Plasma deposited diamond-like carbon films for large neural arrays
To understand how large systems of neurons communicate, we need to develop methods for growing patterned networks of large numbers of neurons. We have found that diamond-like carbon thin films formed by energetic deposition from a filtered vacuum arc carbon plasma can serve as "neuron friendly&...
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| Цитувати: | Plasma deposited diamond-like carbon films for large neural arrays / I.G. Brown, E.A. Blakely, K.A. Bjornstad, J.E. Galvin, O.R. Monteir, S. Sangyuenyongpipat // Вопросы атомной науки и техники. — 2005. — № 1. — С. 152-156. — Бібліогр.: 17 назв. — англ. |
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irk-123456789-789492015-03-25T03:02:06Z Plasma deposited diamond-like carbon films for large neural arrays Brown, I.G. Blakely, E.A. Bjornstad, K.A. Galvin, J.E. Monteir, O.R. Sangyuenyongpipat, S. Low temperature plasma and plasma technologies To understand how large systems of neurons communicate, we need to develop methods for growing patterned networks of large numbers of neurons. We have found that diamond-like carbon thin films formed by energetic deposition from a filtered vacuum arc carbon plasma can serve as "neuron friendly" substrates for the growth of large neural arrays. Lithographic masks can be used to form patterns of diamond-like carbon, and regions of selective neuronal attachment can form patterned neural arrays. In the work described here, we used glass microscope slides as substrates on which diamond-like carbon was deposited. PC-12 rat neurons were then cultured on the treated substrates and cell growth monitored. Neuron growth showed excellent contrast, with prolific growth on the treated surfaces and very low growth on the untreated surfaces. Here we describe the vacuum arc plasma deposition technique employed, and summarize results demonstrating that the approach can be used to form large patterns of neurons. Щоб зрозуміти, як взаємодіють між собою великі системи нейронів, ми повинні розвивати методи вирощування рельєфних структур великого числа нейронів. Ми установили, що алмазоподібні вуглецеві тонкі плівки, що утворюються в результаті могутнього опромінення фільтрованою вуглецевою плазмою вакуумної дуги, можуть бути використані в ролі «нейроно-дружелюбніх» субстанцій для вирощування великих нейронних структур. Літографічні маски можуть бути використані для вормування алмазоподібної вуглецевої структури , а області селективного нейронного приєднання можуть утворювати систематичні нейронні структури. В експериментах, описаних нижче, як підкладку ми використовували предметні стекла мікроскопа, на які наносилися алмазоподібні вуглецеві покриття. Потім на опромінених підкладках були вирощені щурячі нейрони PC-12 і спостерігався ріст кліток. Спостерігався величезний контраст у рості нейронів, від багатого росту на опромінених поверхнях до слабкого на неопромінених. У даній роботі описана використовувана для опромінення вакуумно-дугова установка й узагальнені результати, що показують, що даний метод може бути використаний для формування великих структур нейронів. Чтобы понять, как взаимодействуют между собой большие системы нейронов, мы должны развивать методы выращивания рельефных структур большого числа нейронов. Мы установили, что алмазоподобные углеродные тонкие пленки, образующиеся в результате мощного облучения фильтрованной углеродной плазмой вакуумной дуги, могут быть использованы в качестве «нейроно-дружелюбных» субстанций для выращивания больших нейронных структур. Литографические маски могут применяться для формирования алмазоподобной углеродной структуры, а области селективного нейронного присоединения могут образовывать систематические нейронные структуры. В экспериментах, описываемых ниже, в качестве подложки мы использовали предметные стекла микроскопа, на которые наносились алмазоподобные углеродные покрытия. Затем на облученных подложках были выращены крысиные нейроны PC-12 и наблюдался рост клеток. Отслежен огромный контраст в росте нейронов, от обильного роста на облученных поверхностях до слабого на необлученных. В данной работе описана используемая для облучения вакуумно- дуговая установка и обобщены результаты, показывающие, что данный метод может быть использован для формирования больших структур нейронов. 2005 Article Plasma deposited diamond-like carbon films for large neural arrays / I.G. Brown, E.A. Blakely, K.A. Bjornstad, J.E. Galvin, O.R. Monteir, S. Sangyuenyongpipat // Вопросы атомной науки и техники. — 2005. — № 1. — С. 152-156. — Бібліогр.: 17 назв. — англ. 1562-6016 PACS: 52.77.Dq, 81.05.Uw, 81.15.Jj, 87.80.Rb http://dspace.nbuv.gov.ua/handle/123456789/78949 en Вопросы атомной науки и техники Національний науковий центр «Харківський фізико-технічний інститут» НАН України |
