X-ray microbeam stand-alone facility for cultured cells irradiation

Nuclear Instruments and Methods in Physics Research B 394 (2017) 50–60

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Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb

X-ray microbeam stand-alone facility for cultured cells irradiation _ a,⇑, Jakub Bielecki b, Anna Wiechec´ b, Janusz Lekki b, Zbigniew Stachura b, Sebastian Bozek Katarzyna Pogoda b, Ewelina Lipiec b, Konrad Tkocz b, Wojciech M. Kwiatek b a b

Jagiellonian University Medical College, Department of Pharmaceutical Biophysics, Krakow, Poland Institute of Nuclear Physics Polish Academy of Sciences, PL-31342 Krakow, Poland

a r t i c l e

i n f o

Article history: Received 1 April 2016 Received in revised form 25 November 2016 Accepted 28 November 2016

Keywords: X-ray microbeam construction Cell irradiation model

a b s t r a c t The article describes an X-ray microbeam standalone facility dedicated for irradiation of living cultured cells. The article can serve as an advice for such facilities construction, as it begins from engineering details, through mathematical modeling and experimental procedures, ending up with preliminary experimental results and conclusions. The presented system consists of an open type X-ray tube with microfocusing down to about 2 lm, an X-ray focusing system with optical elements arranged in the nested Kirckpatrick-Baez (or Montel) geometry, a sample stand and an optical microscope with a scientific digital CCD camera. For the beam visualisation an X-ray sensitive CCD camera and a spectral detector are used, as well as a scintillator screen combined with the microscope. A method of precise one by one irradiation of previously chosen cells is presented, as well as a fast method of uniform irradiation of a chosen sample area. Mathematical models of beam and cell with calculations of kerma and dose are presented. The experiments on dose-effect relationship, kinetics of DNA double strand breaks repair, as well as micronuclei observation were performed on PC-3 (Prostate Cancer) cultured cells. The cells were seeded and irradiated on Mylar foil, which covered a hole drilled in the Petri dish. DNA lesions were visualised with c-H2AX marker combined with Alexa Fluor 488 fluorescent dye. Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction The history of research on ionising radiation begins in XIX century, by the time when developed electricity and photography gave the researchers an opportunity for a new kind of experiments. The concern about influence of ionising radiation on human body appeared soon after its discovery. To investigate this problem Pierre Curie attached to his arm a radium product for 10 h, then

Abbreviations: AFM, Atomic Force Microscope; BSA, Bovine Serum Albumin; CC, calibration coefficient; CCD, charge coupled device; cDK, contribution to dose to kerma; DK, dose to kerma; DSB, DNA double strand break; DZ, depth zone; FWHM, [spacial] full width at half of maximum; IFJ PAN, Institute of Nuclear Physics Polish Academy of Sciences; LC, largest cell; LNT, linear no-threshold; OSBS, One Second [duration] Beam Step; PBS, Phosphate Buffered Saline; PC-3, Prostate Cancer [cultured cell line]; PCV, percent of cell volume; PFA, paraformaldehyde; PPS, photon per second; SC, smallest cell; TBP, 0.2% Triton-X and 1% Bovine Serum Albumin in Phosphate Buffered Saline; TID, Total Irradiation Distribution; TIP, Total Irradiation Plateau; VDZ, volume of depth zone. ⇑ Corresponding author at: Jagiellonian University Medical College, Faculty of Pharmacy, Department of Pharmaceutical Biophysics, ul Medyczna 9, 30-688 Krakow, Poland. E-mail addresses: [email protected], [email protected] _ (S. Bozek). http://dx.doi.org/10.1016/j.nimb.2016.11.033 0168-583X/Ó 2016 Elsevier B.V. All rights reserved.

he studied the wound day by day [10]. This harmful experiment was probably the first approach to the research on influence of ionising radiation on living organisms. Presently, biological consequences of such a high-dose irradiation of living tissue are well known [6]. On the other hand, X and gamma-ray radiation, as well as radioactive elements are in common use in medicine, as we believe that low doses of ionising radiation are not harmful. However, an answer to the question whether our beliefs are right is not explicit yet [13]. In the range of low radiation doses, possible clinical effects can appear many years after the irradiation, since they can originate from DNA lesions induced even in a single cell [2,19]. These late aftereffects are called stochastic, because as probabilistic events they can, but does not have, to appear. Moreover, these effects are not unique, i.e. cancerous transformation (the main hazard), can also appear as a consequence of wrong diet, air pollution etc. especially when a long time period is considered. Hence, it is not possible to determine the influence of low doses of ionising radiation on the human body with an input-output like method. Therefore, an indirect and more sophisticated method of research had to be developed. As stochastic consequences can origin from a single irradiated cell, the research at cellular level seems to be

