Micron 38 (2007) 848–853

www.elsevier.com/locate/micron

Scanning transmitted and reflected light microscopy:

A novel microscopy for visualizing biomaterials at interfaces
Ashraf S. Elkady *
Atomic Energy Authority, NRC, Reactor & Neutron Physics Department, Abou Zabal, 13759 Cairo, Egypt

Abstract
Two new types of light microscopy, scanning transmitted light and scanning reflected light microscopy (STLM and SRLM, respectively) were
developed. STLM and SRLM are based on optical density recognition (ODR) of the scanned transmitted or reflected light, respectively, from the
object to be visualized. The obtained image is a result of enhanced interference between the scanning and transmitted/reflected beams from the
object. The new microscopy, in its initial phase, is ideally suited for monitoring macroscopic and sub-millimeter size self-assembly and for
elucidating the connection between the macroscopic and nanoscopic worlds if combined with atomic force microscopy (AFM) or electron
microscopy (EM). The method is demonstrated by monitoring the growth of 3D crystals from their original liquid phase. Some preliminary
measurements carried out using the prototype of the new microscopy are presented and its current and future possible applications are described.
# 2007 Elsevier Ltd. All rights reserved.

Keywords: Light microscopy; Scanning transmitted/reflected light microscopy; Light interference; Macroscopic self-assembly; Crystal growth