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Low temperature plasma and plasma technologies Low temperature plasma and plasma technologies |
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Low temperature plasma and plasma technologies Low temperature plasma and plasma technologies Brown, I.G. Blakely, E.A. Bjornstad, K.A. Galvin, J.E. Monteir, O.R. Sangyuenyongpipat, S. Plasma deposited diamond-like carbon films for large neural arrays Вопросы атомной науки и техники |
| description |
To understand how large systems of neurons communicate, we need to develop methods for growing patterned networks of large numbers of neurons. We have found that diamond-like carbon thin films formed by energetic deposition from a filtered vacuum arc carbon plasma can serve as "neuron friendly" substrates for the growth of large neural arrays. Lithographic masks can be used to form patterns of diamond-like carbon, and regions of selective neuronal attachment can form patterned neural arrays. In the work described here, we used glass microscope slides as substrates on which diamond-like carbon was deposited. PC-12 rat neurons were then cultured on the treated substrates and cell growth monitored. Neuron growth showed excellent contrast, with prolific growth on the treated surfaces and very low growth on the untreated surfaces. Here we describe the vacuum arc plasma deposition technique employed, and summarize results demonstrating that the approach can be used to form large patterns of neurons. |
| format |
Article |
| author |
Brown, I.G. Blakely, E.A. Bjornstad, K.A. Galvin, J.E. Monteir, O.R. Sangyuenyongpipat, S. |
| author_facet |
Brown, I.G. Blakely, E.A. Bjornstad, K.A. Galvin, J.E. Monteir, O.R. Sangyuenyongpipat, S. |
| author_sort |
Brown, I.G. |
| title |
Plasma deposited diamond-like carbon films for large neural arrays |
| title_short |
Plasma deposited diamond-like carbon films for large neural arrays |
| title_full |
Plasma deposited diamond-like carbon films for large neural arrays |
| title_fullStr |
Plasma deposited diamond-like carbon films for large neural arrays |
| title_full_unstemmed |
Plasma deposited diamond-like carbon films for large neural arrays |
| title_sort |
plasma deposited diamond-like carbon films for large neural arrays |
| publisher |
Національний науковий центр «Харківський фізико-технічний інститут» НАН України |
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2005 |
| topic_facet |
Low temperature plasma and plasma technologies |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/78949 |
| citation_txt |
Plasma deposited diamond-like carbon films for large neural arrays / I.G. Brown, E.A. Blakely, K.A. Bjornstad, J.E. Galvin, O.R. Monteir, S. Sangyuenyongpipat // Вопросы атомной науки и техники. — 2005. — № 1. — С. 152-156. — Бібліогр.: 17 назв. — англ. |
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LOW TEMPERATURE PLASMA AND PLASMA TECHNOLOGIES
PLASMA DEPOSITED DIAMOND-LIKE CARBON FILMS
FOR LARGE NEURAL ARRAYS
I.G. Brown, E.A. Blakely, K.A. Bjornstad, J.E. Galvin, O.R. Monteiro
and S. Sangyuenyongpipat
Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
To understand how large systems of neurons communicate, we need to develop methods for growing patterned
networks of large numbers of neurons. We have found that diamond-like carbon thin films formed by energetic
deposition from a filtered vacuum arc carbon plasma can serve as "neuron friendly" substrates for the growth of large
neural arrays. Lithographic masks can be used to form patterns of diamond-like carbon, and regions of selective
neuronal attachment can form patterned neural arrays. In the work described here, we used glass microscope slides as
substrates on which diamond-like carbon was deposited. PC-12 rat neurons were then cultured on the treated substrates
and cell growth monitored. Neuron growth showed excellent contrast, with prolific growth on the treated surfaces and
very low growth on the untreated surfaces. Here we describe the vacuum arc plasma deposition technique employed,
and summarize results demonstrating that the approach can be used to form large patterns of neurons.
