, P. Echlin, D.C. Joy, C.E. Fiori and E. Lifshin, Scanning Electron Microscopy and X-Ray Microanalysis (Plenum, New York, 1981). [9] D. Newbury, D.C. Joy, P. Echlin, C.E. Fiori and J.l. Goldstein, Advanced Scamfing Electron Microscopy and X-ray Microanalysis (Plenum, New York, 1986). [101 H. Shuman, A.V. Somlyo and A.P. Somlyo, Ultramicroscopy 1 (1975/76) 317. [111 T.H. Kitazawa, H. Shuman a~d A.P. Somlyo, Ultramicroscopy 11 (1983) 251. [121 J.A. Chandler, in: Scanning Electron Microscopy/1985, Voi. II, Ed. O. Johari (SEM, AMF O'Hare, IL, 1985) p. 731. [131 P. Echlin, J. Microscopy 140 (1985) 1. [141 A.T. Marshall, in: Scanning Electron Microscopy/1981, Vol. II, Ed. O. Johari (SEM, AMF O'Hare, IL, 1981) p. 327. [151 A.T. Marshall, Scanning Electron Microscopy II (1980) 395. [161 K.T. Izutsu, D. Johnson, M. Schubert, E. Wang, B. Ramsey, A. Tamarin, E. Truelove, W. Ensign and M. Young, J. Clin. Invest. 75 (1985) 1951; K.T. Izutsu and D.E. Johnson, J. Physiology 381 (1986) 297. [17] E. Murphy, D.M. Wheeler, A. LeFurgey, R. Jacob, L.A. Lobaugh and M. Lieberman, Am. J. Physiol. 250 (198t0 C442. [18] A. LeFurgey, Shi Liu, M. Lieberman and P. Ingrarn, in: Microbeam Analysis - 1986, Eds. A.D. Rorrfig and W.F. Chambers (San Francisco Press, San Francisco, CA, 1986) p. 205. [19] B.L. Gupta, M.J. Berridge, T.A. Iiall and R.B. Morcton, J. Exptl. Biol. 72 (1978) 261. [201 A.W. McDowall, J.J. Chang, R. Freeman, J. LePault, C.A. Walter and J. Dubochet, J. Microscopy 131 (1983) 1. [21] J.J. Chang, A.W. McDowaU, J. l.ePault, R. Freeman, C.A. Walter and J. Dubochet, J. Microscopy i32 (i983) 109. [212] P.M. Frederik, hn: Scanning Electron Microscopy/1982, Vol. II, Ed. O. Johari (SEM, AFM O'Hare, IL 1982) p. 709. [23] P.M. Frederik and W.M. Busing, J. Microscopy 121 (1981) 191. [24] G.M. Roomans and J.D. Shelburne, Basic Methods in B~ological X-Ray Microanalysis (SEM, AMF O'Hare, IL, 1983).

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A. LeFurgey et al. / Electron probe microanalysis in cell physioloKv

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