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recommended. To study the influence of low doses of ionising radiation on the human body we have to focus on a one single irradiated cell, and observe the mechanisms of its radiation damage and repair. Such research can be performed in vitro using cultured cell lines [1,31]. A knowledge of the exact dose of radiation delivered to a single cell, as well as experimental repeatability are the crucial factors for this type of experiments. This objective can be achieved using microbeam systems, where the size of beam is comparable to the size of a cell. At these facilities proton, ion, electron or X-ray microbeams are applied for cells irradiation [27,17,19]. Since our bodies are mostly exposed to X-ray radiation from the natural or medical sources, the research using X-ray microbeams seems to be the most interesting. X-ray microbeam systems are mostly constructed as synchrotron experimental lines [1], however X-ray microbeams can also exist as stand-alone facilities based on X-ray laboratory sources (X-ray tubes) [9,19]. This article presents an experimental line dedicated to cultured cells irradiation, which has been set up (designed and constructed) at the Institute of Nuclear Physics of the Polish Academy of Sciences in Krakow (IFJ PAN) [4,5]. Three types of experiments on radiation response at the cellular level were performed: dose-effect dependence, kinetics of DNA double strand breaks (DSB) repair, and micronuclei formation. The experiments were performed using a precise, uniform irradiation method with the beam spot of 20 lm (beam diameter at half of maximum intensity, FWHM)

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Fig. 1. X-ray microbeam line for cultured cells irradiation – system arrangement [5].

2. Materials and methods 2.1. System construction The experimental line dedicated to cells irradiation with the X-ray microbeam at IFJ PAN consists of an X-ray source, an X-ray focusing system, a sample stand, an optical microscope with coaxial light source and a digital scientific camera (Fig. 1). The X-ray source is fixed, while the other elements have 3-dimentional precise movement ability provided by motorized stages. An open type X-ray tube with microfocusing has been chosen as the source of radiation. X-rays emitted from the tube are focused on the sample with the use of the focusing system based on the Bragg reflection rule [4,5,19]. During the focusing process, the image of beam is observed with the X-ray sensitive CCD camera situated on the device holder. When the beam is focused, the camera is replaced by the spectral detector used for spectra and intensity measurements. When the spectrum is measured and the beam is optimized, the spectral detector is replaced by the microscope coupled to a scientific digital camera, which gives the system arrangement as shown in the Fig. 1. The sample stand is located between the focusing system and the microscope. Cells are seeded and irradiated on Mylar foil, which covers a round hole (6 or 10 mm in diameter) made in the Petri dish. The dish is placed vertically on the sample holder. The details of system modules and experimental methods are presented in the next sub-chapters. 2.1.1. Source of radiation An X-ray tube (Hamamatsu L9191) with microfocusing down to about 2 lm [3,4,5,11] (Fig. 2) serves as the source of radiation. The accelerating voltage is in the range of 20–160 kV, while the tube current is in the range of 0–200 lA. However, the actual determinant of the beam intensity is target current, which depends on the accelerating voltage and tube current, and varies from 0 to about 30 lA. Resolution measurements and technical details of the X-ray tube are described by Bielecki et al. [3]. Open type X-ray tubes allow to exchange the anode, and thereby to adjust the beam energy to the sample density and thickness. In the cell

Fig. 2. Hamamatsu L9191 X-ray source [4,5].

irradiation experiment a Titanium anode with 4.51 keV Ka characteristic energy is used. The X-ray beam emitted from the spot of about 2 lm in diameter is formed into a cone of 120 degrees opening angle. In the cells irradiation experiment the tube works at low voltage of 40 kV, which enlarges the emitting spot to about 3.5 lm. 2.1.2. Beam focusing and optimization According to the idea of experiment, the cone beam, which spreads out from the tube spot, should be focused again on the sample i.e. on a single cell. The details on the choice of the focusing system are presented in [4]. The chosen Rigaku focusing system (Fig. 3b) works based on the Bragg reflection rule, and the active module of this system consists of two multilayer elements connected perpendicularly. This kind of the focusing setup is known as the nested or Montel configuration of Kirckpatrick-Baez geometry (Fig. 3c) [30,21,34]. Each element consists of about 80 bilayers of chromium and carbon formed into an elliptical curve, with the average thickness of 3.1 nm in the center of the curve. Thickness of this multilayer changes along the curve in such a way that all X-rays incoming from the source spot and reflected from the multilayer are directed into one common focal spot for every point of reflection along the curve (Fig. 3a). Two elliptical focusing elements connected in the Montel geometry provide this focusing in space (Fig. 3f). Shape of each focusing curve and bilayer average

