2015_X-Ray Micro_rapid probing of features in exposed cells

Prévia do material em texto

Research article
Received: 30 July 2014 Revised: 16 January 2015 Accepted: 16 January 2015 Published online in Wiley Online Library: 6 March 2015
(wileyonlinelibrary.com) DOI 10.1002/xrs.2595
High-resolution scanning transmission soft
X-ray microscopy for rapid probing of
nanoparticle distribution and sufferance
features in exposed cells†
G. Kourousias,a* L. Pascolo,b P. Marmorato,c J. Ponti,c G. Ceccone,c
M. Kiskinovaa and A. Gianoncellia
Refraction and absorption properties of soft X-rays provide distinct advantages for ‘fast’ imaging of matter consisting of light
elements, as biological samples, with sensitivity better than conventional light microscopy. This is very attractive for
nanotoxicology, in particular for rapid screening of nanoparticle distribution and their effects on exposed cells at
submicrometer length scales. In this paper, we report on the first trials demonstrating the potential of scanning transmission
X-ray microscopy to monitor morphological and chemical changes induced in Balb 3T3 mouse fibroblast cells exposed to
CoFe2O4 nanoparticles. This research uses the complementary imaging methods of an advanced synchrotron soft X-ray
microscopy instrument implementing a novel approach based on high-resolution absorption and phase contrast imaging
for the identification of important cellular changes induced by the presence of nanoparticles. Copyright © 2015 John Wiley
& Sons, Ltd.
* Correspondence to: George Kourousias, ELETTRA – Sincrotrone Trieste, Trieste,
Italy. E-mail: george.kourousias@elettra.eu
† Presented at the European Conference on X-Ray Spectrometry, Bologna, Italy,
15-20 June 2014.
a ELETTRA – Sincrotrone Trieste, Trieste, Italy
b Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
c European Commission, Joint Research Centre, IHCP, Ispra, VA, Italy
1
63
Introduction
The attractive potential of using nanoparticles (NPs) in bio-
applications has increased the awareness for their possible health
hazards.[1] In fact, the effect of NPs penetrated inside cells is still
poorly understood and remains a challenge that the relatively
new discipline nanotoxicology has taken up. Nanotoxicology aims
at shedding light on the effects of nanomaterials of various struc-
ture and composition by studying the cellular uptake, toxicity and
effects on intracellular structures. Considering that the dimensions
of the NPs and the cells are in the nanometer to micrometer range,
such investigations require methods that provide information at
relevant length scales. In this respect, X-ray microscopy XRM is
one of the powerful tools for life science applications providing
high-resolution images at subcellular level.[2–5] The X-rays have
shorter wavelengths than visible light and the higher penetration
strength than charged particles, which provide higher spatial reso-
lution than visible light microscopy and the possibility to explore
the invisible-to-light interior of complex matter in two and three
dimensions. Another advantage of using X-rays instead of electrons
is the less demanding sample preparation and the relaxed
requirements for working under vacuum conditions. In addition,
compared with commonly used fluorescence microscopy that
requires labeling with fluorescent stains or expression of a fluores-
cent protein, in the case of biological samples, XRM is a label-free
technique that does not require staining or contrast agents. In
particular, for life science applications, soft X-rays can provide
better contrast in absorption imaging than hard X-rays as a result
of the high absorption cross sections of C, N and O K shells, the
main organic matter constituents.
During the examination of the cells exposed to NPs, one of
the first indication for suffering is the significant changes in
X-Ray Spectrom. 2015, 44, 163–168
cellular morphology. Full-field (TXM) soft XRM has already been
extensively used for high-resolution imaging of cells,[3] exposed
to NPs as well. Some of the examples reported in literature
make use of the soft X-ray tomography setup producing remark-
able results.[5,6] Scanning transmission X-ray microscopy (STXM)
has also been used for life science applications (refer to, for in-
stance, ref.[2] and refs[7–9]), usually with longer acquisition times,
but with the advantage of combining X-ray imaging with spec-
troscopic techniques such as X-ray florescence (XRF) and micro
X-ray absorption near edge structure (XANES). In particular, the
use of a configurable detector for STXM imaging,[10–12] as in
the unique setup of the TwinMic microscopy station, can pro-
vide several simultaneous X-ray imaging modalities, namely
brightfield (or absorption), phase contrast and dark field.
