Microcomposition of Human Urinary Calculi Using Advanced Imaging Techniques

Microcomposition of Human Urinary Calculi Using AdvancedImaging Techniques

Sarah D. Blaschko, Joe Miller, Thomas Chi, Lawrence Flechner, Sirine Fakra,Arnold Kahn, Pankaj Kapahi and Marshall L. Stoller*,†From the Department of Urology, University of California-San Francisco (SDB, JM, TC, LF, MLS), San Francisco, Advanced Light Source, LawrenceBerkeley National Laboratory (SF), Berkeley and Buck Institute for Research on Aging (AK, PK), Novato, California

Purpose: Common methods of commercial urolithiasis analysis, such as lightmicroscopy and Fourier transform infrared spectroscopy, provide limited or noinformation on the molecular composition of stones, which is vital when studyingearly stone pathogenesis. We used synchrotron radiation based microfocusedx-ray fluorescence, x-ray absorption and x-ray diffraction advanced imagingtechniques to identify and map the elemental composition, including trace ele-ments, of urinary calculi on a �m (0.0001 cm) scale.Materials and Methods: Human stone samples were obtained during serialpercutaneous nephrolithotomy and ureteroscopy procedures. A portion of eachsample was sent for commercial stone analysis and a portion was retained forsynchrotron radiation based advanced imaging analysis.Results: Synchrotron radiation based methods of stone analysis correctly iden-tified stone composition and provided additional molecular detail on elementalcomponents and spatial distribution in uroliths. Resolution was on the order of afew �m.Conclusions: Knowledge of all elements present in lithogenesis at this detailallows for better understanding of early stone formation events, which mayprovide additional insight to prevent and treat stone formation.

Key Words: kidney, diagnostic imaging, nephrolithiasis,

Abbreviations

and Acronyms

�XANES � micro-XANES�XAS � microfocused XAS�XRD � microfocused XRD�XRF � microfocused XRFCT � computerized tomographyFTIR � Fourier transform infraredspectroscopyMS � mass spectroscopyPIXE � proton induced x-ray emissionSEM � scanning electron microscopySR � synchotron radiationXANES � x-ray absorption nearedge structureXAS � x-ray absorptionXRD � x-ray diffractionXRF � x-ray fluorescence

Accepted for publication September 18, 2012.Supported by grants from the American Fed-

urolithiasis, radiography

See Editorial on page 417.

726 www.jurology.com

KNOWLEDGE of stone composition di-rects urolithiasis treatment. However,most stones are calcium based and toour knowledge no marker exists to dif-ferentiate subtypes of these stones. Assuch, all calcium stones are approachedin similar fashion surgically and thera-peutically despite differences in com-position. To better understand initialevents in stone formation and determineoptimal medical and surgical manage-ment of urolithiasis, a more thoroughknowledge of all elements present andtheir interactions are required. This in-formation is not available using tradi-
tional stone analysis techniques.
0022-5347/13/1892-0726/0THE JOURNAL OF UROLOGY®

© 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RES

Common methods of clinical stoneanalysis include in vivo imaging tech-niques, such as x-ray, ultrasound andCT, and ex vivo laboratory stone anal-yses, most commonly microscopy, FTIRand XRD. In vivo imaging alludes tostone composition and hardness by ap-pearance on x-ray or by HU on CT butlittle additional information about stonecomposition can be ascertained. FTIRcan determine stone composition andidentify the proportions of constitu-ents present in mixed compositionstones. However, the accuracy of FTIRhas been called into question.1,2 The

eration for Aging Research and California Urol-ogy Foundation, National Institutes of HealthGrants R01 AG031337-01A1, RO1 AG038688,RL1 AAG032113 and P01 AG025901-S1 (PK),Multidisciplinary K12 Urologic Research CareerDevelopment Program Grant K12-DK-07-006 (TC),and a grant from the AUA Foundation ResearchScholars Program and Boston Scientific Corp.,The Endourological Society and the “Friends ofJoe.” The Advanced Light Source is supported bythe Director, Office of Science, Office of BasicEnergy Sciences of the United States Departmentof Energy under Contract DE-AC02-05CH11231.

* Correspondence: Department of Urology,University of California-San Francisco, 400 Par-nassus Ave., A610, San Francisco, California94143 (telephone: 415-476-1611; FAX: 415-476-8849; e-mail: [email protected]).