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Fig. 3. (a) Principle of focusing with multilayer mirror [5,25], (Osmic), (b) Rigaku focusing system with motorized stages for alignment: 1 – perpendicularity; 2, 4 – Bragg angle; 3 – internal aperture; 5 – distance from the source, (c) direct beam formation, (d) X-ray image of all beam elements formed after passage of the incident X-ray beam through the focusing system, (e) singly reflected beam formation, (f) doubly reflected beam formation, (g) spectrum of the direct beam (h) spectrum of the focused beam [5,19].

thickness are optimized for 4.51 keV Titanium Ka characteristic emission line. The source-to-spot distance is 32 mm, while the focusing system is 20 mm thick. According to the Bragg reflection rule, the focusing system works also as a monochromator (Fig. 3g, h), which is an important feature for further evaluation of the dose of radiation absorbed in cells. The image of beam is observed with Photonic Science X-ray sensitive CCD camera (Fig. 1b). The camera has the resolution of 4008
2670 pixels, and each pixel is a single X-ray detector with the size of 14.7 lm in diameter [3]. As the result of X-ray beam passage through the focusing system, 4 elements appear in the beam image. These are: a direct beam (Fig. 3c), two singly reflected beams (Fig. 3e) and the doubly reflected beam (Fig. 3f). The doubly reflected beam, which is the smallest element in the beam image (Fig. 3d), represents the exact focal spot, and appears in the way presented in the Fig. 3f. After focusing process, elements visible in the Fig. 3d have to be obscured, except the doubly reflected beam. In the next step, for spectra and intensity measurements, the CCD camera is replaced

by Amptek XR-100 spectral detector. Due to the Bragg reflection rule, the focused beam is monochromatic (Fig. 3h), and the energy spread of the beam is 313 eV at half maximum. The beam focal spot size was evaluated with the knife-edge method [15]. When a sharp edge is uncovering a Gaussian beam, the intensity of beam as a function of the edge position gives the Gaussian distribuanta (Fig. 4). This function is also given by the formula:

pffiffiffi f ðxÞ ¼ P1 ½1 þ erf ð 2ðx  P2 Þ=P 3 =2

ð1Þ

where P1 is the total power intensity, P2 is the center of the plot, P3 corresponds to 1/e2 radius of the beam and erf denotes the error function. FWHM of the focal spot evaluated from 3 beam shape measurements with the knife-edge method was 19.6 ± 2.5 lm. In the further calculations this parameter is approximated to 20 lm.

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Fig. 4. (a) Principle of the knife-edge measurement, (b) Gaussian distribuanta obtained with the knife-edge method for the focused beam at distance 4 mm, blue dots – experimental data, red line – function fitted according to Eq. (1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.1.3. Calibration of positioning When the beam is focused and optimized, the Optem Zoom 160 optical microscope [28] with Qicam scientific camera [29] is placed on the device holder. The camera has the resolution of 1392
1040 px with the pixel size of 4.65 lm. During the experiments the microscope works at 16
optical magnification with the resolution of 462 lp/mm, and numerical aperture of 0.31. The first step after successful focusing is the calibration of microscope position. The beam can be observed with visible light on P43 scintillator screen (Gd2O2S:Tb) placed on the sample holder. The distance between the microscope and the scintillator is set to the value, where at the maximum microscope magnification scintillator crystal grains are sharply visible. In this arrangement, the microscope and the scintillator maintained at the fixed distance A from each other (Fig. 5a) are moved along the beam axis, searching for the position where the beam image observed on the scintillator screen has the lowest diameter and highest intensity (the distance B in Fig. 5a). When this objective is achieved, the scintillator can be removed. Since then, the microscope is calibrated and its position remains fixed during the experiments. When a sample is placed on the holder, the holder is moved along the beam axis until the cells are sharply visible. According to the previous settings, cells which are situated in the microscope focal plane are also situated in the focal plane of the beam (Fig. 5b). The distance B is about 95 mm, while the difference B-A is about 4–7 mm [5]. Further calibration of the sample positioning system is carried out with JIMA resolution pattern [14]. The pattern consists of a set of lead elements (dots and rectangles) implanted on a silicon substrate (Fig. 6b). Known distances between the lines placed inside the rectangles enable to confirm a calibration coefficient (CC) between the distance in space in micrometers and the distance on a monitor screen in pixels (Fig. 6a, b, here is about 0.3 lm/px), as well as repeatability of actuators movement [5]. With this coefficient, cells marked on an image can be positioned under the beam.1 During all these procedures, and the further experiments as well, the focal plane of the microscope remains fixed. Based on the CC, the focal spot size can be quickly confirmed or corrected just before the experiments (Fig. 6c, d).

