HUMAN TISSUES INVESTIGATION USING PALS TECHNIQUE∗ 
B. Jasi«skaa,†, B. Zgardzi«skaa, G. Chołubekb, M. Gorgola 
K. Wiktorc, K. Wysogl¡da, P. Białasd, C. Curceanue 
E. Czerwi«skid, K. Dulskid, A. Gajosd, B. Głowaczd 
B. Hiesmayrf , B. Jodłowska-J¦drychg, D. Kami«skad 
G. Korcyld, P. Kowalskid, T. Kozikd, N. Krawczykd 
W. Krzemie«h, E. Kubiczd, M. Mohammedd,i 
M. Pawlik-Nied¹wieckad, S. Nied¹wieckid, M. Pałkad 
L. Raczy«skij, Z. Rudyd, N.G. Sharmad, S. Sharmad, R. Shopaj 
M. Silarskid, M. Skurzokd, A. Wieczorekd, H. Wiktork 
W. Wi±lickij, M. Zieli«skid, P. Moskald 
aInstitute of Physics, MariaCurie-Skłodowska University, Lublin,Poland bDiagnosticTechniques Unit,Facultyof Nursing and Health Sciences Medical Universityof Lublin,Poland cDept. of Obstetrics, Gynaecology and Obstetrics — Gynaecological Nursing Facultyof Nursingand Health Sciences, Medical Universityof Lublin,Poland dFacultyof Physics, Astronomyand Applied Computer Science Jagiellonian University, Krakw,Poland eINFN, Laboratori Nazionali diFrascati,Frascati, Italy f Facultyof Physics, Universityof Vienna, Vienna, Austria gChair and Department of Histology and Embryology with Experimental Cytology, Medical Universityof Lublin,Poland hHigh Energy Physics Division, National Centre for Nuclear Research Otwock-‚wierk,Poland iDepartment of Physics, College of Education for Pure Sciences Universityof Mosul, Mosul, Iraq jDepartment of Complex Systems, National Centre for Nuclear Research Otwock-‚wierk,Poland kChair and Department of Gynaecology and Gynaecological Endocrinology Facultyof Nursingand Health Sciences, Medical Universityof Lublin,Poland 
(Received September 6, 2017) 
∗ 2nd 
Presented at the Jagiellonian Symposium on Fundamental and Applied Sub-atomic Physics, Krakw,Poland, June 3–11, 2017. 
† Corresponding author: bozena.jasinska@umcs.pl 
(1737) 
Samples of uterine leiomyomatis and normal tissues taken from pa-tients after surgerywereinvestigated using thePositron Annihilation Life-timeSpectroscopy(PALS). Signifcant di˙erences inallPALS parameters between normal and diseased tissues were observed. For all studied pa-tients, it was found that the values of the free annihilation and orthopositronium lifetime are larger for the tumorous tissues than for the healthy ones. For most of the patients, the intensity of the free annihilation and ortho-positronium annihilation was smaller for the tumorous than for the healthy tissues. For the frst time, in this kind of studies, the 3γ fraction ofpositron annihilation was determined to describe changes in the tissue porosityduring morphologic alteration. 
DOI:10.5506/APhysPolB.48.1737 
1. Introduction 
Forthe lastfew decades,apositronhasbecomeavaluabletoolin mate-rial investigations. Experimental technique based on thepositronbehaviour inthemedium,Positron Annihilation LifetimeSpectroscopy(PALS),is com-monly used in investigations of various kind of materials: from metals to complex mesoporous materials[1–3]. The thermalizedpositron produced initially during β decay can directly annihilate with one of the electrons or createpositronium (Ps) i.e. abound stateofapositron and an electron. Positronium can exist in two di˙erent states: para-positronium (p-Ps) and ortho-positronium (o-Ps),both substates: p-Ps and o-Ps are not stable and annihilate in the vacuum with a mean lifetime values: 125 ps and 142 ns, respectively. In the medium however, the o-Ps lifetime value can be shortenedevenbelow1nsduetothepossibility ofthepositronium trappingin the regions of lower electron density, the so-called free volume or void. In these voids, o-Ps can annihilate not only by intrinsic decay but with one of the electrons from its surroundingsbypick-o˙ process[4]. Shortening of the o-Ps lifetime valueis determinedbythelocal electron density,whichis correlated with the sizeof thevoid where thepositroniumis trapped[5,6]. The equation presentedbelow describes the dependence of the o-Ps lifetime value τpick−off on the void radius R inwhichpositronium istrapped 
 −1 
1 R 12πR 
τpick−off =1 − + sin ,λb =2 ns−1 , (1) 
λb R + Δ 2πR + Δ 
where R is the void radius, λb — averaged Ps decay constant, Δ — empiri-cal parameter refecting Ps interaction with the surroundings. The formulas relatingo-Ps lifetimewiththevoidsize[7–13]allowtousetheo-Ps lifetime value to determine the void sizes in the range from 0.2 to about 100 nm. 