Indeed, although the best 10nm lateral resolution of real space
XRM cannot reach the subnanometer resolution of electron mi-
croscopes, it has been recently demonstrated that the use of
ptychographic methods is able to overcome this limitation.[13,14]
Despite of these interesting features, the use of XRM techniques
in certain groups of medical and life science research is still limited
as it remains relatively unknown and not easily available. Following
Copyright © 2015 John Wiley & Sons, Ltd.
G. Kourousias et al.
1
64
 10974539, 2015, 3, D
ow
nloaded from
 https://analyticalscienc
our previous study, on the chemical transformation of CoFe2O4 NPs
penetrated in different cell compartments of mouse fibroblasts,[15]
this work is focused on another aspect that is the morphological
modifications as a result of biochemical changes in Balb/3T3 cells
induced by these NPs. It explores the possibility to offer fast screen-
ing for the concentration thresholds when apparent suffering of the
cells is evidenced.
ejournals.onlinelibrary.w
iley.com
/doi/10.1002/xrs.2595 by Sincrotrone T
rieste, W
iley O
nline L
ibrary on [15/01/2024]. See the T
erm
s and C
onditions (https://onlinelibrary.w
iley.com
/
Experimental
Materials and methods
Balb/3T3 mouse fibroblast cells were cultured on silicon nitride
Si3N4 100nm thick windows and exposed CoFe2O4 cobalt ferrite
NPs added to the modified Eagle medium culture medium. The
CoFe2O4 NPs were supplied by Colorobbia S.p.A. (Italy) as
suspension in 100% diethylene glycol. Different NP concentrations
were selected, varying from 40 to 1000μM. After 24h of incubation,
both control and NP exposed cells were fixed with 4% formaldehyde
in phosphate-buffered solution. Further details concerning the sample
preparation and toxicity studies are available in Marmorato et al.[15]
The XRM analyses were performed at the TwinMic[16] beamline
(Elettra Synchrotron, Trieste, Italy). The microscope was operated
at 0.9 keV in scanning transmission mode (STXM). The sample was
raster-scanned across a 500nm diameter X-ray probe provided by
a 600μm diameter zone plate diffractive optics with 50nm outer-
most zone. Absorption and phase contrast images were acquired
by a fast readout Andor Technology EMCCD camera[10,12]and were
constructed simultaneously during the scan. The absorption im-
ages are constructed by summing all the photons transmitted by
the specimen point by point in the raster scan, while differential
phase contrast images result from the asymmetrical distribution
of the transmitted light in horizontal and vertical direction on the
charge-coupled device detector, generating X-moment and Y-
moment images respectively.[10,12]
The absorption and phase contrast images were firstly acquired
with a beam size of 500nm and then 135 or 80 nm, as later on
specified in the Results Section.
Visible light microscopy was used to acquire images of cells
stained with red oil solution.
