X-Ray Microscopy and Radiobiology
by Using an Excimer Laser Plasma Source
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1*
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P. Albertano , D. Batani , M. Belli , A. Conti , R. Cotton , F. Flora , A. Grilli ,
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1
1
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1*
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F. Ianzini , P. Di Lazzaro , T. Letardi , M. Moret , A. Nottola , L. Palladino ,
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A. Reale , L. Reale , A. Scafati , M. A. Tabocchini , K. Vigli-Papadaki
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ENEA, C.R.E. Frascati, P.O. Box 65, 00044 Frascati, Italy
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Istituto Sup. di Sanità, (Lab. di Fisica), Romae INFN sez Sanità, Roma, Italy
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Univ. di Tor Vergata, Dip. di Biologia, Roma, Italy
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Univ. dell'Aquila, Dip. di Medicina Interna e Sanità Pubblica, L'Aquila, Italy
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Università de L'Aquila, Dip. di Fisica e INFN g.c. LNGS, L'Aquila, Italy
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Univ. di Milano, Dip. di Fisica, Via Celoria 16, 20133 Milano, Italy
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Dip. di Chimica Strutturale e Stereochimica Inorganica, Univ. di Milano,
Via Venezian 21, 20133 Milano, Italy
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Lab. Naz. Frascati dell'INFN, Frascati, Italy
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guest
Abstract. A soft X-ray plasma source, pumped by a high energy excimer laser
(Hercules), has been successfully applied both to soft X-ray Contact
Microscopy (SXCM) and to radiobiology experiments. Images of cyanobacteria and chlamydomonas, obtained by SXCM for different laser target
materials, are presented. In addition, a new method of obtaining images in a
helium environment at atmospheric pressure is discussed. Regarding to the
radiobiology applications, preliminary results on cells inactivation are also
reported.
1 Introduction
The idea of imaging the internal structure of living cells in their normal life state by
means of an X-ray microscope has been pursued for long time by biologists and
physicists. A particular interest was raised [1], [2], [3], [4], when it was considered
that soft X-rays in a convenient energy range could lead to observation of biological
structures. To obtain better natural contrast of the light internal structures of living
cells, the incident radiation must be limited to the spectral zone between the carbon
and the oxygen X-ray absorption K-edges (280-530eV) which for this reason is
commonly referred to as "water-window". Besides microscopy, the use of soft X-rays
is of great interest for applications in radiobiology research.
In this paper, the experimental results obtained in ENEA national laboratory at
Frascati, with the soft X-ray Contact Microscopy (SXCM) technique based on a
table-top laser plasma source are presented. A new technique for the insertion of
biological specimen into a helium filled interaction chamber has been succesfully
applied. Finally, regarding radiobiological study, preliminary experiments on
inactivation of cultrured mammalian cells are shown.
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P. Albertano et al.
2 Materials and Methods
A high energy (2 J/pulse) excimer laser system, developed at the ENEA Frascati
laboratory [5], was used to pump a soft X-ray plasma source. The 10 ns duration of
the laser pulse, with a beam waist of 20 µm and an intensity of 3x1013W/cm2, was
considered the optimum for generating soft X-ray radiation, peaked at the water
window [6]. The X-ray conversion efficiency in different regions of the spectrum was
measured using fast PIN diodes with various combinations of vanadium and
aluminium filters (to isolate the water window) for various materials. Yttrium,
tantalum and titanium were used as targets, giving the highest ratio between X-ray
emission inside and outside the water window. The dimensions of the X-ray source
were 20-30 µm emitting up to 40 mJ of X-rays with a pulse duration of 8ns in the
water window. For SXCM, the biological samples were placed in vivo in aqueous
medium between a thin X-ray transparent window (100nm thick Si3N4 window made
by FasTec Ltd., UK, with a transmission of approximatey 60% at an X-ray energy of
400eV) and a PMMA resist with a separation gap between the window and the resist
being in the range of 5-15µm. This sandwich was placed at 5mm distance from the
plasma, considering the optimum combination for obtaining an adequate X-ray
fluence while minimizing the penumbra. A more detailed description of the technique
can be found in, for example [7], [8], [9]. For radiobiological experiments, log phase
cells have been irradiated as monolayer through the mylar foil ( some cm2 area)
representing the bottom of the glass irradiation vessel. This was placed 20 cm above
the plasma, partially inserted on the top of the irradiation chamber. In this case a
copper target was used and the chamber was filled with helium at atmospheric
pressure.
