Neutron autoradiography in nuclear track

detectors: simultaneous observation of cells and nuclear tracks from BNC

reaction by UV C sensitization of polycarbonate

1 Comisión Nacional de Energía Atómica, Argentina2 Consejo Nacional de Investigaciones Científicas y

Técnicas, Argentina3 Facultad de Odontología, Universidad de Buenos Aires,

Argentina4 Laboratorio de Microespectrofotometría, CONICET-CNEA

Agustina Portu1,2, Andrés Rossini1,Mario A. Gadan2, Omar A. Bernaola2†, S. I. Thorp1, P. Curotto1, E.C.C Pozzi1,

Rómulo L. Cabrini 2,3,4, Gisele Saint Martin2

Qualitative autoradiograp

hy

QTAHigh

resolution

Tumor and premalingnat tissue sections of hamster cheek pouchand its corresponding autoradiography images. GB-10 (50mg.kg-1)

Quantitative autoradiography

5 ppm 10 ppm 100 ppm

Haematoxylin stained melanoma tumor (NUDE) cryosection and its corresponding qualitative autoradiography image. BPA (350 mg.kg-1), 1013 n.cm-2 . Magnification: 10 X.

S

T

S

T

Conventional autoradiography

QTA

Tissue sectioning

The sections aremounted on

Lexan

Irradiation with thermal neutrons

Tissue removal with trypsin

Etching(PEW 70° C, 2 min)

Track density measurements

Density and evaporation corrections

Quantification of 10B from calibration

curves

Reference system drilled onthe foils

Tissue Resection

and fixation

Boron compoundadministration

Portu et al., ARI 69 (2011) 1698–1701 – Portu et al., Biotech Histochem 88(2013): 217–221

High resolution autoradiography

Simultaneous observation of nuclear tracks and biological material

UV C sensibilization

(CR-39)

K. Amemiya et al., Rad Meas 40(2005) 283 – 288.T. Konishi et al., J. Radiat Res, 48 (2007)255–261.

HRQARHigh resolution

quantitative autoradiography

Solares and Zamenhof, Radiat Res 144 (1995) 50 – 58.Kiger 3rd WS, Micca PL, Morris GM, Coderre JA, Radiat Prot Dosimetry (2002) 99:409 – 12..

To develop a methodology to

produce an “imprint” of cells cultivated on a polycarbonate

detector by exposure of the

detector to UV C radiation…

…in order to observe cells and tracks

simultaneously.

To quantify BPA concentration of

MEL J cells in vitro.

Aim

Effect of UV C on polycarbonate

• Photo-oxidation: polimeric chains clevage

• Photo-Fries: chains re-arragement

UV Spectrum: • UV-A: (320-400) nm• UV-B: (280-320) nm• UV-C: (200-280) nm

yellowing

Irradiation with

ThermalNeutrons

UV-C exposure

Histological analysis

Etching

UV-C irradiation of the assembly sample-detector.

Wavelenght: 254 nm

- Increase of etching velocity- Imprints formation

Proposal

Cells seeding on Lexan

Cells superposition Monolayer

• Detector: circular foils of polycarbonate (Lexan™, 250 μm) on petri dishes (60 mm).

• Cells: human melanoma line MEL J (≈5.105 cells )• Incubation: BPA (10 ppm) for 2 h.• Fixation: Glutharaldehide (5%).

H&E. Magnification: 100x

1

thermal neutron irradiation

2 1012 n.cm-2

1013 n.cm-2

BPA incubation3 0 and 10 ppm

UV-C irradiation4 2, 4 and 6 h Photodegradati

on of

polycarbonate

-15 -10 -5 0 5 10 150,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1.5 cm 3.5 cm 7.5 cm 14.5 cm

Irra

dian

cia

(mW

/cm

2 )

distancia a lo largo de la lampara (cm)

Perfil longitudinal . Lampara 1.

Field uniformity: 15 cm

Irra

dia

nce

(m

W.c

m-

2)

Build-up and characterizati

on of the irradiation

facility

H&E and exploration

5

etching6 2, 3 and 4 min

Optimal conditions?

Methodology (cont)

0 2 4 6 8 10 12 14 16

0,2

0,4

0,6

0,8

1,0

1,2

1,4

distancia (cm)

Irra

dia

nci

a (m

W/c

m2)

Irradiancia en punto central (0,0)

M1M2M3M4M5

Irra

dia

nce

(m

W.c

m-2)

distance (cm)

Irradiance at central point

A 0,0F

UV Lamp

1 2 3 4 5 6

h = 1 cm

h = 14 cm

z x y

4 h, 2 min

2 h UV-C

2 min

3 min

4 min

Etching time

2 min 3 min 4 min

4 h UV-C

Haga clic en el icono para agregar una imagen

4 h 6 h

UV-C time4

min

3 m

in

6 h UV-C

4 min etching

Control BPA

OPTIM

AL

CO

ND

ITIO

NS

No tracks in sample groupsPreferential accumulation

Control – UV 6h – 4 min BPA – UV 6h – 4 min

SEM images

UV-A irradiation(λ=360 nm)

Exposition time: 6 h at a rate of 0.9 mW.cm-2

(same irradiance as UV-C)

10B quantification

11 18 25 33 40 47 54 61 68

grea

ter..

.0

10

20

30

boron concentration (ppm)

327

497

668

839

1009

1180

1351

1521

1692

grea

ter..

.05

101520

Area (µm2)

Mean value: (33±7) ppm

Image ProplusTM

Conclusions

• We developed a

methodology for the

simultaneous

visualization of cell

imprints and tracks

originated from 10B

using polycarbonate

as a nuclear track

detector.

• The best conditions

were established in

order to visualize

imprints and tracks.

• It could be concluded that the

photoinduced damage mechanisms

of the polymeric detector responsible

for the imprint creation, are more

effective for photon energies higher than

that corresponding to UV A radiation.

• We were able to quantify boron concentration

inside the cells.

This methodology will make possible an extensive comparison between cell lines

and the evaluation of different boron compounds under development through

their distribution in vitro.

Tissue imprints

a) Haematoxylin-Eosin stained premalignant tissue section mounted on Lexan (BPA, 1.25x).

b) Autoradiography image of a sucessive section.

1 h UV-C; 2 min etching

40x

Better resolution on epithelium assesment

Improvement of the technique!

If we zoom in …

Thank you very much!