term
s-and-conditions) on W
iley O
nline L
ibrary for rules of use; O
A
 articles are governed by the applicable C
reative C
om
Results and discussion
Cells exposed to different concentrations of CoFe2O4 NPs were im-
aged in scanning transmission mode (STXM). Their morphology
was evaluated through X-ray absorption and phase contrast images
collected by raster-scanningthe specimen with a 0.5μm diameter
probe size over a maximum area of 80 by 80μm. During this first
fast screening, it was possible to notice differences in the morphol-
ogy of the control cells when compared with the cells exposed to
NPs. Figure 1 depicts the absorption and phase contrast images
of a control cell and several cells exposed to different representa-
tive NP concentrations. The study was conducted on a statistically
relevant number of cells exposed to concentration varying be-
tween 40 and 1000μM. The control cell (Fig. 1a and b) exhibits
typical healthy cell morphology with the nucleus appearing darker
in the absorption images, i.e. denser and/or thicker compared with
the surrounding cytoplasm. The phase contrast image (Fig. 1b)
depicts better the topography of the cell, highlighting the presence
of a thicker material on the nucleus and the central part of the cell
when compared with most external cell body. Cells exposed to NPs
wileyonlinelibrary.com/journal/xrs Copyright © 2015 Jo
show instead a different morphology (Fig. 1c–h). Some areas of the
cytoplasm region appear not homogeneous and patchy. The
absorption images show both (i) darker areas (more absorbing
therefore denser and/or thicker), clearly representing clusters of
NPs, reaching dimension up to a few microns in diameters and (ii)
brighter regions that are clearly less absorbing than the sur-
rounding cytoplasm matter. The origin of these brighter areas
will be mentioned in more details later on in the manuscript.
The STXM images of cells exposed to high NP concentrations
(Fig. 1g and h) also show the formation of a white corona sur-
rounding the nucleus. Because all these features are absent in
the control cells, they are clearly attributed to the exposure to
NPs. The presence of NPs evidently induces stress on the ex-
posed fibroblast cells, resulting in an alteration of their morphol-
ogy. Similar behavior was observed on the same cell line
exposed to ZnO NPs (data not shown) and on another cell line,
U87MG glioblastoma-astrocytoma, exposed to CoFe2O4 NPs.
[17]
As previously reported,[15] when Balb/3T3 cell are exposed to
CoFe2O4 NPs, the staining procedure with the red oil probe
reveals a highly increased presence of lipid droplets in the
perinuclear region of the cells (as shown in Fig. 2). By comparing
these images with those obtained by absorption and phase con-
trast modalities, it appears evident that the stained lipid droplets
correspond to the low absorbing and brighter vesicle-like struc-
tures of Fig. 1c–h. Lipids are less dense than the organic cell
matter, therefore they appear less absorbing to X-rays. Comparing
with control images, it is clear that the high presence of lighter
structures and lipid vesicles are definitely associated to NPs and
their presence/dimension increases at increasing NP concentra-
tions (as evident when comparing cells exposed to 40μM in Fig.
1c and d with 250μM in Fig. 1e and f). The maximal stress effect
on the cell morphology was previously noted[15] at NP concentra-
tions equal or above 500μM. Indeed, a bright corona delimitating
the cell nucleus is clearly visible both in absorption and phase con-
trast images in Fig. 1g and h, suggesting the high sufferance state
of the exposed cells, whereas the light compact perinuclear mate-
rial is rarely detectable at lower concentration (Fig. 1c, d, e, f), and
it is completely absent in the control cell (Fig. 1a and b).
For the completeness of the present TXM investigation, higher
resolution images were acquired subsequently in STXM mode for
better revealing lipid accumulation and aggregation as cell suffer-
ance signs consequent to NP accumulation.
Figure 3 shows the absorption (Fig. 3b and d) and phase differen-
tial contrast (Fig. 3c and e) images together with the corresponding
visible light pictures (Fig. 3a) of two fibroblast Balb/3T3 cells
exposed to 500μM concentration of cobalt ferrite NPs. All X-ray
images (Fig. 3b–e) were acquired with a 135nm diameter spot size
at 900 eV incident photon energy.
The high spatial resolution allows for better highlighting the
morphological features induced by the exposure to NPs and at
the same time gives more precise information on the distribution
of NPs. Groups of NPs are clearly visible in the absorption images
as dark spots, located in the cytoplasm region and agglomerated
in clusters of maximum a few microns of diameter. In the phase
contrast images, they appear as small bumps, better delineating
their clustery shape. Moreover, this high spatial resolution absorp-
tion images better show the presence of the circular bright regions
or vacuoles, only partially visible at lower resolution, reaching up to
10 microns in diameter and appearing less dense than the cell
matter itself. The exceptionally high definition of the phase contrast
micrographs better delineates the lipid droplet features, which
appear as hollows or craters, suggesting a vesicle-like shape. NP
hn Wiley & Sons, Ltd. X-Ray Spectrom. 2015, 44, 163–168
m
ons L
icense
Figure 1. Absorption and phase contrast images of a control fibroblast cell (a and b respectively) and of cells exposed to different NP concentration: 40μM (c
and d respectively), 250 μM (e and f respectively) and 500μM (g and h respectively). Imaging parameters: 900 eV incident photon energy, 500 nm spot size,
20ms dwell time/pixel. Image dimensions: (a, b) 50 × 50μm2; (c, d) 44 × 44 μm2; (e, f) 40 × 40 μm2; (g, h) 40 × 40μm2. The images are scaled proportionally.
