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Microradiography of biological samples with Timepix
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2011 JINST 6 C11005
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R ECEIVED: September 16, 2011
ACCEPTED: October 27, 2011
P UBLISHED: November 15, 2011
3–7 J ULY 2011,
Microradiography of biological samples with Timepix
a Institute
of Experimental and Applied Physics, Czech Technical University in Prague,
Horska 3a/22, CZ-12800 Prague 2, Czech Republic
b University Hospital Na Bulovce, Department of Radiological Physics,
Budinova 2, CZ-18081 Prague 8, Czech Republic
c Biological center of the Academy of Sciences of the Czech Republic, Institute of Entomology,
Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic
d Faculty of Science, University of South Bohemia,
Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic
e Charles University in Prague, First Faculty of Medicine,
Salmovsk´a 1, CZ-12000 Prague 2, Czech Republic
E-mail: [email protected]
A BSTRACT: Microradiography is an imaging technique using X-rays in the study of internal structures of objects. This rapid and convenient imaging tool is based on differential X-ray attenuation
by various tissues and structures within the biological sample. The non-absorbed radiation is detected with a suitable detector and creates a radiographic image. In order to detect the differential
properties of X-rays passing through structures sample with different compositions, an adequate
high-quality imaging detector is needed. We describe the recently developed radiographic apparatus, equipped with Timepix semiconductor pixel detector. The detector is used as an imager that
counts individual photons of ionizing radiation, emitted by an X-ray tube FeinFocus with tungsten, copper or molybdenum anode. Thanks to the wide dynamic range, time over threshold mode
— counter is used as Wilkinson type ADC allowing direct energy measurement in each pixel of
Timepix detector and its high spatial resolution better than 1µm, the setup is particularly suitable
for radiographic imaging of small biological samples. We are able to visualize some internal biological processes and also to resolve the details of insects (morphology) using different anodes.
These anodes generate different energy spectra. These spectra depend on the anode material. The
resulting radiographic images varies according to the selected anode. Tiny live insects are an ideal
object for our studies.
K EYWORDS : X-ray detectors; X-ray radiography and digital radiography (DR); Pixelated detectors
and associated VLSI electronics; Inspection with x-rays
1 Corresponding
c 2011 IOP Publishing Ltd and SISSA
2011 JINST 6 C11005
J. Dammer,a,b,1 F. Weyda,c,d J. Benes,e V. Sopko,a,b J. Jakubeka and V. Vondracekb
1.1 Timepix detector
1.2 The X-ray microimaging system
Several recent techniques based fully or partially on digital technology were implemented into biology within last several years. We demonstrate advantages of technique called ,,Microradiography”,
still not generally used in biology research. This method provides great and almost new way to
study structure of organisms [1].
Currently used imaging detectors as radiography film or scintillator detectors, can not visualize
required information about inner structure of scanned sample. But semiconductor pixel detectors
count single particle of radiation and their non-limited dynamic range allows appropriate imaging of soft tissues by X-ray radiation. Detectors Timepix have not so called dark current (noise)
and work in non-limited dynamic range. These features of detectors confer high quality and high
contrast of final images. Timepix detectors allow to measure the spectra of X-rays source. The
selected spectra of X-ray tube and voltage have effect on the resulting image. Modification of Xray spectra is achieved with different anode. This article describes the images options of biological
samples in its structure modification on anode X-ray tube voltage, and taken images at different
X-ray spectra [2].
Timepix detector
The Timepix detector consists of two chips: the sensor chip and a CMOS readout chip containing
the counting electronics [3]. The sensor is a semiconductor detector which can be manufactured
of different materials (Si, GaAs, CdTe) and of thickness e.g. 300 µm. The rear contact of the sensor is divided into a matrix of 256 × 256 cells (pixels) with the pixel pitch of 55 µm. The size
of the active detector area is 14.11 × 14.11 mm (see figure 1). Each Timepix pixel is equipped
with a counter operating in one of the three modes: Medipix mode (counting of incoming particles), Timepix mode (measurement of particle detection time) and Time over threshold (TOT)
mode allowing direct energy measurement in each pixel [4].
2011 JINST 6 C11005
The X-ray microimaging system
The system we used (figure 1) contains the µ-focus FeinFocus FXE-160.51, spot <1 µm operated
at 10–160 kV and 50 µA–1 mA (tube with tungsten, copper or molybdenum anode), a sample
holder and the pixel detector Timepix. The Timepix detector is used as a spectra measurement and
an imager whose detection threshold is adjustable and which is above 5 keV [5]. The whole setup
for X-ray microradiography is shown in figure 2. The source and the detector are kept fixed in one
position, while the sample is placed on a rotating stage. Both the detector and the holder for the
sample have three freedom degrees for movements in the Cartesian axes x, y, z, by means of motorized stages, while the X-ray tube can be positioned manually. By altering distance between the
sample and the detector, an additional (geometric) magnification from 1× to 30× could be set. The
exposure time of one frame is from 100 ms to 30 s, depending on the type of the object, source to
object and object to detector distances, beam parameters and detector settings. The quality of radiographic images moreover so-called strongly depends on the beam hardening effect [6]. The correction of the acquired data consists of the calibration of each detector pixel at different levels of hardening. The calibration is measured using a set of aluminum absorbers of various thicknesses [6].