1

See the video in the supplementary materials (or on the website http://www. microbeam.eu/cells-irradiation.html).

2.2. Model of cell irradiation In the cell irradiation experiment the energy of radiation deposed in cells is usually the fundamental issue for further analysis [19]. For evaluation of this energy, beam and cell models should be created and then used in computations of the X-rays interaction with cellular matter. 2.2.1. Cell modelling The PC-3 cell model was developed based on PC-3 cell topography measurements performed using an Atomic Force Microscope (AFM, [18,26]). Through the analysis of 25 cell profiles (Fig. 7b) the independent ranges of cell height (hk = 3.44 ± 0.56 lm and radius (Rk = 15.38 ± 3.88 lm) were evaluated with the WSxM software (Fig. 7a, [12,5]. The cell profile (Fig. 7b) was approximated by the positive part of parabola

hðxÞ ¼ ax2 þ b;

ð2Þ

where x and h(x) are given in micrometers, and a and b parameters were calculated from (hk, Rk) values for the created models of the largest (LC) and the smallest (SC) cell. Hence, the models of PC-3 cell geometry were approached by the positive part of a paraboloid (Fig. 7c).

Hðx; yÞ ¼ aðx2 þ y2 Þ þ b

ð3Þ

Density of the cell was approximated by the density of water, thus the calculation of cell mass mk was proceeded by the calculation of its volume. The volume of positive part of paraboloid that models a single cell can be calculated as the following integral:

V k ¼ 2p

Z

Rk

ð4Þ

xhðxÞdx 0

The parameters of the largest (LC) and the smallest cell (SC) are presented in the Table 1.

Table 1 Largest (LC) and smallest (SC) cell characteristics. Cell model

a

b/hk [lm]

Rk [lm]

Vk [lm3]

mk [pg]

LC SC

0.0108 0.0217

4.0 2.88

19.26 11.5

2329 597

2.33 0.6

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Fig. 5. (a) Searching for the smallest size of the spot using the scintillator screen. Microscope and scintillator are moved along the beam axis maintaining at the fixed distance A from each other, (b) optimal position of the microscope at the distance B, where focal plane of the beam and focal plane of the microscope overlay [5].

Fig. 6. (a) CCD pixels readout across the pattern structure, (b) part of JIMA resolution pattern, (c) beam image on scintillator screen, (d) CCD pixels readout of the beam image on scintillator screen [5], (e) sum of RGB distributions from the plot ‘‘d” (blue curve) with the Gaussian distribution (red curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.2.2. Beam modelling The beam profile was approximated by the normal distribution multiplied by a constant:

pffiffiffiffiffiffiffi gðxÞ ¼ M  expðx2 =2r2 Þ=ð 2prÞ

ð5Þ

pffiffiffiffiffiffiffiffiffiffiffiffi where r ¼ FWHM=ð2 2 ln 2Þ is the standard deviation of normal distribution (JQC), and M is a constant [5]. Similarly to the Eq. (4), the total intensity of beam can be calculated as:

I0 ¼ 2p

Z 3r

x  gðxÞdx;

ð6Þ

0

which can be conceptualized as the ‘‘beam volume”. Thus

I0 ¼ 2M  p 

Fig. 7. (a) PC-3 cells image obtained with Atomic Force Microscope, (b) cell profile obtained with WSxM software, (c) the largest, the average and the smallest PC-3 cell models (in scale) presented with Mathematica software [5].