The 5 μm scale bar is valid for all images.
High-resolution STXM for probing NP distribution and sufferance in cells
1
65
 10974539, 2015, 3, D
ow
nloaded from
 https://analyticalsciencejournals.onlinelibrary.w
iley.com
/doi/10.1002/xrs.2595 by Sincrotrone T
rieste, W
iley O
nline L
ibrary on [15/01/2024]. See the T
erm
s and C
onditions (https://onlinelibrary.w
iley.com
/term
s-and-conditions) on W
iley O
nline L
ibrary for rules of use; O
A
 articles are governed by the applicable C
reative C
om
clusters and lipid droplets are in clear connection but only
marginally co-localizing, indicating that NPs are not captured
by the lipid structures; they seem to aggregate in proximity of
the lipid droplets. The presence of vacuoles is hinted by the
visible light images (Fig. 3a and d) but with really poor definition
and ambiguity.
X-Ray Spectrom. 2015, 44, 163–168 Copyright © 2015 John W
While the cells exposed to 500μM concentration of NPs seem to
maintain an intact cell shape, especially visible in the phase contrast
images, the morphology of cells exposed to higher concentration
appear greatly disturbed. High-resolution images of cells exposed
to 1000μM concentration of NPs were taken on different fibroblast
cells: A representative cell is shown in Fig. 4.
iley & Sons, Ltd. wileyonlinelibrary.com/journal/xrs
m
ons L
icense
Figure 2. Visible light images of two fibroblast cells incubated for 24 h with 500 μM NP concentration. The red spots represent lipids stained by red oil O
solution while the nuclei are stained in blue by Hoechst. The 5 μm scale bar is valid for all images.
Figure 3. Visible light (a) and absorption (b, d) and phase contrast (c, e) images of two fibroblast cells exposed to 500 μM NP concentration. Imaging
parameters: 900 eV incident photon energy, 135 nm spot size, 50ms dwell time/pixel. Image dimensions: (b, c) 60 × 40μm2; (d, e) 32 × 60 μm2. The images
are scaled proportionally. The 10μm scale bar is valid for all images except for the visible light one.
G. Kourousias et al.
1
66
 10974539, 2015, 3, D
ow
nloaded from
 https://analyticalsciencejournals.onlinelibrary.w
iley.com
/doi/10.1002/xrs.2595 by Sincrotrone T
rieste, W
iley O
nline L
ibrary on [15/01/2024]. See the T
erm
s and C
onditions (https://onlinelibrary.w
iley.com
/term
s-and-conditions) on W
iley O
nline L
ibrary for rules of use; O
A
 articles are governed by the applicable C
reative C
om
The micrographs in Fig. 4a and b were acquired with the
same image parameters as Fig.3 (at 135nm spot size and step
size and at an energy at 900eV). Again, the absorption images
show the NP clusters distributed in the perinuclear region and
the presence of lipid droplets, a few of them even crossing
the white corona and extending in the cell nucleus region. As
mentioned in the preceding texts, the white corona is clearly vis-
ible in all the analyzed cells exposed to 1000μM NP concentra-
tion. In order to increase the contrast and better delineate the
cell morphology at this high NP concentration, the images 4c,
d were acquired at 707 eV (corresponding to the Fe L3 edge,
where Fe has a high absorption contrast) with a reduced beam
size (and the step size) to 80nm. The optimization of the
imaging parameters allows to better delineate the sufferance
state of the cell that is manifested not only with lipid droplet
formation but also as flattening of the shape, visible in both
wileyonlinelibrary.com/journal/xrs Copyright © 2015 Jo
absorption and phase contrast micrographs (Fig. 4d). In particu-
lar, the nucleoli appear less contrasting and the cellular matter
thinner and/or less dense, making the lipid droplets less visible.