Our microradiography system can be used for spectra measurement with different anode (tungsten,
copper and molybdenum) with calibrate Timepix detector in Time Over Threshold (TOT) mode [4].
Timepix detector was calibrated in keV [7]. Compare these spectra in figure 3, 5, 6. The measured
spectra were motivation for microradiographics imaging of biological samples. The difference of
anode spectrum should be visible in microradiographics images.
In case of detector Timepix was working in medipix mode. Each microradiographics image
are aquired on greater wax moth larva of Galleria melonella (Insecta: Lepidoptera; well known
insect modell species), see figure 6. Here we are able to recognize details of fat body, digestive
and tracheal system, spinning glands and structures inside head capsule, where biggest difference
in comparison with another anodes and voltage is apparent in molybdenum anode at 80 kV and
2011 JINST 6 C11005
Figure 1. Timepix detector.
it is evident especially on arrangement of fat body. In addition to greater wax moth larva, we
have studied larval and pupal stages of horse chestnut leafminer, Cameraria ohridella (Insecta:
Lepidoptera) and imagoes of woodlouse isopod (Crustacea: Oniscoidea) with similar results.
It was shown that the detector allows measurement of X-ray spectra with Timepix in TOT mode.
The microradiographic imaging with detector Timepix in Medipix mode represents modern noninvasive and non-destructive method of investigation especially suitable for inspection opaque biological objects. Hereby, we demonstrate results with different X-ray’s anodes (tungsten, molybdenum, copper). The choice of appropriate anode improves microradiographic study of internal
structure of biological samples with different composition.
This work was realized in the frame of the Medipix Collaboration and was supported in part by the
Research Grant Collaboration of the Czech Republic with CERN (No.1P04LA211), by the Fundamental Research Center Project (LC06041), the Research Programs 6840770029 and 6840770040,
and the Grants No. 2B06005 and 2B06007 of the Ministry of Education, Youth and Sports of the
Czech Republic. The Grant Agency of the Academy of Sciences of the Czech Republic is also
acknowledged (No. IAA600550614).
2011 JINST 6 C11005
Figure 2. Experimental table-top setup for X-ray microradiography and microtomography: microfocus
FeinFocus X-ray tube, calibration carrousel, automatic sample holder and Timepix detector.
Figure 4. X-ray spectra of Cu, W, Mo — 60 keV.
Figure 5. X-ray spectra of Cu, W, Mo — 80 keV.
2011 JINST 6 C11005
Figure 3. X-ray spectra of Cu, W, Mo — 40 keV.
2011 JINST 6 C11005
Figure 6. Microradiography of greater wax moth larva of Galleria melonella. Anatomical details like arrangement of fat body, digestive system, tracheal system, spinning glands and structures inside head capsule
differ more or less at different X-ray’s anodes. Compare for instance W 40 keV and Mo 80 keV.
[1] P. Frallicciardi et al., Real-time X-ray 2D and 3D micro-imaging of living animals with Medipix2
single photon counting detector, IEEE Conf. Proc. NSS/MIC 2008 (2008) 4760.
[2] L. Navratil et al., Medicinsk´a biofyzika (in Czech), Grada, Prague Czech Republic (2005), pg. 304.
[3] X. Llopart et al., Timepix, a 65k programmable pixel readout chip for arrival time, energy and/or
photon counting measurements, Nucl. Instrum. Meth. A 581 (2007) 485.
[4] J. Zemlicka et al., Analysis of painted arts by energy sensitive radiographic techniques with the pixel
detector Timepix, 2011 JINST 6 C01066.
[6] J. Jakubek, D. Vavrik, T. Holy, M. Jakubek and Z.Vykydal, Experimental system for high resolution
X-ray transmission radiography, Nucl. Instrum. Meth. A 563 (2006) 278.
[7] J. Jakubek, Precise energy calibration of pixel detector working in time-over-threshold mode, Nucl.
Instrum. Meth. A 633 (2011) S262.
2011 JINST 6 C11005
[5] J. Jakubek, Data processing and image reconstruction methods for pixel detectors, Nucl. Instrum.
Meth. A 576 (2007) 223.