† Financial interest and/or other relationshipwith Bard, Cook, Boston Scientific, EM Kineticsand Ravine Group.

presence of trace elements and their

http://dx.doi.org/10.1016/j.juro.2012.09.098Vol. 189, 726-734, February 2013

EARCH, INC. Printed in U.S.A.

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MICROCOMPOSITION OF HUMAN URINARY CALCULI USING ADVANCED IMAGING TECHNIQUES 727

location in the stone cannot be identified by FTIRor XRD.

Urolith morphology has been studied extensivelywith transmission electron microscopy and SEM.3

Elemental composition can be determined with highprecision and sensitivity using MS.4 However, MSdestroys the sample and does not provide informa-tion on the spatial distribution of elements. Elemen-tal mapping has been performed using scanned elec-tron5 and proton beams,6 which generate XRFradiation with element specific energy.

To our knowledge this study introduces a newtype of advanced imaging technique to the urologicalcommunity. It can identify the presence, locationand chemical environment of elements, includingtrace elements, in uroliths with �m (10–6 m) resolu-tion. An intense, tightly focused x-ray beam gener-ated by a synchrotron particle accelerator is used asa probe and the emitted or transmitted x-rays areanalyzed. The results of 5 stone samples are pre-sented, and different analytical and imaging tech-niques are compared.

MATERIALS AND METHODS

Uroliths were obtained from human subjects during serialureteroscopy and percutaneous nephrolithotomy proce-dures. Portions of the stones were sent for standard labo-ratory microscopic and FTIR analysis. Other sampleswere analyzed using synchrotron based �XRF, �XAS and�XRD at the Advanced Light Source beamline 10.3.2 inBerkeley, California. Examined stone samples includedcomposite calcium phosphate, calcium oxalate, uric acidand cystine calculi (see table).

Fourier Transform Infrared SpectroscopyStones were analyzed at commercial laboratories by mi-croscopic and FTIR identification. FTIR measures the ab-sorption or emission of infrared light from a sample. The

Composition of study samples on commercial FTIR and synchr

Sample No.* FTIR Composition (%)

1 Calcium oxalate dihydrate (15), calciumoxalate monohydrate (70), calciumphosphate (15)

Calcium, ironphosphoruspotassium,potassium,

2 Calcium phosphate (90), calciumoxalate dihydrate (10)

Calcium, ironphosphorus

3:Subsection 1 Uric acid (80), ammonium acid

urate (20)Calcium, ironphosphorus

Subsection 2 Calcium oxalate monohydrate (80),calcium phosphate (20)

4† Calcium phosphate (100) Calcium, ironphosphorus

5 Cystine (100) Sulfur, iron,

* Typically several sites were probed per sample using microfocused x-ray beam. Cwere collected on bulk stones (figs. 3 and 5).
† On �XRD hydroxyapatite � 2.
vibrational and rotational modes of atoms in a moleculecan be excited by infrared radiation. Infrared absorptionor emission can be used to study the nature of molecularbonds. Since different substances have distinct absor-bance patterns and the degree of absorption is propor-tional to concentration,7 FTIR can be used as a routinefingerprinting method by comparing measurements withknown standards.8 Unlike traditional spectroscopy, themain advantage of FTIR is that it simultaneously collectsall frequencies in the infrared spectrum using a movableand a fixed mirror inside the interferometer, which signif-icantly accelerates sample analysis. FTIR is the leadinganalytical technique for clinical laboratory kidney stoneanalysis.

X-Ray FluorescenceXRF imaging uses an x-ray beam to ionize the atoms of asample. The x-ray ionization process causes 1 inner shellelectron of the atom to be emitted as a photoelectron,leaving an ion in an excited state and a core hole behind it.To relax to a lower energy state, an electron from an outershell fills the core hole and the excess energy is emitted asfluorescence radiation (fig. 1). The energy of the emittedphotons is the difference of the electron binding energiesof the shells between which the transition occurred. Elec-tron binding energies of various elements are different sothat each atom can be identified uniquely. Furthermore,core shell ionization and relaxation are independent ofvalence electrons that form chemical bonds. Thus, atomscan be reliably identified in different molecular environ-ments. However, this also means that XRF does not allowthe study of interactions between atoms.