In some areas of the cytoplasm, the cellular matter seems
almost indistinguishable from the Si3N4 support, except for the
presence of NPs. This can be confirmed by comparing the cell
transmission for different NP concentrations by means of the
Beer–Lambert–Bougerlaw,[18] line profiles and statistical measure-
ments such as standard deviation. Similar stress-induced effects
were observed on other cell lines and with other nanomaterials
as well (data not shown). It is also worth noticing that the
imaging performed at this energy and with this spatial resolution
provides an exceptional definition of the NP clusters, some of
them with only a few 100nm size.
The effects of NP presence are even better highlighted when
comparing the exposed cells with control ones (Fig. 5a and b); the
hn Wiley & Sons, Ltd. X-Ray Spectrom. 2015, 44, 163–168
m
ons L
icense
Figure 4. Absorption (a, c) and phase contrast (b, d) images of two fibroblast cells to 1000 μM NP concentration. Imaging parameters: 900 eV (a, b) and
707 eV (corresponding to the L3 Fe edge) (c, d) incident photon energy, 135 nm (a, b) and 80 nm (c, d) spot size, 350ms dwell time/pixel. Image
dimensions: (a, b) 30 × 30μm2; (c d) 35 × 35μm2. The images are scaled proportionally. The 5μm scale bar is valid for all images.
Figure 5. Absorption (a) and phase contrast (b) images of a control fibroblast cell (not exposed to NPs). Imaging parameters: 900 eV incident photon energy,
135 nm spot size, 120ms dwell time/pixel. Image dimensions: 41.5 × 45 μm2. The 5μm scale bar is valid for all images.
High-resolution STXM for probing NP distribution and sufferance in cells
1
67
 10974539, 2015, 3, D
ow
nloaded from
 https://analyticalsciencejournals.onlinelibrary.w
iley.com
/doi/10.1002/xrs.2595 by Sincrotrone T
rieste, W
iley O
nline L
ibrary on [15/01/2024]. See the T
erm
s and C
onditions (https://onlinelibrary.w
iley.com
/term
s-and-conditions) on W
iley O
nline L
ibrary for rules of use; O
A
 articles are governed by the applicable C
reative C
om
cell material appears homogenous without white corona or lipid
droplets. The cell is meatier, and the nucleus is well embedded in
the cytoplasmic material.
The presence of lipid droplet, associated to NP exposure in
fibroblast Balb 3T3 cells, has already been observed through visible
light microscopy combined with red oil staining.[15]
X-Ray Spectrom. 2015, 44, 163–168 Copyright © 2015 John W
Another work[19] reports on the identification of vacuolated
swelling of the cytoplasm in hepatocytes from rat liver exposed
to different size and concentrations of gold NPs. Hyaline
vacuolation was observed and assessed through visible light
microscopy on conventional histological sections stained with
hematoxylin and eosin.
iley & Sons, Ltd. wileyonlinelibrary.com/journal/xrs
m
ons L
icense
G. Kourousias et al.
1
68
 10974539, 2015, 3, D
ow
nloaded from
 https://analyticalsciencejournals.onlinelibrary.w
iley.com
/doi/10.1002/xrs.2595 by Sincrotrone T
rieste, W
iley O
nline L
ibrary on [15/01/2024]. See the T
erm
The present work shows that NP presence and its effects on the
cell morphology, particularly the cell shape and the presence of
lipid droplets, can be easily identified with submicron soft TXM
imaging, without the need of labeling or dyeing the specimen.