PACS: 52.77.Dq, 81.05.Uw, 81.15.Jj, 87.80.Rb
1. INTRODUCTION
The study of the functional unit of the nervous
system, the neuron, has been an active field of
investigation both at the single-cell level and at the level
of large numbers of interconnected neurons, for example
within the human brain [1]. The behaviour of individual
neurons has been studied using microelectrodes to
monitor the electrical signals (“action potentials”)
generated within the neuron and along its dendrites (the
branch-like arms that carry signals toward the neuron cell
body) and axons (the long “tail” that carries the neuron
output signal to other cells). One can think of the single-
cell electrical behaviour as the performance at the
“device level” [2], and at this level much is known. At
the “system level”, however, much less is known − we
know very little about how large numbers of neurons
communicate among themselves. There has been good
progress made in the growth of neuron cultures in vitro.
The neurons grow, extend dendrites and axons, form
synapses, and create neural networks [2]. In order to
explore the electrical characteristics of large numbers of
associating neurons, however, we need first to develop
techniques for forming 2-dimensional patterned arrays of
neurons. The pattern parameters should all be under
control of the experimenter, including geometry of the
pattern, line width, and pattern size (number and density
of neurons). Several approaches to patterning have been
explored [3], including mechanical fabrication of troughs
and ridges [4], laser micromachining [5], surface
photochemical methods [6], photoresist methods, among
others. These methods work and have been used to grow
neural arrays. Here we describe some exploratory work
investigating the suitability of vacuum-arc-plasma based
methods of surface modification as a tool for forming
large patterned neuronal arrays.
2. EXPERIMENTAL METHOD
Plasma deposition was done using a filtered vacuum
arc system that has been described in detail elsewhere
[7,8,9]. The vacuum arc is a high current discharge
between two electrodes in vacuum [9]. Metal (or carbon)
plasma is produced in abundance from the cathode
material, and it is this plasma that carries the arc current.
For the work described here, a repetitively pulsed
vacuum arc plasma source was used; the pulse length was
5 ms and the repetition rate was 1 pulse per second. A
90o magnetic filter was used to remove the 'macroparticle'
flux (tiny droplets of cathode material) from the plasma
stream [10]. The plasma stream exiting the magnetic duct
was allowed to deposit onto a 1" x 3" glass substrate
mounted on a grounded holder positioned about 10 cm
from the duct exit. A simplified schematic of the filtered
vacuum arc plasma deposition system is shown in Fig. 1,
and a photograph of the plasma stream in Fig. 2.
Fig. 1. Simplified schematic of the filtered vacuum arc
plasma deposition set-up
Films of thickness in the approximate range 30–1000
Е were formed on glass microscope slides. We
investigated neuron growth on films formed from a wide
range of materials, including C, Mg, Ti, Pd, Ta, Ir, Pt and
Au; and by depositing at a somewhat elevated
background pressure we also formed and explored a
number of metal oxides, e.g. aluminum oxide, titanium
oxide and tantalum oxide were also deposited.
152 Problems of Atomic Science and Technology. 2005. № 1. Series: Plasma Physics (10). P. 152-156
Fig. 2. Plasma produced by the vacuum arc plasma gun
at the lower left streams through the magnetic filter duct
and is deposited onto a substrate at the upper right
A characteristic feature of vacuum-arc-produced
plasmas is the relatively high directed energy of the ions,
in the range 20–150 eV depending on the ion species
[11]. The film deposition is thus an energetic deposition
(even for the case of zero substrate bias), and for the case
of carbon this results in the film material formed being a
high quality, hydrogen-free, diamond-like carbon (DLC)
[12,13], as opposed to amorphous carbon or graphite. As
described below, we found that the carbon films were
particularly advantageous for enhanced neuron growth.
All of the neuron growth work described in the following
was done with DLC.
