Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy
The potential of surface plasmon resonance-enhanced total internal reflection microscopy for visualization of submicron particles has been demonstrated using submicron-sized silicon rods as a test object. Submicron Si-rods were deposited onto the surface of a plasmon-supporting gold film by sedim...
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Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України
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irk-123456789-1184172017-05-31T03:06:28Z Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy Rengevych, O.V. Beketov, G.V. Ushenin, Yu.V. The potential of surface plasmon resonance-enhanced total internal reflection microscopy for visualization of submicron particles has been demonstrated using submicron-sized silicon rods as a test object. Submicron Si-rods were deposited onto the surface of a plasmon-supporting gold film by sedimentation from suspension, and their images were obtained using optical microscope with SPR excitation. Quality of images obtained in this way was compared with images viewed from the prism side in the SPR microscopy configuration. Specific features of light scattering from filiform objects are discussed. The study was aimed at development of a novel type of SPR-based biosensor relied upon direct count of biological species of interest (bacteria, viruses, large biomolecular complexes). 2014 Article Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy / O.V. Rengevych, G.V. Beketov, Yu.V. Ushenin // Semiconductor Physics Quantum Electronics & Optoelectronics. — 2014. — Т. 17, № 4. — С. 368-373. — Бібліогр.: 31 назв. — англ. 1560-8034 PACS 73.20.Mf http://dspace.nbuv.gov.ua/handle/123456789/118417 en Semiconductor Physics Quantum Electronics & Optoelectronics Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України |
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English |
| description |
The potential of surface plasmon resonance-enhanced total internal reflection
microscopy for visualization of submicron particles has been demonstrated using
submicron-sized silicon rods as a test object. Submicron Si-rods were deposited onto the
surface of a plasmon-supporting gold film by sedimentation from suspension, and their
images were obtained using optical microscope with SPR excitation. Quality of images
obtained in this way was compared with images viewed from the prism side in the SPR
microscopy configuration. Specific features of light scattering from filiform objects are
discussed. The study was aimed at development of a novel type of SPR-based biosensor
relied upon direct count of biological species of interest (bacteria, viruses, large
biomolecular complexes). |
| format |
Article |
| author |
Rengevych, O.V. Beketov, G.V. Ushenin, Yu.V. |
| spellingShingle |
Rengevych, O.V. Beketov, G.V. Ushenin, Yu.V. Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy Semiconductor Physics Quantum Electronics & Optoelectronics |
| author_facet |
Rengevych, O.V. Beketov, G.V. Ushenin, Yu.V. |
| author_sort |
Rengevych, O.V. |
| title |
Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy |
| title_short |
Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy |
| title_full |
Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy |
| title_fullStr |
Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy |
| title_full_unstemmed |
Visualization of submicron Si-rods by SPR-enhanced total internal reflection microscopy |
| title_sort |
visualization of submicron si-rods by spr-enhanced total internal reflection microscopy |
| publisher |
Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України |
| publishDate |
2014 |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/118417 |
| citation_txt |
Visualization of submicron Si-rods
by SPR-enhanced total internal reflection microscopy / O.V. Rengevych, G.V. Beketov, Yu.V. Ushenin // Semiconductor Physics Quantum Electronics & Optoelectronics. — 2014. — Т. 17, № 4. — С. 368-373. — Бібліогр.: 31 назв. — англ. |
| series |
Semiconductor Physics Quantum Electronics & Optoelectronics |
| work_keys_str_mv |
AT rengevychov visualizationofsubmicronsirodsbysprenhancedtotalinternalreflectionmicroscopy AT beketovgv visualizationofsubmicronsirodsbysprenhancedtotalinternalreflectionmicroscopy AT usheninyuv visualizationofsubmicronsirodsbysprenhancedtotalinternalreflectionmicroscopy |
| first_indexed |
2025-07-08T13:56:35Z |
| last_indexed |
2025-07-08T13:56:35Z |
| _version_ |
1837087328321929216 |
| fulltext |
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2014. V. 17, N 4. P. 368-373.