The o-Ps lifetime in the living organism should be connected to the mor-phology of the cells and may be used as an indicator of the stage of the development of metabolic disorders[14]. 
The other parameter that can be applied to study the porosity of the material/tissues modifcation is the fraction fo−Ps−3γ of o-Ps atoms anni-hilating with the emission of 3γ quanta[15,16].Takinginto accounttwo processes leading to o-Ps decay: intrinsic decay and pick-o˙ process, the 3γ fraction canbe described as fo−Ps−3γ = τo−Ps/τT , where τT denotes the o-Ps lifetime value in the vacuum, equal to 142 ns. In the case when only one kindof freevolumes/pores existsin the material, the 3γ fraction in the whole spectrum canbe e˙ectively describedbythe dependence 
� 
1 − 4 
3 Po−Ps τo−Ps 
f3γ =+ Po−Ps , (2) 
372 τT 
where, Po−Ps denotes the ortho-positronium formation probabilitydependingonthe molecular structureoftheinvestigatedobject.Itwas successfully applied to investigate high-porosity materials [17–20]. In this article, we describe an application ofo-Ps lifetime and intensity as well as the frst ap-plication of the 3γ fraction for investigation of the diseased tissues ofhuman body. 
ThePALSwas successfully appliedin studiesof manyclassesof materials but onlyinavery limitednumberof papers concerningliving biological systems. Some applications were describedbyJean andAche in 1977[21]. Theyfocused on studiesof healthyand abnormal skin samples[22,23]and reported that the S parameterfrom the Doppler broadening of annihilation line is correlated with a broadly defned level of skin damage. For the last fewyears, biological systems have aroused the interest of annihilation tech-niquesagain[24]. The preciseinvestigationsofhuman tissues seemstobea very complex problembecause of the presence of biofuids wherepositronium can also undergo annihilation. However,hydrated solid materialswere successfully studied usingPALS. In thepaperbyHugenschmidt et al. [25], some experiments concerning the behaviour of the free volume on water loading, drying, and uniaxial pressure on glucose–gelatine compounds were performed. The dynamics of the water sorption by hydrophilic lyophilised yeast cellswas studied[26]. The precise understandingofvarious processes in biological systemsisveryimportant,asthey canbe appliedto increase thepossibilitiesofhuman body diagnosis using thePositron EmissionTomography(PET). One of the innovative scanner (referred to as J-PET) has been constructed at the Jagiellonian University, Krakw,Poland[27]. One oftheadvantagesof J-PETisapossibilityofmulti-photonimaging[28,29] whichenables the diagnosis based onpositron andpositronium lifetime[14] as well as based on the ratio of 3γ and 2γ annihilation rates[15]. 
In this paper, the human normal and diseased tissues are investigated usingPALS in order to compare modifcations in the tissue structure during tumour progression. 
2. Experimental 
2.1. Materials 
Uterine leiomyomas are the most common benign uterine tumours in women. Histologically, they consist mainly of smooth muscle cells and con-tain di˙erent amounts of fbrous tissue. Literature data have shown that more than 30%ofwomenhavemyomas[30]. 
In our study, patients with myomas were qualifed to a surgery on the basis of symptoms, history, clinical examination and ultrasonography. Each patient underwent hysterectomy with or without adnexa. The study was approved by the Bioethics Committee of the Medical University of Lublin. Each patient agreed to a clinical trial. Samples were taken directly from the organ, just after removal of the uterus from the surgical feldby one of thetwo experienced researchers inthe operating room. Tissue fragments were taken from the sites rated as macroscopically altered as well as from the normal tissues and placed directly into a transporting chamber. In any case, the time of delivery of the tissue to testing, in the same stable temperature(23 ± 1◦C)was less than1 hour. Eachof the examined tissues was subsequentlysubjectedtoa histopathological examination(TableI). 