Z 3r 0

pffiffiffiffiffiffiffi x  expðx2 =2r2 Þ=ð 2prÞdx

ð7Þ

On the other hand, from the integration of the monoenergetic beam spectrum presented in Fig. 3h, performed with the results of 5 independent spectra measurements, the beam intensity was determined as I0 = 6.83 ± 0.17104 photon/s (pps). Substituting this value into Eq. (7) we obtain the constant M, and the beam profile in the focal spot given by the formula gðxÞ ¼ 152  3:8  expð0:0069  x2 Þ, where x is given in micrometers, and g(x) is given in pps/lm (Fig. 8a). Hence, the 3D model of beam distribution is estimated by the following formula:

Gðx; yÞ ¼ 152  expð0:0069  ðx2 þ y2 ÞÞ;

ð8Þ

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where G(x, y) is the beam intensity in pps through 1 lm2 specified by the (x, y) coordinates (Fig. 8b). 2.2.3. Irradiation schemes Two schemes of irradiation were realized: (i) one by one irradiation method of selected cells, and (ii) a method of fast raster irradiation of a rectangular area that contained a chosen group of cells [5]. In the first method, the cells dedicated for irradiation were marked on their image acquired with the microscope digital camera (Fig. 9a). The beam position marked before (on the beam image obtained with the scintillator screen Fig. 6c) was also imported into the cells image (the point P1 in the Fig. 9a). An input file with positions of the beam and positions of the marked cells was generated to be used by the sample actuators control software. Based on the calibration coefficient CC (Chapter 2.1.3) the cells were positioned one by one in the beam focus. The procedure is presented in the video clip included in the supplementary materials. The one by one irradiation method is advantageous over widebeam irradiations and represents the essentials of microbeam idea. However, this method faces two significant problems related to cells and their environment. The first problem stems from the fact that cells have different sizes (Figs. 7, 9a) and hence different masses. When the beam energy deposited in every cell is assumed to be the same, the absorbed dose may vary for different cells. The second issue is connected with the refraction of visible light in cell medium liquid. Fig. 9b illustrates this effect with an example of a golden pattern placed on the dish with cellular medium liquid. The image shift caused by light refraction at cellular medium

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surface can be even up to 60 lm, while the diameter of average cell is about 30 lm. Therefore, for a precise targeting of cells the amount of medium should be reduced. However, the removal of the medium makes the cells environment more hostile and unnatural. Then, in these conditions, the cells response can be also affected by drying or malnutrition effects. The second method of irradiation is conceptually simpler, however in certain types of experiments, when non-targeted effects (e.g. bystander response) are not studied, this easy method seems to be even more accurate and effective. In this method, a rectangular area containing a chosen group of cells is irradiated uniformly with the step corresponding to the FWHM value of the focal spot [5]. At a certain step, the Total Irradiation Distribution (TID) obtained from summation of these beam distributions (Fig. 8b, d) is uniform at a certain area (Fig. 10f, g). The step was optimized by home-developed Mathematica code [35]. The unit of beam distribution function is photon per second (pps), hence the function TID(x, y) represents the total number of photons incident per one square micrometer of the sample area defined by the (x, y) coordinates during the whole irradiation process, proceeded with One Second [Duration] Beam Step (OSBS). The optimal step for the 2D model equals 0.7 FWHM. In this case the sum of sequent profiles gives the planar distribution in a certain interval (Fig. 10d). We define this interval as Total Irradiation Plateau (TIP). In the 3D case, the planar irradiation distribution is obtained for the step equal to FWHM value or even greater (1.6 FWHM Fig 10g), up to 1.8 FWHM. This uniform ‘‘photon rain” leads to uniform distribution of the dose absorbed in cells, which

Fig. 8. (a) Beam profile and (b) 3D model of the beam [5].

Fig. 9. (a) Cells marked for irradiation in the Image-Pro software [22] (Mediacy), (b) composition of two photographs of a part of golden pattern (in the right corner) situated on a sample dish. One image was obtained when the pattern was covered with cellular medium liquid, the second after drying up. The image shift caused by light refraction is about 60 lm [5].

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Fig. 10. (a) Beam profiles separated by the step equal to FWHM value [5], (b) 2D Total Irradiation Distribution (TID) with One Second duration Beam Step (OSBS) equal to FWHM value, (c) beam profiles separated by the beam step of 0.7 FWHM value, (d) 2D TID with OSBS of 0.7 FWHM value, (e) image of cell area irradiated with OSBS equal to 1.6 FWHM, (f) 3D TID for the 11
11 grid of beam distributions with OSBS equal to 1 FWHM, (g) 3D TID for the 11
11 grid of beam distributions with OSBS equal to 1.6 FWHM, (h) 3D TID for the 11
11 grid of beam distributions with the OSBS equal to 2 FWHM.