The current report intends to suggest submicron soft TXM as an
effective tool for identifying important cellular changes caused by
the interaction with nanomaterials. Following our protocol, the
appearance of lipid droplet can be monitored by soft TXM absorp-
tion and phase contrast images. With this approach, there is an
increased resolution inmonitoring the toxic effect of NPs compared
with the conventional red oil staining procedure. In fact, this ap-
proach allows also revealing other concomitant cellular changes
as cell material loss in greatly suffering cells.
From a microscopy point of view, the imaging parameters are of
significant importance. Usually, in TXM mode, the cells are imaged
in the water window to enhance the contrast between water and
organic matter, especially for hydrated samples. In our case, the
incident energy was 900 eV because the reported measurements
are part of a more extensive study that originally involved not only
X-ray imaging but also low-energy XRF mapping.[15] This study
shows that energies around 1 keV seem to give an adequate
contrast especially in the absorption images. Preliminary test made
at 2 keV produced instead weakly contrasting absorption images,
because of the fact that at this energy, the cell organic matter be-
haves more transparently to X-ray photons. Moreover, by imaging
the cell close to iron (or alternatively cobalt) edge, the contrast be-
come even stronger, as shown in Fig. 4c and d.
Cell morphology could be investigated conveniently on fixed
cells, without the need of staining or dye, therefore avoiding the
possibility of creating artifact that could alter the outcomes of
spectroscopic analyses such as XRF and XANES, or even FTIR.
s and C
onditions (https://onlinelibrary.w
iley.com
/term
s-and-conditions) on W
iley O
nline L
ibrary for rules of use; O
A
 articles are governed by the appl
Conclusions
To our knowledge, this is the first time that vacuolation and lipid
droplet development in cells have been identified simply by high-
resolution soft X-ray scanningmicroscopy. The advantage for simul-
taneously monitoring absorption and differential phase contrast
images appears to be a very powerful approach for investigating
morphological changes and stress in biological samples. While
absorption images permit to discriminate the presence of thicker
and/or denser areas, the phase contrast images highlight topo-
graphical information such as edges, border, bumps and hollows.
Even though most of the stress-related features are visible in both
absorption and phase contrast images, it is clear that the combina-
tion of the two imaging modes can provide insightful information
on the cell reaction to the NP presence. For instance, the borders
of the cell membrane, of the white corona, of the NP cluster and
of the lipid droplets are much clearer in the phase contrast mode
and leave no doubts about the developed cell morphology. Phase
contrast imaging becomes essential at high energy, where absorp-
tion contrast of organic tissue is very low or negligible. In our case,
both absorption and phase contrast images provide enough con-
trast, but as we stressed in the previous paragraph, we believe that
the combination of them can offer an even more powerful tool.
Moreover, the reported study shows that even energies higher
than the water window, required for simultaneous soft X-ray XRF
mapping for characterization of chemical state of many transition
wileyonlinelibrary.com/journal/xrs Copyright © 2015 Jo
metal containing NPs, allow collection of imageswith a contrast
good enough to identify different subcellular structures.
Thus the presented work demonstrates the potentiality of this
microscopy technique for advanced imaging in life science
applications, complementing conventional visible light microscopy
but with higher spatial resolution.
icab
Acknowledgements
This work has been carried out within the ‘Nanobiosciences’ Joint
Research Centre action and European Project ‘CellNanotox’ (FP6-
2004-NMP-TI-4-032731) and part of it within SENSE project founded
by INAIL – sede provinciale di Trieste. European Commission is
gratefully acknowledged for financial support. The authors thank
Dr G. Baldi, Dr. D. Bonacchi and Dr G. Lorenzi for providing the
cobalt ferrite NPs.
References
[1] M. Auffan, J. Rose, J.-Y. Bottero, G. V. Lowry, J.-P. Jolivet, M. R. Wiesner.
Nat. Nanotechnol. 2009, 4, 634.
[2] J. Kirk, C. Jacobsen, M. Howells. Q. Rev. Biophys. 1995, 22, 33.