© 2014, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
368
PACS 73.20.Mf
Visualization of submicron Si-rods
by SPR-enhanced total internal reflection microscopy
O.V. Rengevych, G.V. Beketov, Yu.V. Ushenin
V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine,
45, prospect Nauky, 03028 Kyiv, Ukraine;
Phone: +38(044) 525-40-20; e-mail: o.v.shynkarenko@gmail.com
Abstract. The potential of surface plasmon resonance-enhanced total internal reflection
microscopy for visualization of submicron particles has been demonstrated using
submicron-sized silicon rods as a test object. Submicron Si-rods were deposited onto the
surface of a plasmon-supporting gold film by sedimentation from suspension, and their
images were obtained using optical microscope with SPR excitation. Quality of images
obtained in this way was compared with images viewed from the prism side in the SPR
microscopy configuration. Specific features of light scattering from filiform objects are
discussed. The study was aimed at development of a novel type of SPR-based biosensor
relied upon direct count of biological species of interest (bacteria, viruses, large
biomolecular complexes).
Keywords: surface plasmon resonance, total internal reflection microscopy, submicron
Si-rods, counting biosensor, pathogen detection.
Manuscript received 03.02.14; revised version received 18.07.14; accepted for
publication 29.10.14; published online 10.11.14.
1. Introduction
Introduction of SPR biosensors more than 2 decades ago
provided a strong impact on progress in biochemical
science and related disciplines, allowing label-free real-
time characterization of biomolecular interactions [1-3].
By now, this technique has also found numerous
applications in many other areas, from electrochemical
studies to food quality control and environmental
monitoring. Growing use of SPR has stimulated research
focused on developing new SPR-related techniques:
label-enhanced SPR, surface plasmon fluorescence
spectroscopy, SPR microscopy (SPRM), also referred to
as SPR imaging (SPRI), etc. [4-12]. SPRM was first to
demonstrate by B. Rothenhäusler and W. Knoll for
imaging of low-contrast samples with high lateral
resolution [13]. Currently this method is related mainly
to accessing multi-element arrays of macroscopic
sensitive areas at the sensor surface and represents just a
high-throughput multichannel version of the classical
SPR-biosensor. Recent studies aimed at SPR-assisted
visualization of single cells and viruses utilize optical
arrangement very similar to that used in SPR imaging
[14-16]. General drawback of this technique is strong
interference effects arising from light diffraction on
particles, which size is comparable with the wavelength.
Interference patterns result in splattering of the image,
thus hampering true visualization of the particles.
In our study, we demonstrate advantages of
alternative approach, based on scattering of
electromagnetic field associated with surface plasmons
into the external medium. Similar principle lies at the
core of the Total Internal Reflection Microscopy (TIRM)
[17]. SPR results in enhancement of the evanishing
electromagnetic field compared to ordinary TIR,
therefore this method can be referred to as an SPR-
enhanced TIRM. The standard SPR biosensor utilizes a
total internal reflection (TIR) prism for optical excitation
of surface plasmons (SP) in thin (usually close to 50 nm)
Au film, deposited onto surface of a transparent
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2014. V. 17, N 4. P. 368-373.
© 2014, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
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substrate. This method of SP excitation is known as the
Kretchmann configuration [18]. At a specific angle of
incidence, the so-called resonant angle, the intensity of
the light reflected from the Au film, drops down to a
minimum due to the resonant excitation of SP. The SPR
biosensor exploits dependence of the resonant angle on
the adsorption of arbitrary substance on the Au surface.
The feasibility of using this effect for biosensing was
based on development of functionalization technologies
for gold. They allowed to immobilize on the Au surface
biomolecules capable of selective binding to specific
species (the analyte), which are present in the liquid
probe. The output signal of the biosensor is then
dependent on the averaged number of specifically bound
molecules of the analyte. This method finds the most
straightforward applications for label-free immuno-
assaying [19-22].