�XRF uses a microfocused x-ray beam with a focal spotof a few �m (fig. 2, A). The energy of the incoming x-rayradiation must be higher than the binding energy of theinner shell electrons for ionization to occur. This providesthe flexibility to selectively ionize elements by choosingthe appropriate x-ray energy. However, certain elementsmight be excluded by the limitations of the x-ray source. Ifthe energy of emission lines of certain elements is closerthan the energy resolution of the detector, these elements

based techniques

XRF (No. sites) Calcium �XANES (No. sections)

trontium, potassium, chlorine, lead,ium, iron, zinc, strontium,(1), calcium, iron, zinc, strontium,(1)

Calcium oxalate (1), hydroxyapatite (1)

trontium, potassium, chlorine, Hydroxyapatite

trontium, potassium, chlorine,

Calcium oxalate (1), hydroxyapatite (1)

trontium, potassium, chlorine,

assium, chlorine, copper, chromium

XANES measurements were performed on sample thin sections and all other data

otron

�

, zinc, s(1), calcchlorinechlorine, zinc, s

, zinc, s

, zinc, s(2)

zinc, pot

alcium

MICROCOMPOSITION OF HUMAN URINARY CALCULI USING ADVANCED IMAGING TECHNIQUES728

cannot be distinguished. The low energy cutoff of the x-raydetector (1,750 eV) limits the detection of elements tothose with an atomic number of 14 or greater.

For all x-ray measurements stone samples were placedon Kapton® tape (which has negligible x-ray absorption)attached to an aluminum frame. Sample data were col-lected at room temperature. Cystine crystals were placedon molybdenum foil and mounted on a cooling stage. Datawere collected at �30C. �XRF spectra were typically ob-tained in 30 to 40 seconds (fig. 3).

Synchrotron RadiationX-ray generators are widely used in medicine. In an x-raygenerator accelerated electrons hit a metal surface andionize the atoms of the target. The subsequent relaxationprocess leads to the emission of characteristic x-rays, sim-ilar to fluorescence radiation, as described. The energy ofthis radiation depends on the target metal and can bechanged only by replacing the target. This is not practicalfor routine medical or scientific applications.

Synchrotrons are another type of x-ray source based onparticle accelerators, which were originally built for highenergy physics experiments. A synchrotron is a circularaccelerator with a diameter of tens to hundreds of meters.When a charged particle, eg an electron, is forced onto acircular path by a magnetic field in an accelerator, it emitsSR. SR energy covers a broad range of the electromagneticspectrum, including infrared, visible, ultraviolet, and softand hard x-rays. SR is several orders of magnitude moreintense than x-rays from a generator. It can be focused toa �m sized spot, while maintaining low divergence. SRfrom the accelerator is monochromatized by crystals andfocused on a sample by mirrors. The collection of mono-chromator, mirrors, apertures and other components iscalled a beamline, which is typically tens of meters long

Figure 1. XRF in calcium. Incoming x-ray photon excites coreshell electron of atom, in this case K-shell electron. Core hole isfilled by 1 outer electron and extra energy, which is bindingenergy difference of 2 shells, is released as fluorescence radia-tion. Since electron binding energies are element specific, ana-lyzing fluorescence energy can be used to determine atomiccomposition of its source. Calcium photoionization and subse-quent fluorescence emission shown resulted in calcium K peakof 3,690 eV energy (K and L electron binding energies were4,038.5 and 348.5 eV, respectively). To generate calcium K peakenergy of incoming x-ray beam must be higher than 4,038.5 eV(fig. 3). X-ray emission peaks are labeled based on shell fromwhich they originated. L, M and N, electron shells.

and connects the exit port of the synchrotron to the

sample area. The properties of SR, such as tunabilityand high brightness, make it an indispensable tool forscientific research. Changing the x-ray energy enablesthe selective ionization of different electron shells andthe high intensity allows the study of small samples.These unique capabilities created a new field of analyt-ical techniques, including the elemental mapping ofsmall specimens.

�XRF Elemental MapsA microfocused stationary x-ray beam with a samplemounted on an x-y stage enables the scanning of thesurface of a sample. Collection of a �XRF spectrum at eachscan point yields an elemental map of the sample surface,which shows the spatial distribution of different atoms(fig. 4). Typically, a low resolution, coarse map is collectedfirst with subsequent fine maps focusing on interestingareas. Data collection time is typically a few hours permap.