We used PC-12 neurons derived from a transplantable
rat pheochromocytoma from the adrenal gland. The cells
are grown in RPMI 1640 media with 2 gm/L glucose
(Invitrogen), 10% heat-inactivated horse serum
(Invitrogen), 5% fetal bovine serum (HyClone), 2 mM L-
glutamine, 1.5 g/L sodium bicarbonate, pen strep at 370C,
7.5% CO2 on Type I Collagen coated BiocoatTM (Becton
Dickinson) plastic 100 mm petri plates. Stock cultures
were fed every three days with 2/3rds fresh media, and
subcultured every 9 days with a 1:4 cell split ratio. Nerve
Growth Factor (NGF) 2.5S (Invitrogen) was added to cell
media at concentrations of 50 ng/ml. On a collagen-
coated substrate, neurite elongation proceeds at an
average rate of ~50 µm/day for at least 10 days. After 2
weeks of NGF exposure, the cultures generated a dense
mat of neuritic processes.
PC-12 cells were inoculated at 1 x 105 cells/ml onto
sterile glass slides that were pre-cleaned, then coated by
plasma deposition with DLC, and then coated with Type
I collagen. Cells were allowed to adhere to the slide in a
7.5% CO2 incubator at 37 oC, for 3 hours, and then gently
flooded with growth media. Cell growth was monitored
by phase light microscopy. After 3-6 days of cell growth,
NGF was added to the media at 50 ng/ml. After the
addition of NGF, cell division stops and differentiation
begins. PC-12 cells double every 96 hours. Cultures were
visually monitored daily and images captured every other
day, up to 1.5 months after initiation of the cultures.
3. RESULTS
3.1. DLC CHARACTERISTICS
The quality of a DLC sample can be quantified in a
number of ways depending on the particular application
for which the material will be used, but one way that is
general useful is to specify the ratio of diamond-bonded
carbon atoms to graphitically-bonded carbon atoms in the
material. This is called the sp3:sp2 ratio, referring to the
coupling between carbon atoms; alternatively the sp3
fraction can be stated, this being a measure of the fraction
of diamond-bonded carbon atoms in the sample. A
second important characteristic of a particular DLC
sample is whether or not it contains hydrogen. DLC films
can be deposited in a variety of ways. A common method
of forming DLC is to use a hydrocarbon precursor gas,
and in this case the material is hydrogenated; the
hydrogen content can be as high as several tens of
percent.
The kind of DLC that is formed by a carbon vacuum
arc is of high quality in both of these respects, since the
precursor material is purely carbon, and the energetic
deposition (high ion streaming energy) leads to high sp3
fraction. Fig. 3 shows the sp3 content as measured by an
EELS (electron energy loss spectrometry) technique
taken for the case of deposition onto a metallic substrate,
for which case the substrate can be pulse-biased so as to
control the ion deposition energy. The diamond-bonded
fraction maximizes at about 85% for a carbon ion energy
of about 100–150 eV; this result has been confirmed by a
number of groups around the world. For the case of a
glass (insulating) substrate, it is not possible to bias the
substrate, and the ion deposition energy for a carbon
plasma is about 20 eV, the directed energy with which
the vacuum arc carbon plasma is formed. Importantly, we
see from Fig. 3 that even for this case (zero bias), the sp3
fraction is very high, about 80%.
We conclude that the DLC formed by our technique
and used in this work for neuron growth experiments is
of high quality – high diamond-bonded fraction (about
80%) and hydrogen-free (about <1%).
153
Fig. 3. sp3 content of DLC films deposited by filtered
vacuum arc plasma deposition as a function of energy of
the carbon ion flux arriving at the substrate
3.2. NEURON GROWTH
Neurons grew on all the processed substrates, but there
was a wide variation observed in the total number of
attached cells and their morphology. We found that the
metals provided a generally positive growth enhancement
and that all of the metal oxides were generally negative
in their effect. The single film material that stood out as
providing vastly enhanced growth was carbon, which as
described above is deposited in the form of hydrogen-
free diamond-like carbon, or DLC. We therefore chose to
investigate neuron growth on diamond-like carbon
surfaces in more detail. Variation of DLC film thickness
indicated that about 100–300 Е was near optimum. With
thinner films, the neuron “contrast ratio” − ratio of
neuron growth density on the DLC-coated region to
density on the non-DLC-coated region − was less, and
thicker films tended to delaminate from the substrate.