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is then nearly independent on their sizes and masses. Precise targeting of cells is then not required and the cells can remain submerged in the medium liquid. Consequently the experiment duration is limited mainly by medium droplet drying or streaming down (droplet remains in the dish hole for a long time due to the surface tension). 2.2.4. Kerma and absorbed dose In the experiments presented later in the article, the method of uniform irradiation of a chosen sample area was applied. Rectangular areas were irradiated with the step equal to the FWHM value of focal spot with the intensity at Total Irradiation Plateau of ITIP ¼ 956 pps=lm2 . Before these photons reach the cells, they pass through dM = 1.5 lm thick Mylar foil, where cells are situated during irradiation process. The linear attenuation coefficient of Mylar is rM ¼ 5:37  103 =lm [23]. Applying the Lambert-Beer formula, the beam intensity after passing through the foil is:

IM ¼ ITIP  expðrM  dM Þ ¼ 948 pps=lm2

ð9Þ

According to the cell model evaluated before, the total irradiation intensity at the cell base is ICB ¼ IM pR2k . The intensity Ipp of point-to-point passage of the photon beam through the cell can be calculated in a similar way as in the Eq. (4), where the function h(x) is replaced by the intensity of the beam after passing the distance. Hence Ipp ¼ IM  expðrw  hðxÞÞ; where rw = 5.8  103/lm [23] is the linear attenuation coefficient for 4.51 keV photons in water (Fig. 11) [5,23], and the total intensity of the beam after passing the cell is

IPC ¼ 2pIM

Z

Rk

0

x  expðrw  hðxÞÞdx

ð10Þ

In our case expðrw  hðxÞÞ ’ 1  rw  hðxÞ, hence

IPC ¼ 2pIM ¼ 2pIM

Z

Rk

0

Z

0

Rk

x  ð1  rw  hðxÞÞdx xdx  2pIM

Z 0

Rk

rw  xhðxÞdx ¼ ICB  IM  rw V k ð11Þ

where IM  rw  V k ¼ IDC is the intensity of radiation distracted in the cell. The results of kerma and dose rate calculations are presented in the following table (Table 2). The calculation of dose rate is supported with Monte Carlo simulations of delta electrons passage

Fig. 11. Principle of calculation of a number of photons interacting in the cell volume.

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Table 2 Parameters of irradiation at OSBS equal to 1 FWHM for the largest (LC) and the smallest (SC) cell. ICB, IPC, IDC correspond to irradiation intensity at cell basement, passing the cell and distracted in the cell respectively. K is kerma per OSBS (One Second duration Beam Step), and D. is dose per OSBS.

LC SC

ICB [kpps]

[kpps]

IDC [kpps]

K [Gy/osbs]

D [Gy/osbs]

1131 403

1118 399.7

12.99 3.33

3.97 3.97

2.56 2.12

in water performed with the Casino software [8]. Calculation details are presented in the supplementary materials. 2.3. Experimental procedures The experiments were performed on a cultured line of human Prostate Cancer cells (PC-3) [16]. After preparation, about 16–18 h before the experiments, cells were seeded on a Mylar foil, which covered a 6 or 10 mm round hole in the bottom of Petri dish (Fig. 12) [31,5,19]. Until irradiation, the dish filled with cellular medium was incubated at the temperature of 37 °C and 5% CO2. During the irradiation, the dish was placed in the vertical position of sample holder. Cells were irradiated with the uniform method, with the step equal to the FWHM value of the focal spot (20 lm). Two types of cellular irradiation determinants were analysed: DNA double strand breaks and micronuclei formation. The two staining and visualisation procedures are described below. 2.3.1. DNA double strand breaks (DSB) visualisation After irradiation, cells were incubated for a time period determined by experimental objectives. Next, cells were rinsed in PBS (Phosphate Buffered Saline) and fixed in 1.5% PFA (paraformaldehyde) solution. Cells fixed in 70% ethanol solution, were incubated at the temperature of 20 °C for the time period from 2 days up to 2 weeks until the day of immunostaining. The DSBs immunostaining began with permeabilisation of cellular membranes proceeded with Triton-X solution. After another PBS rinsing, the cells were incubated for 2 h in a solution contained Anti-Phospho-Histone c-H2AX antibody [1]. Anti-Phospho-Histone c-H2AX antibody attach to phosphorylated histone H2AX, which appears the vicinity of DNA double strand break. After rinsing with TBP (0.2% Triton-X and 1% n (BSA) in PBS) solution, cells were immersed in a solution contained Alexa Fluor 488 (secondary antibody with fluorescent dye). After another TBP rinsing, the fluorescent dye DAPI (40 ,6-dia midino-2-phenylindole) was used. DAPI dye attaches to all DNA molecules and activates in UV light, while Alexa Fluor 488, which appears only in DSB locations, activates in 488 nm light. DAPI and Alexa Fluor images combined together make the visualisation of DSB lesions in cells (Fig. 13a). These images were acquired with epifluorescence microscope Olympus BX51 [24], with the same

Fig. 12. Petri dish with a 10 mm drilled hole filled with cellular medium [5].