If �XRF spectra or elemental maps are to be used foraccurate quantitative analysis of sample composition, sev-eral points must be considered. The probability of theionization of an atom depends on the x-ray energy and thecore shell being ionized. These probabilities are knownand tabulated in the literature. The emitted fluorescenceintensity depends on the volume hit by the incomingbeam. This can be calculated from the beam size and thepenetration depth of the x-rays. Finally, self-absorption ofemitted fluorescence by the sample must be considered.Absorption related complications can be overcome usingthinly sliced samples, similar to those prepared for trans-mission electron microscopy.

�XRF elemental mapping is possible with an x-raygenerator. However, its flexibility is limited to the fixedx-ray energy and the significantly lower beam intensity,especially when focused on a small spot. XRF can also begenerated through the impact of high energy electrons orprotons on the sample. An SEM equipped with a fluores-cence detector can also be used for elemental mapping.5

The greatest advantage of an SEM is the high spatialresolution achievable (approximately 1 nm). However, ex-tensive sample preparation, limited penetration depth ofthe electron beam (a few nm) and limited detection sensi-tivity from electron deceleration (bremsstrahlung) limitsits usefulness. A PIXE instrument can also generateelemental maps with a reported beam spot size of 5 to 10�m if the proton beam is sufficiently focused.6,9 PIXErequires sample preparation and beam penetrationdepth is a few �m.

XAS and XANESThe �XRF experiments described were performed with anx-ray beam at fixed energy while the energy of the emittedfluorescence radiation was analyzed. In an x-ray absorp-tion experiment the energy of the x-ray beam was changedand the intensity of the beam transmitted through thesample was recorded (fig. 2, B). This type of experimenttakes advantage of the energy tunability of x-rays from asynchrotron, which is not feasible with x-ray generators.The strongest absorption happens when the x-ray energyis near the ionization energy of an atomic shell becausethe x-ray photons are destroyed in the process. The ion-
ization energy of an atomic shell is the same as the bind-

on me


ing energy of the electrons in that shell. This is oftencalled an absorption edge based on the shape of the ab-sorption spectrum (fig. 5).

The near edge x-ray absorption spectrum of an ele-ment is influenced by the chemical state of the atomunder investigation, and by the species, distances andchemical states of surrounding atoms. Modulations thatarise in the absorption spectra are due to interactions ofthe emitted photoelectrons with neighboring atoms. Us-ing this method, the oxidation state and coordinationenvironment of atoms can be determined nondestruc-tively.

Due to its sensitivity to the molecular environment,XANES is well suited to be a fingerprinting method. Usinga database of previously measured spectra, the composi-

Figure 2. Some analytical methods used for stone analysis. A, tbeam of single energy hits sample mounted on x-y stage. EmitAs stage is scanned, elemental map of sample can be generatemeasured in XAS experiment. Microfocused beam and x-y staoccurs in crystalline samples where monochromatic x-ray beamsurface area. Microfocused beam and x-y stage enable diffracti

tion of samples can be accurately determined (fig. 5).

X-Ray DiffractionIn XRD an x-ray beam is focused at a sample. Theperiodicity of a crystalline structure causes interferenceeffects, which results in a diffraction pattern (fig. 2, C).The diffraction pattern is characteristic and can be usedas a fingerprint for matching against known patternsfrom a database.8 Under the right circumstances thepositions of all atoms in the sample can be determinedwithout comparison to previously obtained structures.The greatest drawback of XRD is that the sample mustbe crystalline.

We performed �XRD using a microfocused x-ray beam,which allowed the diffraction analysis of small areas ofour samples to investigate differences in composite stones(fig. 6). XRD experiments can be performed with labora-

�XRF measurement setup. Focused (few �m in diameter) x-rayorescence is detected and energy is analyzed in x-ray detector.ansmission of x-ray beam of varying energy through sample isble spectroscopy on different parts of sample (�XAS). C, XRD

ates diffraction pattern, which is recorded on detector with largeasurements on different parts of sample (�XRD).

ypicalted flud. B, trge enagener

tory based instruments based on x-ray generators. How-


ever, to achieve a small focal point of the x-ray beam forsmall samples or for spatial scanning, a high performancerotating anode generator with advanced optics is required.