Phenomenon underlying the proposed approach
consists in scattering of light into the external medium
from relatively large particles placed on the surface of
the SPR sensor. Surface plasmons are associated with
the evanescent electromagnetic field, spreading outside
the Au surface over the distance of 1/4 of the
wavelength. When the surface is microscopically
homogeneous, no light is transmitted into the ambience.
However, local inhomogeneities with size, comparable
to or less than the wavelength, distort the space
distribution of the evanescent field, which makes them
the effective emitters of light. Exploration of confined
electromagnetic fields and their interactions with
conduction electrons at metallic interfaces or small
metallic nanostructures has become now a subject of the
emerged field of nanophotonics and plasmonics [23].
SPR-assisted visualization of small particles by light
scattering may appear a promising concept for
development of fast, sensitive, and robust method for
revealing pathogenic microorganisms and viruses.
2. Sample preparation and experimental techniques
Feasibility of visualization of submicron particles
located at the surface of the plasmon-supporting metal
film by light scattering was investigated using
submicron-sized silicon rods as a test object. This choice
was made owing to their easily recognizable shape, as
well as high index of refraction (n = 3.865) and
relatively low optical absorption (k = 0.015) for λ =
0.65 m, which were conductive to high intensity of
light scattering. The rods were grown by VLS process in
a closed volume using the chemical gas-conveying
reaction with Au nanodroplets as a catalyst [24]. This
process resulted in both velvet-like coverage formed by
Si-rods on the walls of the silica ampoule and the
entangled clots of Si filiform crystals (FC) in the
ampoule volume (Fig. 1). To avoid mechanical
destruction, the as-grown tangles of Si-rods were
disengaged by stirring in ethanol until a homogeneous
suspension is formed. Ethanol was found to be the most
suitable solvent for this purpose, yielding high enough
stability of the suspension without addition of
surfactants. The Si-rods were then separated by size
using sedimentation method. The diameter of separated
Si-rods was evaluated with the scanning probe
microscopy (SPM). The submicron fraction (Fig. 2) was
then taken for the experiment to mimic the pathogenic
microorganisms.
Plasmon-supporting sensor chips were
manufactured by thermal evaporation of Au onto F1
glass substrates 20201 mm in size with a Cr adhesive
sublayer. Optimal thickness of Au film for efficient
excitation of surface plasmon resonance is 50 nm.
Submicron Si-rods were deposited onto the Au surface
of the sensor chips from the ethanol suspension. Special
measures were taken to avoid conglutination of Si-rods
during deposition.
Fig. 1. Growth of submicron Si-rods by using VLS process.
Appearance of as-grown clots of Si filiform crystals in the
unsealed ampoule (above).
Fig. 2. AFM image of typical submicron Si-rod after
separation by size using the sedimentation method.
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2014. V. 17, N 4. P. 368-373.
© 2014, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
370
Fig. 3. Experimental setup for visualization of submicron
particles by SPR-enhanced total internal reflection microscopy.
Microscopic images of Si-rods deposited onto Au
layer were obtained using custom-built experimental
setup based on the model Plasmon-6 biosensor (ISP
NASU, Ukraine) [25] (Fig. 3). The optical layout of the
setup is shown in Fig. 4. The Kretchmann configuration
was used for optical excitation of SPR with the 3-mW
GaInP/AlGaInP semiconductor laser diode ( = 650 nm)
serving as a light source and a total internal reflection
(TIR) prism as a coupler. The laser radiation was
focused into 51 mm spot. Measurements were carried
out in air environment with TIR prism made from K8
(BK7) optical glass. Specific features of images
retrieved using scattered and reflected light beams have
been compared for different orientations of the substrate.
3. Experimental results and discussion
For K8 glass prism (n = 1.5141 at = 0.65 m) and Au
thickness 45 nm, the calculated value of the resonant
angle, r , is 43.368. For calculation, the optical
constants of Au were assumed to be 0.2 + i 3.8. Precise
positioning of the prism at the resonant angle was made
under control of both the intensity of the reflected beam
and the visual brightness of the specimen surface.