3 Results
3.1 Biological Images in Vacuum
As it is well known, the X-rays in the water window, are highly absorbed by air. For
this reason, the interaction chamber is held in vacuum with the Si3N4 window bearing
the atmospheric pressure of water. The elapsed time from initially loading the cells in
the environmental holder, insertion into the chamber, evacuation and subsequent Xray exposure is typically 15-30 minutes. In Fig.1 an image of a green alga,
Leptolyngbya is shown. The image was obtained using an yttrium target, with an Xray flux in the water-window of 50 mJ/cm2.
3.2 Biological Images Obtained in a He-Filled Chamber
at Atmospheric Pressure
The new method for obtaining X-ray microscopy images, is based on the use of a
helium filled interaction chamber held at atmospheric pressure (Fig.2). This is
possible since the transmission of helium at the water window through the 5mm
separation between the plasma and the Si3N4 window is more than 80%. The
insertion of the sample is simply done through the pipe (Fig.2), so no chamber
X-Ray Microscopy and Radiobiology
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evacuation is needed. Furthermore, with this method the size of the Si3N4 window is
not limited by pressure effects. The first image obtained in the helium filled chamber
was of Leptolyngbya and can be seen in Fig.3. The image was obtained using a
tantalum target with an X-ray flux in the water window of 35 mJ/cm2. The quality of
the image was limited by resist contamination (this factor though, had no connnection
with the applied method to obtain the image). Finally, an important point to be
mentioned is that in the helium environment the debris bombardment emitted by the
plasma are significantly slown down.
Fig. 1. X-ray microscopy image of leptolyngbya obtained in vacuum.
3.3 Radiobiology Applications
At the energies typical of soft X-rays the photons are absorbed by the cells by
photoelectric effect and the photoelectrons energy is released over dimensions
comparable with those of the DNA (few nm). This allows to analyse the biological
effects of very localized deposition events in cultured mammalian cells. The fluences
appropriate for this study were obtained with few tens of laser shots on a copper tape
target moved by a step motor (Fig.2) at a photon energy of 1-1.5 keV. First results on
inactivation of LN12 mouse cells are shown in Fig.4. Work is in progress to achieve
an accurate dose calibration in order to extract the relevant radiobiological
parameters.
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Fig. 2. Experimental set-up of the interaction chamber when filled with helium and operating at
atmospheric pressure.
µm
Fig. 3. Image of Leptolyngbya, obtained
at atmospheric pressure.
Fig. 4. Surviving fraction versus X-ray
fluence for LN12 cells.
X-Ray Microscopy and Radiobiology
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4 Conclusions
Two different applications of X-rays obtained from a plasma source, pumped by an
excimer laser, were presented. In the microscopy application, a comparison between
the standard technique used up to now and a newly developed one was presented.
With the new method, the use of a helium filled interaction chamber at atmospheric
pressure enables a straightforward insertion of the samples in the chamber. As such,
the long evacuation time needed for each sample to be imaged was succesfully
avoided. As it has been mentioned above, it is evident that the advantages of this new
method are significant, with its potentials in need for further investigation. Moreover,
the irradiation set up of the plasma source has been succesfully applied for
radiobiological studies. Future research on SXCM is planned in three directions.
Increasing the size of Si3N4 windows to 1mm2, so as to obtain images of more and
larger size cells, like sperm ones. Shortening of the laser pulse to increase the
conversion efficiency from laser energy to X-rays pulse energy and hence to enhance
the X-ray fluence. Finally, the use of a thinner Yttrium target for an expected
reduction of the debris bombardment is planned. Some specific improvements are
planned also for the radiobiology applications. The reduction of the laser pulse
duration will mainly enhance the emission of X-rays at 1 keV. This will allow a
reduction of the band width ∆λ/λ to 10% and thus enable a more accurate relation
between X-ray fluence and dose to be achieved.
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