Although XRD, XRF and elemental mapping can bedone using smaller, separate, laboratory based instru-ments, XAS/XANES can only be performed using SR. SRalso allows all techniques to be used on the same samplein short succession without further handling. Results canbe combined in a meaningful way to guide subsequenttesting.

Synchrotrons are large-scale scientific instrumentsthat are built and operated at government research labo-ratories. The beamlines and end stations installed at syn-chrotrons are shared among many research groups. Ac-cess is limited, although it is free for approved academic

Figure 3. XRF spectra of representative stone samples collectedK peak at 3,690 eV as long as exciting x-ray energy was above K-and third spectra showed no calcium signal because x-ray excdeliberately excluding calcium measurement. Purpose was to swhich would be masked by strong calcium peak. Cystine sampleelements chlorine, potassium, iron, zinc, copper and strontium werscattered without energy loss.

researchers. For this reason, the SR based analytical

methods described can be performed only intermittently.They are not suited for clinical analysis of a large numberof stones on a regular basis.

RESULTS

Laboratory and synchrotron based methods of stoneanalysis were completed on mixed composition cal-cium oxalate, calcium phosphate, uric acid and cys-tine stones. The results of x-ray analysis techniqueswere concordant with those of commercial FTIR (seetable).

Fourier Transform Infrared Spectroscopy

FTIR results included information on stone type and

ray energies. Calcium containing stones showed strong calciumnization threshold, also known as K-edge, of 4,038.5 eV. Secondenergy was below calcium core shell ionization energy, thus,al from other elements, such as phosphorus for apatite stone,strong sulfur K peak. Due to �XRF method high sensitivity, tracebserved. Elastic peak represents incoming x-ray beam, which was

at 3 x-shell ioitationee signshowse also o

the percent of different components in mixed com-

.


position stones (see table). No information was pro-vided on trace elements.

Microfocused XRF

�XRF measurements were made on all samples.Various x-ray energies were used to distinguishamong the K-shells of different elements. In thefluorescence spectra of the cystine stone sulfur is thedominant emission peak. For calcium based stonesthe calcium peak dominates and for calcium phos-phate stones phosphorus is also strongly present(fig. 3).

Figure 4. A, detailed image of stone samples mounted on beamwith image brightness proportional to calcium signal strength.15 � 6 �m beam spot size. Left edge shows weaker calcium flexperimental geometry. Similarly, signal from indentation (arrocomposite stone (see table), resulting in uneven calcium distribScale bars indicate 600 (sample 1) and 500 (samples 2 to 4) �m

Figure 5. Calcium XANES spectra (solid lines) of composite scalcium innermost absorption edge (K-edge) occurred at 4,038portions. XANES spectra from calcium compound library were
lines). A, hydroxyapatite stone portion. B, calcium oxalate stone porti
The sensitivity of �XRF allowed trace elements tobe identified in all samples (see table). All stonescontained small amounts of iron, zinc, chlorine andpotassium. Sample 1 contained lead, whereas thecystine sample contained small amounts of copperand chromium. Calcium based stones also containedstrontium, which is known to incorporate into calci-fying structures and tissues.10–12

Composite sample 1 had a strong phosphorus sig-nal in the calcium phosphate region, a weak phos-phorus signal at a border zone and no phosphorus

-y stage. B, �XRF elemental maps reveals calcium distributionere collected at 1,6205 eV x-ray energy, 20 �m pixel size and

ence signal for all 4 maps due to shadowing effect caused bysample 4 was weakened by protrusion at right. Sample 3 wasNote strong calcium signal on upper and lower edges (arrows).

sample 3 containing hydroxyapatite and calcium oxalate. ForData were collected with 9 � 3 �m beam at 2 distinct stone

ared to our measurements for compound identification (dotted

line xData wuorescw) in

ution.

tone.5 eV.comp

on.


signal in the calcium oxalate portion of the stone.Sample 3, a composite stone, also demonstrated astrong phosphorus peak in the calcium phosphatearea (see table).