Presence of the nanorods at the Au/air interface results
in breaking the momentum conservation for surface
plasmons, thus letting the electromagnetic waves to be
emitted into the external medium (air). The scattered
light was focused by the objective lens onto the solid-
state imaging device, and the images were acquired
using standard software. All the images, except that
shown in Fig. 7a, were obtained using the objective lens
with numerical aperture 0.11.
Typical images of Si-rods are shown in Fig. 5. The
Si-rods look like bright touches on a dark background,
whose length generally does not exceed 100 m. Ideally,
at the resonance angle no radiation should be transmitted
into the air through the Au layer. Nevertheless,
numerous luminous spots and faintly glowing nebulas
can also be observed in the background. This radiation
was attributed to initially existing surface defects
resulted from contamination or imperfect technology.
These defects can clearly be seen in Fig. 2. Noteworthy
is pronounced difference in scattering intensity between
the rods oriented in a direction perpendicular to the
direction of SPs propagation, and the other randomly
oriented rods. In principle, the scattering intensity can be
influenced by several factors. In particular, in case of
evanescent field, it is dependent on the distance between
the scattering particle and the surface [17]. To ascertain
that scattering is really orientation-dependent, images of
the same Si-rod acquired at different angular orientations
were compared (Figs 6a and 6b). It is clearly seen that
for the Si-rod the angular deviation of only 10 causes
drastic drop of light scattering intensity, while brightness
of dot-like scatterers remains practically unchanged.
Under conditions of directional illumination, strong
dependence of visibility for long, thin objects on the
direction and polarization of the incident light was also
observed in regular light microscopy [26]. Theoretical
analysis of this effect shows that intensity of light
scattering by infinite homogeneous cylinders is a
function of illumination angle, polarization of the
incident light, and the angle of observation [27].
Though this analysis is not directly applicable to the
scattering of light by the particle interacting with the
evanescent field, dependence of intensity on the
observation angle can be conjectured also for this case.
To verify this assumption, images of the same area of
the surface were obtained using objective lenses with
different numerical aperture (Figs 7a and 7b). The
image obtained with the numerical aperture 0.4
evidently contains more details than that obtained with
the numerical aperture 0.11. This is in agreement with
the above assumption, because larger aperture admits
larger angular cone of the scattered light. Nevertheless,
this does not rule out the existence of other physical
effects influencing the visible brightness of the rods in
dependence of their orientation. Thus, considering the
rod as a limiting case of a thin strip of transparent
material at the Au surface, it could be surmised that the
total internal reflection also contributes to this effect. It
is evident that a wide strip oriented along the direction of
SPs propagation, will not transfer light into the external
medium, when its angle of incidence exceeds the critical
one. Unfortunately, this possibility could not be explored
with the experimental setup used in this study and will
be the subject of further researches.
The image of the sample obtained in the SPRM
configuration is presented in Fig. 8. The contrast of this
picture is much lower in comparison with the images
obtained with the SPR-enhanced TIRM, though no
dependence of brightness of Si-rods on orientation can
be observed. Highly heterogeneous brightness of the
background also hampers visualization of the point-like
scatterers, which are clearly resolved with the use of the
SPR-enhances TIRM arrangement.
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2014. V. 17, N 4. P. 368-373.
© 2014, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
371
Fig. 4. Layout of optical configuration employed for
visualization of submicron particles by SPR-enhanced total
internal reflection microscopy (a) and surface plasmon
resonance microscopy (b): 1 – total internal reflection prism,
2 – plasmon-supporting Au layer, 3 – test objects, 4 –
microscope lens, 5 – solid-state focal plane.
Fig. 5. Typical images of Si-rods obtained by the SPR-
enhanced total internal reflection microscopy. The thick arrow
shows direction of SPs propagation; small arrows indicate
weak images of Si-rods misoriented against the perpendicular
to this direction.
The experimental results presented above
demonstrate advantages of the SPR-enhanced TIRM for
imaging of submicron objects located at the surface of
the plasmon-supporting film. This method can find
applications in biosensor technology for detection of
trace amounts of infectious microorganisms in various
media. Feasibility of pathogens detection with the SPR-
based biosensors has already been demonstrated [28-31].