2.2. Methods 
To perform PALS measurements and compare the results for samples with tumor and the reference one,two slicesof eachsample, separatedby a plexiglass partition element, were placed inside the steel chamber presented in Fig. 1. The total volume of the sample was about 1.5 cm3 . The 22Na positron source with the activityof 700kBqwas placed inside thepartition and inserted between the slices of the sample directly before the measurement. The steel seal assured that the sample was closely attached to the source. The sample chamber was placed between two scintillation heads, equipped with BaF2 scintillators, used as STARTand STOP counters. The detectors collected the γ quanta corresponding topositron creation inside the source andpositron/positronium annihilation inside the sample, respectively. The electrical impulses from the heads were processed with the use of standardFast–Slow delayed coincidence spectrometer [1,3]. The time intervalsbetweenSTART and STOP eventswere measured with the useof time-to-amplitude converter with the time range of 100 ns. The resolution curveof thespectrometer was approximatedby a single Gaussian with the 
TABLEI 
Comparison of the tested samples. 

Fig.1. Open chamber with tissue sample. The 22Na source was placed in the hole in the plexiglass plate and then inserted into the sample area (in this case: left-hand side). 
full widthathalf maximum(FWHM)of230ps.Suchparametersallowedto perform precise measurements ofpositron/positronium lifetimes from about 100 ps, up to few ns. 
Inorderto excludethe infuenceofsampleagingonPALS results,thereference sample, and the one with the tumor were measured at the same time, withthe useoftwoPALSspectrometersof almost identical parameters and thepositron sourcesof similar activities. After 1.5 hoursofmeasurements, the sampleswereswapped,andthen,thedatafromtwospectrometers were compared. The analysis of the results showed that: 
—PALS parameters do not depend on the spectrometer; 
— In the interval of a few hours, no infuence of sample aging on PALS results was noticed. 
The lifetime spectra were analyzed with the use of LT 9.2 program[31] and threediscrete components ascribed to annihilation of para-positronium 
+ 
(p-Ps, τ1), unboundpositrons(e, τ2)and ortho-positronium (o-Ps,τ3)were found, respectively, for each sample. 
3. Results and discussion 
For eachpatient, a pair of samples was investigated: one normal (N) and one tumor-altered (A). Exemplary microscopic images for one of the pair of tissues arepresentedinFig. 2. The di˙erencesin organization and density between the healthyand tumorous tissues are well-visible in the micrometer scale. 

Fig.2. Linearmuscle cellsofa normal uterus (left) andchaotic fbroid cells (right) 
— both images from the tested tissues. Magnifcation is equal to 100. Figure length: 0.62 mm and 1.2 mm, respectively. 
The results of PAL spectra analysis for normal (diamond) and altered (circle) tissues areshowninFig. 3.For comparison,the referencepointsfor water(crosses), whichis the main constituentof biofuids in living organisms is added.Waterwas extensively testedbythePALS technique[32–38],but for the purpose of this study, additional measurement for deionized water was carried out under the same measurement conditions as for human tissues. During the LT analyses,all threecomponentswereassumedas free.For human tissues, there are no justifed reasons to fx the ratioI1 : I3 =1:3 or tofxthep-Psvalueat125ps(it wasfoundthatthe useof suchprocedures signifcantly modifes the results). This kind of spectra analysis leads to some parameter misrepresentation, especially the frst component intensity is unviable high, however, it is a justifed method in this stage of investigation. Moreover, the main purpose of this paper is a comparison between normal and altered tissues taken from the same patient and to study the patient-to-patient variations. From the results presentedin Fig. 3, one can conclude that there are signifcant di˙erences of results obtained for normal (and also altered) tissues between di˙erent female patients. Therefore, it is notpossible to determine the state of health of the tissue from a single PAL spectrum measurement, and it is necessary to correlate two measurementsperformed for normal andaltered tissues from the same patient. The 

(a) (b) (c) 
Fig.3. (Colour on-line) The lifetime andintensity valuesofPALspectra components for normal (blue diamonds) and altered (red circles) uterine tissue. From left to right: p-Ps, free annihilation, o-Ps. In addition: uterine leiomyomas tissue after 16 hours storage in formalin (empty circles) and deionized water at room temperature (crosses).For the frst patient,both sampleswere classifed as altered by means of the microscopic images. 
di˙erencesinthe absolute valuesofthePALspectra parameters aresmall butdetectable. While correlating the respective parametersvalues obtained for normal and altered tissues from the same patient, a clear trend can be observed: the lifetime values of all three components in altered tissue are higher than those measured in normal tissue from the same patient. Similar relationwas observed previouslyin the literature[22,39]. 