XRF Elemental Maps

Calcium �XRF maps demonstrated a strong calciumsignal across all calcium based samples (fig. 4). Sam-ple 3, a uric acid/calcium composite stone, had anuneven calcium distribution. The upper and loweredges clearly contained more calcium than the cen-tral part (fig. 4, B, arrows). This indicates the spe-cific micro architecture of this sample, where thelocalization of the calcium oxalate/calcium phos-phate portion on the edges can be distinguishedfrom the uric acid portion in the middle.

Due to the experimental geometry used for �XRFmapping (fig. 2, A), the surface topography of sam-ples can result in absorption of the emitted fluores-cence radiation. This causes intensity variations(shadows) in the signal, which must be consideredwhen interpreting elemental distributions (fig. 4).

X-Ray Absorption Near Edge Structure

Calcium �XANES spectra were collected at multiplelocations of stones containing calcium phosphateand calcium oxalate components (see table). Using�XANES as a fingerprinting method and comparingour results to a database of XANES spectra, calciumoxalate and hydroxyapatite were identified (fig. 5).

X-Ray Diffraction

�XRD measurements were performed on calciumphosphate stone and used as a fingerprintingmethod. Diffraction patterns were compared toknown patterns. An excellent match was found for

Figure 6. �XRD of calcium phosphate stone sample 4. Twodistinct locations of same sample were analyzed by focusedx-ray beam with 15 � 6 �m spot size and 16,000 eV energy.Inset, powder diffraction pattern corresponding to blue curve.Diffraction intensity for each site matched hydroxyapatitediffraction.

hydroxyapatite (fig. 6).

DISCUSSION

All analytical methods used in the current studyyielded consistent results, validating the newly in-troduced microfocused x-ray beam techniques as anexcellent tool for investigating renal stone forma-tion. These x-ray techniques enabled high resolutionimaging of uroliths, determination of chemical com-position and detection of numerous trace elementsthat are integral parts of kidney stones. The study ofthese trace elements, particularly zinc, and theirpivotal role in stone formation is promising (Chi etal, unpublished data).

All stone analysis methods have limitations.Gross examination and light microscopy may pro-vide sufficient topographic information for the pre-liminary identification of characteristic pure stonesbut they cannot consistently identify compositestones. The constituents of composite stones can bedetermined by FTIR but the presence of trace ele-ments and the spatial distribution of elements can-not be determined. XRD provides elemental compo-sition but cannot evaluate amorphous materials,such as matrix stones. MS can identify elementalcomposition and trace elements but the sample isvaporized during evaluation so that elemental spa-tial distribution cannot be evaluated. Electron mi-croscopy provides high spatial resolution but no infor-mation on the elemental or molecular composition ofsamples (see Appendix).

The integrated experimental setup that we usedallowed us to perform �XRF, �XAS and �XRD mea-surements in short succession without removingsamples from the mounting. Thus, the advantages ofall 3 methods could be exploited by combining sen-sitive elemental and chemical composition measure-ments with �m scale imaging.13 The microfocusedx-ray beam necessary for these experiments is madepossible by the unique properties of SR, includingenergy tunability, low divergence and high inten-sity.

Recent advances in stone composition specifictherapy have been enabled by detailed knowledge ofstone formation and composition. For example, theadministration of methylated cystine interrupts thenormal crystallization of cystine stones.14 Moreover,cystine stones have differing susceptibility to litho-tripsy based on their relative roughness or smooth-ness, suggesting differences in crystallization.15 Onthe other hand, the lower amount of trace elementsin calcium oxalate monohydrate stones makes themmore resistant to lithotripsy.16 Additional focus hasbeen placed on identifying the trace elements in-volved in lithogenesis and the role that these ele-ments may have in stone formation.10–12 SR tech-
niques provide detailed data on the presence and


interactions of elements in uroliths that are notavailable using other analytical techniques.