Fig. 4 shows the effect of DLC on neuron growth. Fig.
4(a) shows neurons grown on a glass substrate, and Fig.
4(b) shows the growth for the same period of time for a
DLC substrate. Clearly the DLC provides superbly
enhanced growth.
Fig. 4. Neuron growth under similar conditions for
growth on a glass substrate (upper), and growth on a
DLC-coated substrate (lower)
The photograph in Fig. 5 shows clearly how a kind of
"fence" is provided by a DLC/non-DLC boundary. One
can see that (i) neuron growth is healthy on the upper
DLC-coated region, with virtually no growth on the
lower uncoated region, (ii) in the DLC region, neurons
grow extended processes (axons and neurons), (iii) the
neuron extensions show a pronounced tendency to
confine their growth to the DLC region.
Fig. 5. Selective neuron growth on DLC-coated
substrate. Neuron growth after 15 days on a glass slide
onto which a 100 Е thick film of DLC was deposited.
The DLC region can be seen as a slightly darker region
occupying the upper 80% of the photograph; there was
no DLC coating on the lower part of the image. The
whole slide was coated with Type I Collagen
The results of another growth experiment are shown
in Fig. 6. Here the neuron density is prolific, much
greater than would be chosen for a controlled experiment.
But the point is made clear that the growth is limited to
only the DLC-coated region. Neuron growth is on a glass
substrate processed by plasma deposition of ~100 Е
coating of diamond-like carbon (DLC) film. The plasma
deposition was such that the lower part of each photo is
the DLC-treated region, and the upper part is not DLC-
treated. The entire substrate was collagen coated, and the
neurons were seeded over the entire surface. The upper
photo shows the delicate neurite growth that develops on
the DLC-treated region; the lower photo shows that the
neuron growth in the DLC-treated region continues to a
dense and prolific neuron density. These results indicate
that neurons grow selectively on the lower DLC-treated
regions and not on the upper untreated regions. The
contrast (ratio of neuron density in the treated region to
neuron density in the untreated region) is very high, and
neuron growth in the treated region is healthy.
The results of our first attempt at neuron patterning
are shown in Fig. 7. Neuron growth is on a glass
substrate processed by plasma deposition of ~150Е
diamond-like carbon (DLC) film. Prior to deposition,
“LBNL” was written on the glass slide using a fine-point
marker pen, and then the DLC was deposited. After DLC
deposition, the ink was removed with alcohol, thus
leaving “LBNL” patterned in negative in the DLC film.
The slide was then coated with Type I Collagen and
seeded with PC-12 rat neurons. The neurons were
154
allowed to grow for 3 days, at which point NGF (Nerve
Growth Factor) was added. The micrographs shown in
Fig. 7 were taken after a growth period of 6 days after
initiation of the cultures.
Fig. 6. Selective neuron growth on DLC coated surface. The lower part of each photograph shown was DLC coated
and the upper part not coated; the entire substrate was then collagen coated, and neurons were then seeded over the
entire surface. A delicate neurite growth develops on the DLC-treated region (left photo), which subsequently develops
into a dense and prolific neuron field (right photo). (Scale: the width of each photograph is several hundred microns)
Fig. 7. Patterned growth of neurons to form “LBNL” (in negative)
4. DISCUSSION AND CONCLUSION
As discussed, the DLC film is deposited first onto the
glass substrate, subsequent to which a collagen film is
added; we estimate the collagen to be at least several
hundred Angstroms thick. Then the neurons are cultured
on the collagen. The precise mechanism that promotes
preferential growth in the presence of DLC is not known,
but we suggest some mechanisms that may play a role.
Collagen has a fibrous structure that is not impervious
to cell growth. The intertwined fibrils of the collagen
film do not form a solid barrier between the neurons and
the DLC, but present an open fibril matrix that is
conducive to cell growth. The biocompatibility of DLC is
known from a sizeable body of prior work that has been
reported in the literature having to do with DLC coating
of various kinds of prostheses and devices implanted into
the body [14-16], and in this sense our results are
consistent with the larger body of reported work and not
unexpected.