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10 lg/ml respectively). As cytochalasins disable cytokinesis, a pair of nuclei was visible inside every cell membrane as the result of incomplete cell division. In some cases a small micronucleus was visible in the neighbourhood of two bigger ones (Fig. 14).

Fig. 13. CCD pixels readout in the form of RGB color distributions along the cell [5].

Qicam camera. Images were also acquired with the Image-Pro software. The graphical visualisation was practically used to evaluate the total intensity of DSB lesions in cells. The Fig. 15 presents an example of CCD pixels readouts along cells, which show RGB color distributions of the cell image. For the red and the blue distributions, the integrals along cell intervals were calculated (sum of color intensity for every pixel in the interval). A ratio of the red to the blue integral was adopted as a determinant of DSB intensity [5]. With this method the dose-effect relationship as well as kinetics of DSB repair were analysed.

2.3.2. Micronuclei visualization Some misrepaired or unrepaired DNA lesions can lead to DNA fragmentation. As a consequence, free-floating parts of DNA chains appear in a cell. During cell division, these separated parts of DNA form micronuclei [32]. The number of micronuclei in a cell population is related to the total irradiation that the population was exposed to in a certain time interval. For micronuclei visualisation, after irradiation cells were filled with medium doped with cytochalasins, and incubated for the time periods of 24, 48 and 72 h. After incubation period the medium was removed, and the cells were rinsed with PBS [32]. Next, they were fixed in a solution of methanol and acetic acid (9:1 proportion). After removal of the fixative, the cells were rinsed with distilled water. In the next step, they were stained with a solution of propidium iodide and fluorescein, diluted in PBS (5 lg/ml and

Fig. 14. Micronuclei observed in PC-3 cells after 4.51 keV X-ray irradiation [5].

3. Results The primary objective of the preliminary experiments presented in this article was to test the system, mainly in terms of the irradiation precision. Thus for the clean-cut visualisation of irradiated area (Fig. 10e) high doses of radiation were applied. The experiment conditions were tailored to correspond with another, parallel experiment, performed on the same PC3 cell line irradiated with targeted protons [20]. In this study, cells were exposed to variable (50-8000) number of 2 MeV protons, which corresponded to doses of 1.3–209 Gy/cell. Those experiments revealed a dependence of cell response on population passage number. About 93% of ‘‘youngest cells” (2–4 passages) survived irradiation with 4800 protons per cell (125 Gy).

Fig. 15. (a) Dose-effect relationship for PC-3 cells irradiated with 4.51 keV X-ray, (b) kinetics of DSB repair for PC-3 cells after irradiation with the dose of 75 Gy [5].

S. Boz_ ek et al. / Nuclear Instruments and Methods in Physics Research B 394 (2017) 50–60 Table 3 Micronuclei experiment (4.51 keV X-ray). Dose per cell (Gy)

Cells analysed

Cells with micronucleus

Density of micronuclei (%)

0 70 93.5

63 105 45

3 7 2

4.8 6.7 8.9

59

investigation of resonance energies, where the dose-effect gradient is highest, and the DSB recovery time is longest for the same dose per cell. Such resonance effects were described by Watanabe et al. [33], among others. The research on resonance effects in cancer cells irradiation studies might provide guidance for radiation therapy improvement. 4. Conclusions The X-ray microbeam line for irradiation of living cultured cells was designed and constructed. The preliminary experiments on dose-effect relationship, micronuclei formation, and kinetics of DSB repair were successfully performed, as well as one by one irradiation of single cells. However, due to many challenges of single cells irradiation, this method was not applied in the long run, as it was not strictly required for the conducted experiments. A method of uniform irradiation with an exact absorbed dose per cell was developed and successfully applied. The experiments confirmed the cells ability to survive and recovery after one-time irradiation with the dose of dozens of grays. The presented method does not require the focusing of the beam on a single cell, hence the application of a simpler focusing system is also possible. It could be also considered, that the focusing system is a module, which can be replaced by more advanced optics when necessary. Acknowledgements

Fig. 16. Inspection of the beam shutter speed with the microscope. The semicircle is the focusing system window (Fig. 3b) obscured by the external aperture (vertical copper element in the Fig. 1). The irradiation time was set to zero. The recorded video (supplementary material or the website http://www.microbeam.eu/ beamshutter.html) analysis (Corel Video Studio) indicates the shortest irradiation time of about 30 ms.