The image quality obtained with the SPR-enhanced
TIRM was sufficient for counting the scattering particles
of submicron size. Transfer of counting methods to
biosensor technology may result in considerable
improvement of sensitivity, as compared to measuring
the integral effect of specific binding exploited in current
SPR-based biosensors.
Fig. 6. Images of Si-rod obtained by the SPR-enhanced total
internal reflection microscopy with different orientations
against the perpendicular to the direction of SPs propagation.
Fig. 7. Images of Si-rod obtained using objective lenses with
numerical aperture 0.4 (a) and 0.11 (b).
Semiconductor Physics, Quantum Electronics & Optoelectronics, 2014. V. 17, N 4. P. 368-373.
© 2014, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
372
Fig. 8. Typical images of Si-rods obtained using surface
plasmon resonance microscopy configuration.
4. Conclusions
Advantages of the novel method, the SPR-enhanced
TIRM, for imaging of submicron particles immobilized
on the surface of the plasmon-supporting film over the
surface plasmon resonance microscopy has been
demonstrated. Usage of submicron-sized silicon rods as
a test object allowed for clear distinguishing their images
from extraneous defects usually present at the surface.
The pronounced difference in scattering intensity
between the rods oriented in the direction perpendicular
to that of SPs propagation and other randomly oriented
rods have been observed. Experimental evidence was
obtained that this behaviour can be partially attributed to
angular dependence of the scattered intensity on the
observation angle. Other possible mechanisms of this
dependence have been also discussed.
This study was aimed at development of a novel
type of SPR-based technique relied upon direct count of
biological species of interest (bacteria, viruses, large
biomolecular complexes), rather then measuring the
integral effect of specific binding exploited in current
SPR-based biosensors.
The search for innovative principles of
nanodiagnostic assays for detection of infectious
pathogens is an active area of investigation. It is
expected that the proposed method could find
applications in biochemistry, biomedicine, food safety
inspection, and environmental monitoring, especially for
fast detection and identification of trace amounts of
pathogens in the natural water resources.
Acknowledgements
The authors would like to extend their most sincere
appreciation to Dr. Prof. A. Klimovskaya for providing
the submicron-sized silicon nanorods for this study.
This work was partially supported by Swiss
National Science Foundation through SCOPES JRP
IZ73Z0_152661.
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Semiconductor Physics, Quantum Electronics & Optoelectronics, 2014. V. 17, N 4. P. 368-373.
PACS 73.20.Mf
Visualization of submicron Si-rods
by SPR-enhanced total internal reflection microscopy
O.V. Rengevych, G.V. Beketov, Yu.V. Ushenin
V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine,
45, prospect Nauky, 03028 Kyiv, Ukraine;
Phone: +38(044) 525-40-20; e-mail: o.v.shynkarenko@gmail.com
Abstract. The potential of surface plasmon resonance-enhanced total internal reflection microscopy for visualization of submicron particles has been demonstrated using submicron-sized silicon rods as a test object. Submicron Si-rods were deposited onto the surface of a plasmon-supporting gold film by sedimentation from suspension, and their images were obtained using optical microscope with SPR excitation. Quality of images obtained in this way was compared with images viewed from the prism side in the SPR microscopy configuration. Specific features of light scattering from filiform objects are discussed. The study was aimed at development of a novel type of SPR-based biosensor relied upon direct count of biological species of interest (bacteria, viruses, large biomolecular complexes).
Keywords: surface plasmon resonance, total internal reflection microscopy, submicron Si-rods, counting biosensor, pathogen detection.
Manuscript received 03.02.14; revised version received 18.07.14; accepted for publication 29.10.14; published online 10.11.14.