CONCLUSIONS

Only the advanced imaging techniques used in thisstudy allow for the identification and spatial resolu-tion of trace elements in stones. Since no new uroli-thiasis medications have been developed in approx-imately 20 years, additional information aboutelemental interactions in stones is clearly needed for
drug discovery. Knowledge of trace elements that
augment stone formation or make stones more re-sistant to lithotripsy may lead to the development ofdirected chelators or the modification of trace ele-ments to help prevent stone formation or cause theformation of stones that are more easily fragmented.Identifying trace elements and their interactions instones may be essential for urolithiasis treatment.Until we better understand stone composition, wewill make little headway in improving stone therapyand initiating preventive measures against stone
formation.
APPENDIXExperimental stone analysis techniques according to spatial resolution

Method (characteristics) Spatial Resolution (nm) Advantages Disadvantages Description

FTIR* (spectroscopic, laboratory� synchrotron based,transmission)

Not applicable Mature technique, available at manylaboratories, inexpensive,determines sample molecularcomposition

No elemental composition Infrared spectroscopy excitesmolecular bond vibrational� rotational modes,detecting specific bondtypes, eg C-C, C-O

MS (spectroscopic, laboratorybased)

Not applicable Mature technique, available at manylaboratories, sensitive � accuratecomposition determination,including atoms, molecules �fragments

Sample is vaporized Measures charged particlemass-to-charge ratio

Transmission electronmicroscopy (imaging,diffractive, laboratory based,transmission)

Greater than 0.2 Highest spatial resolution analyticaltechnique, diffraction � elementalanalysis possible

No elemental mapping,large, expensive,extensive samplepreparation, limitedsample types

Contrast originates fromdifferences in electronabsorption electronsthrough thin sample

SEM (imaging, diffractive,laboratory based, reflection)

Greater than 1 High spatial resolution, elementalmapping (Z greater than 5) �diffraction possible

Typically samplepreparation is necessary,large, expensive

Secondary electronsreflected from samplesurface provide topology.Adding XRF detectorenables elementaldetermination/mapping

Scanning transmission x-raymicroscopy (imaging,spectroscopic, synchrotronbased, transmission)

Greater than 10 �XAS with high spatial resolutionyields elemental maps � chemicalenvironment information

Synchrotron based, noteasily accessible, thinlysectioned samplesneeded

X-ray absorption is measuredthrough thin sample �x-ray energy tunabilityyields elemental sensitivity

Bright field microscopy/lightmicroscopy* (imaging,laboratory based,transmission, reflection)

Greater than 200 Easily accessible, inexpensive,stereo microscope gives 3D image

Information limited bymagnification � visiblelight use

Contrast originates fromvisible light absorption/reflection differences

MicroCT (imaging, laboratoryand synchrotron based,transmission)

Greater than 200 Lab based, medium sized, nondestructive, yields 3-dimensionalimage of sample internal structure

No elementalcomposition, expensive

Contrast originates fromx-ray absorption differences

PIXE, microPIXE Greater than 1,000 Provides elemental composition (Zgreater than 11), can detect traceelements with high sensitivity onabsolute scale, spatial elementalmapping possible withmicrofocused beam

Large, expensive, samplepreparation needed

Sample irradiation with highenergy protons leads toXRF radiation emission, ofwhich energy is elementspecific

XAS, extended x-ray absorptionfine structure, XANES,�XAS,* micro-extended x-rayabsorption fine structure,�XANES (spectroscopic,synchrotron based,transmission, reflection)

Greater than 2,000 Can determine chemical environmentof specific elements

Synchrotron based X-ray absorption is measuredover small x-ray energyrange near absorption edge


APPENDIX (continued)

Method (characteristics) Spatial Resolution (nm) Advantages Disadvantages Description

XRF, �XRF* (imaging,spectroscopic, laboratory �synchrotron based, reflection)†

Greater than 2,000 Provides elemental composition (Zgreater than 13), can detect traceelements, laboratory basedinstruments common, spatialelemental mapping possible atsynchrotron with microfocusedbeam

No information onelement molecularenvironment, highresolution elementalmapping requiressynchrotron

Sample x-ray irradiationleads to fluorescenceradiation emission, ofwhich energy is elementspecific

XRD, �XRD* (diffractive,laboratory � synchrotronbased, transmission)

Greater than 2,000 Mature technique, available at manylaboratories, can accuratelydetermine crystalline materialcomposition

Sample must be in crystalform, high intensitymicrofocused beamrequires synchrotron

Crystalline materialdiffraction pattern providescrystal fingerprinting or3-dimensional atomicstructure

* Technique was used in study.
† For electron and proton induced XRF see SEM and PIXE.
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Microcomposition of Human Urinary Calculi Using Advanced Imaging Techniques

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