The PALS parameters measured in tissues are similar, but not equal, to those known for water. While the intensity of the components in these two media are very close, di˙erences are noticeable in the values of all three lifetimes — in tissues all lifetimes are longer. The greatest discrepancies were found for the p-Ps lifetime:(0.188 ± 0.006)ns and(0.272 + 0.008)ns for water and normal tissue sample, respectively. 
The comparison of PALS parameters for fresh tissue samples and the ones conservedin formalinwas alsoperformed.The altered tissue from the second patientafter the standard measurement was placed for16hin formalin at 23◦Cand thenmeasured again with the useofPALStechnique. Itwas found that the formalin storage signifcantly infuenced the studied param-eters, especially the o-Ps intensity(an increase from the initial value 19.5% upto23%was observed)andtheaverage lifetimeofthe secondcomponent related to the annihilation of freepositrons. Based on the measurement presentedabove,itwas concluded that samplesintendedforPALS measurements should not be modifed in advance to obtain characteristics for the possible futurein vivo imaging as results should refect composition of living organisms. 
From data presentedin Fig. 3(a) thevoid radius (assuming spherical shape of void) and (b) the fraction of 3γ positron annihilation were calcu-lated according to equations(1)and(2), respectively (Fig. 4). The di˙er<BR>ence inboth parametersbetween the normal and diseased tissues are small and varies from patient to patient: in the altered tissues, voids are sparingly larger, while 3γ fraction change non-monotonically. The 3γ fraction depends onboth:lifetimeandintensityofo-Pscomponentwhichrefectthevoidsize and the concentration, respectively. In e˙ect, it depends on the porosity structure of the investigated material. In the case of our measurements, it maysuggest that theobserved di˙erenceinPALS parametersbetweennor-mal and altered tissues refects the degree or the kind of tissue pathogenic modifcation. Thisconclusion seems tobe confrmedby measurementsperformed on the frst sample: the material microscopically considered to be a normal tissue after the histological examination was qualifed as diseased (Table I); therefore,PALS results refect the degree of tissue deformation in one patient(involvedwith the experimental uncertainty). 

Fig.4. (Colour on-line) Thevoid radius (left), and 3γ fraction (right), as estimated fromPALS, determinedfor normal (blue diamonds) andaltered (red circles) tissues. 
4. Conclusions 
The preliminary results of PALS measurements for pairs of samples: normal and diseased, taken from patients’ uterus just after surgery and not solidifed show smallbut meaningful di˙erences inside the pairandbetween the patients. For all studied patients, the free annihilation and orthopositronium lifetimevalueswere found tobe larger for the diseasedtissues with respect to the lifetime of the normal ones. At the same time, the in-tensity of both components, generally, was found to be smaller for altered tissues than for the normal ones. Our results thatprovide the information on the molecular scale of the tissues structure justify the expectation that PALS measurementsofpositron andpositronium parameters couldbeuseful in the cancer/tumour diagnostics. They constitute the frst step on a wayto establish correlationsbetweenpositronium properties in the tissue and the staging of tumours for the elaboration of the in vivo morphometric imaging proposed in[14, 15]. 
However, to unequivocally conclude the existence of such correlations, a largenumberofpatientsand di˙erentkindsof tumours/cancers havetobe analyzed. 
We acknowledge the support by the National Science Centre, Poland (NCN) through grant No. 2016/21/B/ST2/01222, by the Ministry for Science and Higher Education through grant 7150/E-388/SPUB/2017/1, and Polish National Centrefor Researchand DevelopmentthroughgrantLIDER274/L-6/14/NCBR/2015. B.C.H. acknowledges the Austrian ScienceFund FWF-P26783. 
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