1. Introduction
Introduction of SPR biosensors more than 2 decades ago provided a strong impact on progress in biochemical science and related disciplines, allowing label-free real-time characterization of biomolecular interactions [1-3]. By now, this technique has also found numerous applications in many other areas, from electrochemical studies to food quality control and environmental monitoring. Growing use of SPR has stimulated research focused on developing new SPR-related techniques: label-enhanced SPR, surface plasmon fluorescence spectroscopy, SPR microscopy (SPRM), also referred to as SPR imaging (SPRI), etc. [4-12]. SPRM was first to demonstrate by B. Rothenhäusler and W. Knoll for imaging of low-contrast samples with high lateral resolution [13]. Currently this method is related mainly to accessing multi-element arrays of macroscopic sensitive areas at the sensor surface and represents just a high-throughput multichannel version of the classical SPR-biosensor. Recent studies aimed at SPR-assisted visualization of single cells and viruses utilize optical arrangement very similar to that used in SPR imaging [14-16]. General drawback of this technique is strong interference effects arising from light diffraction on particles, which size is comparable with the wavelength. Interference patterns result in splattering of the image, thus hampering true visualization of the particles.
In our study, we demonstrate advantages of alternative approach, based on scattering of electromagnetic field associated with surface plasmons into the external medium. Similar principle lies at the core of the Total Internal Reflection Microscopy (TIRM) [17]. SPR results in enhancement of the evanishing electromagnetic field compared to ordinary TIR, therefore this method can be referred to as an SPR-enhanced TIRM. The standard SPR biosensor utilizes a total internal reflection (TIR) prism for optical excitation of surface plasmons (SP) in thin (usually close to 50 nm) Au film, deposited onto surface of a transparent substrate. This method of SP excitation is known as the Kretchmann configuration [18]. At a specific angle of incidence, the so-called resonant angle, the intensity of the light reflected from the Au film, drops down to a minimum due to the resonant excitation of SP. The SPR biosensor exploits dependence of the resonant angle on the adsorption of arbitrary substance on the Au surface. The feasibility of using this effect for biosensing was based on development of functionalization technologies for gold. They allowed to immobilize on the Au surface biomolecules capable of selective binding to specific species (the analyte), which are present in the liquid probe. The output signal of the biosensor is then dependent on the averaged number of specifically bound molecules of the analyte. This method finds the most straightforward applications for label-free immuno-assaying [19-22].
Phenomenon underlying the proposed approach consists in scattering of light into the external medium from relatively large particles placed on the surface of the SPR sensor. Surface plasmons are associated with the evanescent electromagnetic field, spreading outside the Au surface over the distance of (1/4 of the wavelength. When the surface is microscopically homogeneous, no light is transmitted into the ambience. However, local inhomogeneities with size, comparable to or less than the wavelength, distort the space distribution of the evanescent field, which makes them the effective emitters of light. Exploration of confined electromagnetic fields and their interactions with conduction electrons at metallic interfaces or small metallic nanostructures has become now a subject of the emerged field of nanophotonics and plasmonics [23]. SPR-assisted visualization of small particles by light scattering may appear a promising concept for development of fast, sensitive, and robust method for revealing pathogenic microorganisms and viruses.
2. Sample preparation and experimental techniques
Feasibility of visualization of submicron particles located at the surface of the plasmon-supporting metal film by light scattering was investigated using submicron-sized silicon rods as a test object. This choice was made owing to their easily recognizable shape, as well as high index of refraction (n = 3.865) and relatively low optical absorption (k = 0.015) for λ = 0.65 (m, which were conductive to high intensity of light scattering. The rods were grown by VLS process in a closed volume using the chemical gas-conveying reaction with Au nanodroplets as a catalyst [24]. This process resulted in both velvet-like coverage formed by Si-rods on the walls of the silica ampoule and the entangled clots of Si filiform crystals (FC) in the ampoule volume (Fig. 1). To avoid mechanical destruction, the as-grown tangles of Si-rods were disengaged by stirring in ethanol until a homogeneous suspension is formed. Ethanol was found to be the most suitable solvent for this purpose, yielding high enough stability of the suspension without addition of surfactants. The Si-rods were then separated by size using sedimentation method. The diameter of separated Si-rods was evaluated with the scanning probe microscopy (SPM). The submicron fraction (Fig. 2) was then taken for the experiment to mimic the pathogenic microorganisms.