In our X-ray irradiations, three types of radiation response were examined: intensity of c-H2AX signal as a function of radiation dose (dose-effect relationship), intensity of c-H2AX signal as a function of recovery time (kinetics of DSB repair) and micronuclei formation. In the dose-effect experiment 32 cells in 3 samples were irradiated with the doses of 23.5 Gy, 47 Gy and 70 Gy (10, 20 and 30 OSBS respectively) and fixed 30 min after irradiation (Fig 6a). In the kinetics of DSB repair experiment 43 cells in 4 samples were irradiated with the same dose (70 Gy) and fixed in different periods of time after irradiation (Fig 6b). In the micronuclei formation experiment 213 cells were analysed for presence of micronuclei (Table 3). The experiment on kinetics of DNA DSB repair shows cells ability to recovery of DSB lesions within several hours after irradiation with the dose of 70 Gy (the 3rd point in the Fig. 6a). However, with the presented system low doses are also achievable. The shortest irradiation time determined by the home-made beam shutter is about 30 lS2 (Fig. 16). Even for the highest beam intensity, for OSBS equal to 1.8 FWHM, we obtain TIP function of 297 pps/lm2, which leads to the kerma per cell of 1.23 Gy/OSBS and the average dose per cell equal to 0.73 Gy/OSBS. Hence, in 30 ms of irradiation we obtain the dose of 22 mGy/cell. Reducing the beam intensity (Fig. 3h) to 30% of the maximum value, we obtain the dose of 6.5 mGy/cell. The absorbed dose, even if given in arbitrary units, is still useful when certainly determined and repeatable. Comparison of the gradient in dose-effect dependence and DSB recovery time for previously irradiated cells and unexposed cells from the control group would examine the radiation hormesis theory [7]. Another possible application of the X-ray microbeam facility can be devoted to the 2

See the website http://www.microbeam.eu/beamshutter.html.

The work and equipment was supported by Foundation for Polish Science (grant No. 222/FNiTP/119/2005), Ministry of Science and Higher Education – Republic of Poland (grant No. DPN/N15/ COST/2010) and National Science Centre – Poland (grant No. NN 518295540). Cells AFM images were acquired in the Laboratory of Biophysical Microstructures (IFJ PAN). We also want to acknowledge Professor Alan Michette and the participants of European MP0601 COST Action (Short Wavelength Laboratory Sources) for precious exchange of experience. The online dictionaries bab.la and glosbe.com were very useful for writing this article. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.nimb.2016.11.033. References [1] D. Anderson et al., Spatial and temporal distribution of c-H2AX fluorescence in human cell cultures following synchrotron-generated X-ray microbeams: lack of correlation between persistent c-H2AX foci and apoptosis, J. Synchrotron Radiat. 21 (2014) 801–810, http://dx.doi.org/10.1107/S1600577514011424. [2] J.S. Bertram, The molecular biology of cancer, Mol. Asp. Med. 21 (2001) 167– 223. _ [3] J. Bielecki, S. Bozek, J. Lekki, Z. Stachura, W.M. Kwiatek, Applications of the Cracow X-ray microprobe in tomography, Acta Phys. Polonica A 115 (2009) 537–541. _ [4] S. Bozek, J. Bielecki, J. Baszak, H. Doruch, R. Hajduk, J. Lekki, Z. Stachura, W.M. Kwiatek, X-ray microprobe – A new facility for cell irradiations in Krakow, Nucl. Instrum. Methods Phys Res. B 267 (2009) 2273–2276, http://dx.doi.org/ 10.1016/j.nimb.2009.03.032. _ [5] Sebastian Bozek, Construction and implementation of X ray microbeam for radiobiological research at cellular level (PL: Konstrukcja i wykorzystanie mikrowia˛zki promieniowania X do badan´ radiobiologicznych na poziomie komórkowym), PhD thesis in Polish under the supervision of Wojciech. M. Kwiatek, Institute of Nuclear Physics Polish Academy of Sciences, 2012. http:// www.biofizyk.pl/napromienianiekomorek.pdf. [6] H.M. Cullings, Impact on the Japanese Atomic Bomb Survivors of Radiation Received From the Bombs, Health Phys. 106 (2014) 281–293, http://dx.doi.org/ 10.1097/HP.0000000000000009. [7] Doss. Mohan, Linear no-threshold model VS. Radiation hormesis, Dose Response 11 (2013) 480–497, http://dx.doi.org/10.2203/dose-response.13005.Doss.

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