Another possible explanation for the observed
beneficial effect of DLC resides in the effect of the DLC
on a likely ordering of the long collagen molecules on the
substrate. The DLC surface consists of carbon atoms and
dangling bonds [17]. We speculate that this surface may
promote ordering of the collagen molecules parallel to
the glass surface, and that this molecular ordering may
favour neuron growth as opposed to a direct interaction
between neuron and DLC.
The work described here demonstrates the suitability
of filtered-vacuum-arc deposition of diamond-like carbon
for forming patterned arrays of large numbers of live
neurons. We have shown that energetic plasma
deposition of carbon to form an ultra-thin layer of DLC
on the substrate surface provides a means for selective
neuron attachment, growth, and differentiation on that
155
surface. The neuron growth contrast ratio (ratio of neuron
density on plasma-treated regions to neuron density on
untreated regions) can be very high, adequate for the
fabrication of large patterned arrays of neurons.
ACKNOWLEDGEMENTS
One of us (S.S.) is indebted to the Royal Golden Jubilee
program of the Thailand Research Fund for support at
Lawrence Berkeley National Laboratory. This work was
supported by the U.S. Department of Energy under
Contract Number DE-AC03-76SF00098.
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ПЛАЗМЕННОЕ НАНЕСЕНИЕ АЛМАЗОПОДОБНЫХ УГЛЕРОДНЫХ ПЛЕНОК
НА БОЛЬШИЕ НЕЙРОННЫЕ СТРУКТУРЫ
Я. Браун, Э. Блэкли, К. Бьйорнстад, Й. Гэлвин, О. Монтейро
Чтобы понять, как взаимодействуют между собой большие системы нейронов, мы должны развивать
методы выращивания рельефных структур большого числа нейронов. Мы установили, что алмазоподобные
углеродные тонкие пленки, образующиеся в результате мощного облучения фильтрованной углеродной
плазмой вакуумной дуги, могут быть использованы в качестве «нейроно-дружелюбных» субстанций для
выращивания больших нейронных структур. Литографические маски могут применяться для формирования
алмазоподобной углеродной структуры, а области селективного нейронного присоединения могут
образовывать систематические нейронные структуры. В экспериментах, описываемых ниже, в качестве
подложки мы использовали предметные стекла микроскопа, на которые наносились алмазоподобные
углеродные покрытия. Затем на облученных подложках были выращены крысиные нейроны PC-12 и
наблюдался рост клеток. Отслежен огромный контраст в росте нейронов, от обильного роста на облученных
поверхностях до слабого на необлученных. В данной работе описана используемая для облучения вакуумно-
дуговая установка и обобщены результаты, показывающие, что данный метод может быть использован для
формирования больших структур нейронов.
ПЛАЗМОВЕ НАНЕСЕННЯ АЛМАЗОПОДІБНИХ ВУГЛЕЦЕВИХ ПЛІВОК
НА ВЕЛИКІ НЕЙРОННІ СТРУКТУРИ
Я. Браун, Э. Блэкли, К. Бьйорнстад, Й. Гэлвин, О. Монтейро
Щоб зрозуміти, як взаємодіють між собою великі системи нейронів, ми повинні розвивати методи
вирощування рельєфних структур великого числа нейронів. Ми установили, що алмазоподібні вуглецеві тонкі
плівки, що утворюються в результаті могутнього опромінення фільтрованою вуглецевою плазмою вакуумної
дуги, можуть бути використані в ролі «нейроно-дружелюбніх» субстанцій для вирощування великих нейронних
структур. Літографічні маски можуть бути використані для вормування алмазоподібної вуглецевої структури , а
області селективного нейронного приєднання можуть утворювати систематичні нейронні структури. В
експериментах, описаних нижче, як підкладку ми використовували предметні стекла мікроскопа, на які
наносилися алмазоподібні вуглецеві покриття. Потім на опромінених підкладках були вирощені щурячі
нейрони PC-12 і спостерігався ріст кліток. Спостерігався величезний контраст у рості нейронів, від багатого
росту на опромінених поверхнях до слабкого на неопромінених. У даній роботі описана використовувана для
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опромінення вакуумно-дугова установка й узагальнені результати, що показують, що даний метод може бути
використаний для формування великих структур нейронів.
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