Plasmon-supporting sensor chips were manufactured by thermal evaporation of Au onto F1 glass substrates 20(20(1 mm in size with a Cr adhesive sublayer. Optimal thickness of Au film for efficient excitation of surface plasmon resonance is (50 nm. Submicron Si-rods were deposited onto the Au surface of the sensor chips from the ethanol suspension. Special measures were taken to avoid conglutination of Si-rods during deposition.
Fig. 1. Growth of submicron Si-rods by using VLS process. Appearance of as-grown clots of Si filiform crystals in the unsealed ampoule (above).
Fig. 2. AFM image of typical submicron Si-rod after separation by size using the sedimentation method.
Fig. 3. Experimental setup for visualization of submicron particles by SPR-enhanced total internal reflection microscopy.
Microscopic images of Si-rods deposited onto Au layer were obtained using custom-built experimental setup based on the model Plasmon-6 biosensor (ISP NASU, Ukraine) [25] (Fig. 3). The optical layout of the setup is shown in Fig. 4. The Kretchmann configuration was used for optical excitation of SPR with the 3-mW GaInP/AlGaInP semiconductor laser diode (( = 650 nm) serving as a light source and a total internal reflection (TIR) prism as a coupler. The laser radiation was focused into 5(1 mm spot. Measurements were carried out in air environment with TIR prism made from K8 (BK7) optical glass. Specific features of images retrieved using scattered and reflected light beams have been compared for different orientations of the substrate.
3. Experimental results and discussion
For K8 glass prism (n = 1.5141 at ( = 0.65 (m) and Au thickness 45 nm, the calculated value of the resonant angle, (r, is 43.368(. For calculation, the optical constants of Au were assumed to be 0.2 + i (3.8. Precise positioning of the prism at the resonant angle was made under control of both the intensity of the reflected beam and the visual brightness of the specimen surface. Presence of the nanorods at the Au/air interface results in breaking the momentum conservation for surface plasmons, thus letting the electromagnetic waves to be emitted into the external medium (air). The scattered light was focused by the objective lens onto the solid-state imaging device, and the images were acquired using standard software. All the images, except that shown in Fig. 7a, were obtained using the objective lens with numerical aperture 0.11.
Typical images of Si-rods are shown in Fig. 5. The Si-rods look like bright touches on a dark background, whose length generally does not exceed 100 (m. Ideally, at the resonance angle no radiation should be transmitted into the air through the Au layer. Nevertheless, numerous luminous spots and faintly glowing nebulas can also be observed in the background. This radiation was attributed to initially existing surface defects resulted from contamination or imperfect technology. These defects can clearly be seen in Fig. 2. Noteworthy is pronounced difference in scattering intensity between the rods oriented in a direction perpendicular to the direction of SPs propagation, and the other randomly oriented rods. In principle, the scattering intensity can be influenced by several factors. In particular, in case of evanescent field, it is dependent on the distance between the scattering particle and the surface [17]. To ascertain that scattering is really orientation-dependent, images of the same Si-rod acquired at different angular orientations were compared (Figs 6a and 6b). It is clearly seen that for the Si-rod the angular deviation of only (10( causes drastic drop of light scattering intensity, while brightness of dot-like scatterers remains practically unchanged. Under conditions of directional illumination, strong dependence of visibility for long, thin objects on the direction and polarization of the incident light was also observed in regular light microscopy [26]. Theoretical analysis of this effect shows that intensity of light scattering by infinite homogeneous cylinders is a function of illumination angle, polarization of the incident light, and the angle of observation [27]. Though this analysis is not directly applicable to the scattering of light by the particle interacting with the evanescent field, dependence of intensity on the observation angle can be conjectured also for this case. To verify this assumption, images of the same area of the surface were obtained using objective lenses with different numerical aperture (Figs 7a and 7b). The image obtained with the numerical aperture 0.4 evidently contains more details than that obtained with the numerical aperture 0.11. This is in agreement with the above assumption, because larger aperture admits larger angular cone of the scattered light. Nevertheless, this does not rule out the existence of other physical effects influencing the visible brightness of the rods in dependence of their orientation. Thus, considering the rod as a limiting case of a thin strip of transparent material at the Au surface, it could be surmised that the total internal reflection also contributes to this effect. It is evident that a wide strip oriented along the direction of SPs propagation, will not transfer light into the external medium, when its angle of incidence exceeds the critical one. Unfortunately, this possibility could not be explored with the experimental setup used in this study and will be the subject of further researches.