[3] W. Meyer-Ilse, D. Hamamoto, A. Nair, S. A. Lelievre, G. Denbeaux,
L. Johnson, A. L. Pearson, D. Yager, M. A. LeGros, C. A. Larabell.
J. Microsc. 2001, 201, 395.
[4] A. Ide-Ektessabi, Applications of Synchrotron Radiation: Micro Beams in
Cell Micro Biology and Medicine, Springer, Verlag Berlin Heidelgberg,
2007.
[5] C. A. Larabell, K. A. Nugent. Curr. Opin. Struct. Biol. 2010, 20(5), 623.
[6] M.A. Le Gros, C.G. Knoechel, M. Uchida, D.Y. Parkinson, G. McDermott, C.
A. Larabell, Comprehensive Biophysics, 2012, 2, 90, Biophysical
Techniques for Characterization of Cells, Petra Schwille. Oxford:
Academic Press.
[7] M. Howells, C. Jacobsen, A. Warwick, A. Principles and Applications of
Zone Plate X-ray Microscopes, Springer, Berlin, 2006.
[8] L. Finney, S. Mandava, L. Ursos, W. Zhang, D. Rodi, S. Vogt, D. Legning,
J. Maser, F. Ikpatt, O. I. Olopade, D. Glesne. ProcNatlAcadSci U S A. 2007,
104(7), 2247.
[9] B. Kaulich, A. Gianoncelli, A. Beran, D. Eichert, I. Kreft, P. Pongrac,
M. Regvar, K. Vogel-Mikus, M. Kiskinova. M. J. R. Soc. Interface 2009, 6,
S641.
[10] G.R. Morrison, A. Gianoncelli, B. Kaulich, D. Bacescu, J. Kovac, Proc. 8th
Int. Conf. X-ray Microscopy IPAP Conf. Series, 2006, 7, 377.
[11] M. Feser, B. Hornberger, C. Jacobsen, G. De Geronimo, P. Rehak, P. Holl,
L. Strüder. Nucl Instrum Meth A 2006, 565, 841.
[12] A. Gianoncelli, G. R. Morrison, B. Kaulich, D. Bacescu, J. Kovac. Appl. Phys.
Lett. 2006, 89, 251117.
[13] A. Sakdinawat, D. Attwood. Nature Photon. 2010, 4, 840.
[14] B. Kaulich, P. Thibault, A. Gianoncelli, M. Kiskinova. J. Phys. Condens.
Matter 2011, 3(8), 083002.
[15] P. Marmorato, G. Ceccone, A. Gianoncelli, L. Pascolo, J. Ponti, F. Rossi,
M. Salomé, B. Kaulich, M. Kiskinova. Toxicol. Lett. 2011, 207(2), 128.
[16] B. Kaulich, D. Bacescu, J. Susini, C. David, E. Di Fabrizio, G.R. Morrison,
P. Charalambous, J. Thieme, T. Wilhein, J. Kovac, D. Cocco, M. Salome,
O. Dhez, T. Weitkamp, S. Cabrini, D. Cojoc, A. Gianoncelli, U. Vogt,
M. Podnar, M. Zangrando, M. Zacchigna, M. Kiskinova, Proc. 8th Int.
Conf. X-ray Microscopy IPAP Conf. Series, 2006, 7, 22.
[17] A. Gianoncelli, P. Marmorato, J. Ponti, L. Pascolo, B. Kaulich, C. Uboldi,
F. Rossi, D. Makovec, M. Kiskinova, G. Ceccone. Interaction of
magnetic nanoparticles with U87MG cells studied by synchrotron
radiation X-ray fluorescence techniques. X Ray Spectrom. 2013, 42(4),
316–320.
[18] Beer "Bestimmung der Absorption des rothen Lichts in farbigen
Flüssigkeiten" Annalen der Physik und Chemie, 1852, 26, 78.
[19] M. A. Abdelhalim, B. M. Jarrar. Lipids Health Dis. 2010, 10, 166.
le C
re
hn Wiley & Sons, Ltd. X-Ray Spectrom. 2015, 44, 163–168
ative C
om
m
ons L
icense