The image of the sample obtained in the SPRM configuration is presented in Fig. 8. The contrast of this picture is much lower in comparison with the images obtained with the SPR-enhanced TIRM, though no dependence of brightness of Si-rods on orientation can be observed. Highly heterogeneous brightness of the background also hampers visualization of the point-like scatterers, which are clearly resolved with the use of the SPR-enhances TIRM arrangement.
Fig. 4. Layout of optical configuration employed for visualization of submicron particles by SPR-enhanced total internal reflection microscopy (a) and surface plasmon resonance microscopy (b): 1 – total internal reflection prism, 2 – plasmon-supporting Au layer, 3 – test objects, 4 – microscope lens, 5 – solid-state focal plane.
Fig. 5. Typical images of Si-rods obtained by the SPR-enhanced total internal reflection microscopy. The thick arrow shows direction of SPs propagation; small arrows indicate weak images of Si-rods misoriented against the perpendicular to this direction.
The experimental results presented above demonstrate advantages of the SPR-enhanced TIRM for imaging of submicron objects located at the surface of the plasmon-supporting film. This method can find applications in biosensor technology for detection of trace amounts of infectious microorganisms in various media. Feasibility of pathogens detection with the SPR-based biosensors has already been demonstrated [28-31]. The image quality obtained with the SPR-enhanced TIRM was sufficient for counting the scattering particles of submicron size. Transfer of counting methods to biosensor technology may result in considerable improvement of sensitivity, as compared to measuring the integral effect of specific binding exploited in current SPR-based biosensors.
Fig. 6. Images of Si-rod obtained by the SPR-enhanced total internal reflection microscopy with different orientations against the perpendicular to the direction of SPs propagation.
Fig. 7. Images of Si-rod obtained using objective lenses with numerical aperture 0.4 (a) and 0.11 (b).
Fig. 8. Typical images of Si-rods obtained using surface plasmon resonance microscopy configuration.
4. Conclusions
Advantages of the novel method, the SPR-enhanced TIRM, for imaging of submicron particles immobilized on the surface of the plasmon-supporting film over the surface plasmon resonance microscopy has been demonstrated. Usage of submicron-sized silicon rods as a test object allowed for clear distinguishing their images from extraneous defects usually present at the surface.
The pronounced difference in scattering intensity between the rods oriented in the direction perpendicular to that of SPs propagation and other randomly oriented rods have been observed. Experimental evidence was obtained that this behaviour can be partially attributed to angular dependence of the scattered intensity on the observation angle. Other possible mechanisms of this dependence have been also discussed.
This study was aimed at development of a novel type of SPR-based technique relied upon direct count of biological species of interest (bacteria, viruses, large biomolecular complexes), rather then measuring the integral effect of specific binding exploited in current SPR-based biosensors.
The search for innovative principles of nanodiagnostic assays for detection of infectious pathogens is an active area of investigation. It is expected that the proposed method could find applications in biochemistry, biomedicine, food safety inspection, and environmental monitoring, especially for fast detection and identification of trace amounts of pathogens in the natural water resources.
Acknowledgements
The authors would like to extend their most sincere appreciation to Dr. Prof. A. Klimovskaya for providing the submicron-sized silicon nanorods for this study.
This work was partially supported by Swiss National Science Foundation through SCOPES JRP IZ73Z0_152661.
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© 2014, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
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