1. Introduction that appropriate conditions are met (Boncheva et al., 2002,

Though being purely based on physical principles in its
function, optical microscopy is being used by a large
community of researchers from different scientific disciplines,
e.g. biologists, material scientists, metallurgists, physicists and
many others. The rapid and continuing development in the field
of microscopy allowed for multidisciplinary applications well
beyond the scope of physics, chemistry and biology. In order to
face major challenges, these sciences should combine their
efforts with many other sciences, e.g. convergence of
nanotechnology and biotechnology resulted in the growth of
nanobiotechnology. This interdisciplinary combination can
create many innovative tools.
One of the most important applications in this context is the
use of microscopy with its different kinds (e.g. optical
microscopy and atomic force microscopy) in studying the
structural peculiarities of self-assembling processes in con-
densed matter. Self-assembly is a process in which components,
either separate or linked, spontaneously form ordered
aggregates. Self-assembly can occur with components having
sizes from the molecular to the macroscopic scale, provided
* Tel.: +20 102849049; fax: +20 244620778.
E-mail address: ashraf_elkady@yahoo.com.
0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2007.06.004
2003; Clark et al., 2002).
Of special interest is the process of molecular self-assembly,
in which molecules or parts of them spontaneously form
ordered aggregates without any human intervention, thus
introducing a ‘bottom-up’ approach to the fabrication of objects
specified with nanometre precision. The molecular structures
and intermolecular interactions of macromolecules (DNA,
proteins and lipids) are particularly amenable to the design and
synthesis of complex molecular objects (Winfree et al., 1998).
Particularly, the self-assembly of amphiphilic molecules
constitute one of the most fundamental mechanisms for the
construction of soft condensed matter biomaterials (Wong
et al., 2000). It is, also, well known that lipids, as well as
mixtures of anionic and cationic single chain surfactants, can
readily form bilayers (Sackmann and Lipowsky, 1995; Kaler
et al., 1989) that can adopt a variety of distinct geometric forms:
they can fold into soft vesicles or random bilayers (the so-called
sponge phase) or form ordered stacks of flat or undulating
membranes (Dubois and Zemb, 1997). However, the majority
of modern optical microscopes for investigating the micro-
scopic and semi-macroscopic structure of the above mentioned
condensed matter systems are not only bulky but expensive as
well. The need for a cheap portable optical microscope was the
motivation for this work. In the present work I introduce a novel
approach for a portable and less expensive optical microscopy
 A.S. Elkady / Micron 38 (2007) 848–853
that could be used for studying self-assembled structures at the
macroscopic and sub-millimeter length scales. The new
microscopy can be used as sample inspection facility for in
situ pre-nanostructure characterization and as a complementary
facility for atomic force and electron microscopy.
The emphasis in the present article will be on the current and
future potential of the new microscopy in the field of
nanobiotechnology, as well as the possibility of applying the
followed approach to another radiation-based microscopy, e.g.
neutron microscopy.
2. Materials and methods
The STLM/SRLM was developed at the condensed matter
laboratory, NRC, Egyptian Atomic Energy Authority, from a
conventional optical scanner with high resolution. Its optical
layout is shown in Fig. 1. The specimen could be attached either
directly to the modified optical scanner stage in the low
magnification mode, or directly after a magnifying lens above
the scanner in the higher magnification mode.
A diode laser (l = 650 nm) with maximum output power
5 MW is used as a coherent radiation source along with
attached lens system in scanning transmitted mode for
translucent and weakly reflecting specimens with big difference
in its compartments optical density. LED, argon, neon and
Fig. 1. A schematic diagram of the STLM/SRLM. The sample in a liquid cell is
placed either in direct contact with the modified optical scanner in the low
magnification mode or directly after a magnifying lens above the scanner. The
sample is illuminated either by a diffracted laser beam, LED or another suitable
illumination source. The reflection of the transmitted scanning light is produced
using a mirror above the sample (see text for more details).
849
another conventional illumination could also be used with
transparent specimens. The specimen can be deposited either
on a reflecting or polished smooth surface inside a liquid quartz
cell in the reflecting mode, or on ordinary glass slides that can
be fixed in the liquid cell in transmission mode. Alternatively,
ordinary glass slides could also be used for measurements
carried out in reflection mode along with a reflecting mirror
above the specimen.
A sensitive photodetector and electronic unit are used for
recording the scanned transmitted/reflected signal and its
analog-to-digital conversion for local optical density recogni-
tion (ODR). Analysis of the image and local fluctuations in
optical density is performed under PC control. Different color
filters are used for suppressing the excitation wavelengths when
the facility is used in fluorescence mode. The scanner is
designed in a way such that to be compact and light for a
portable microscope. Larger scanners are available for bigger
samples.
Suitable software, e.g. FemtoScan online (Filonov and
Yaminsky, 2006) could be used along with PC for image
processing. Two- and three-dimensional images can be
analyzed to obtain, e.g. size of particles, Fourier transforma-
tion, intensity profiles, texture values, etc. Also, it is possible to
do several scans in few seconds for an arbitrary point in sample
to predict kinetics and spatiotemporal changes.
Black cumin seed oil, used in specimens preparation, was
purchased from Isis Company (Cairo, Egypt) and used without
further purification. Neomycin drug was a kind gift from El-
Nasr Pharmaceuticals Chemicals Co., Cairo, Egypt.
3. Results and discussions
STLM and SRLM are characterized by a simple alignment
design and inexpensive components that makes them advanta-
geous over other sophisticated light microscopy. Another
advantage is that the new microscopy can be used for
visualizing micro- and macro-sized objects (Whitesides and
Boncheva, 2002) in their native environment at both solid and
liquid interfaces. It could also be used to monitor in situ kinetics
and temporal structural changes that take place in few seconds
in large macromolecular aggregates. STLM and SRLM can also
be applied successfully as a sample inspection facility for the
pre-nanostructure characterization. The resolution and magni-
fication of the new microscopy is mainly limited by the scanner
resolution and the phase coherence of the scanning and
transmitted/reflected light beams. We are working towards
improving the technical parameters for higher resolution
images. In this section, we shall emphasize on some
preliminary measurements carried out, using the prototype
of the new microscopy.
Black cumin seed, or black seed for short, is believed to be
indigenous to the Mediterranean region but has been cultivated
into other parts of the world including the Arabian Peninsula,
northern Africa, parts of Asia and Australia. Indeed, I have
chosen samples prepared from the black seed oil as it represents
a rich object from both the medical and physical viewpoints.
From the medical viewpoint, black Cumin is considered to be
850 A.S. Elkady / Micron 38 (2007) 848–853
one of the greatest healing herbs of all times (Salomi et al.,
1992; Demir et al., 2006). From the physical viewpoint, it is an
optically dense matter that is suitable for visualizing different
aspects at different conditions by registering the optical density
of different compartments in sample volume.
Fig. 2 shows a low magnification image of a test tube,
containing a suspension of black cumin seed oil in distilled
water. A higher magnification image of the suspension particles
in reflection mode is displayed in Fig. 3. The image was taken
after adding a cationic drug (neomycin) to the suspension and
followed with time.
Interestingly, the skeletons of some solid crystals begin to
appear after 2 days and were visualized by the new microscopy
(see Fig. 3). After about 10 days a complete crystal growth has
Fig. 2. SRLM image for a test tube (diameter = 1 cm), containing a suspension
of black cumin seed oil. The image is obtained at low magnification. The
emulsion particles as well as layers in the suspension are visible.
Fig. 3. SRLM of black cumin seed suspension, the arrows show the appearance
of skeletons of solid crystals growing from the liquid suspension. Scale
bar = 1000 mm.
taken place and 3D crystals in the order of mm size were
visualized arising from the original liquid phase (see Fig. 4).
Thus, the new microscopy proved to be efficient to visualize the
coexistence of two phases, the liquid and solid phases
simultaneously. This allows for in situ characterization of both
soft and hard condensed-matter systems at interfaces. Accord-
ingly, it could be applied successfully for studying the crystal
growth of macromolecules and their complexes with drugs and
other ligands (Stanley et al., 2006).
The interfacial properties such as the ordered aggregation
behavior and nucleation mechanism as well as the mutual
relationship between morphology of the complexes and the
local crystalline structure could be successfully studied with the
aid of such facility and be utilized, e.g. in drug design.
Moreover, the possibility of using polarized light in the new
microscopy proved to be useful in studying anisotropy of liquid
crystals and gels (data not shown). It could also be used for
imaging and studying fluorescently labeled samples (see
Fig. 5(a)). The aggregation of the black cumin seed suspension
particles resulted in their fusion and subsequent construction of
a nano-lamellar phase (Fig. 5(b)). Thus, the full picture for the
aggregation behavior could only be obtained by integrating the
two facilities, namely AFM and STLM.
Integrating the current microscopy with atomic force
microscopy, electron microscopy and/or Raman microscopy
would enhance the overall imaging capabilities and allow for
comprehensive studies for biomaterials at interfaces. Further-
more, the successful application of fluorescence labeling in the
current facility will allow for applications in biochips, DNA
microarrays (Sabanayagam and Lakowicz, 2007), as well as in
semiconductor multicolor quantum dots that are used recently
in diagnostics (Smith et al., 2006; Michalet et al., 2005). Thus,
the multimode facilities that could be used along with STLM/
SRLM due to its open architecture give the instrument much
capability for imaging different samples with different optical
properties. Besides, the production of scanners with multi-
angle response (Forrest, 2007) would allow for 3D stereoscopic
images if adapted to current facility. Another perspective
development of the microscopy is going on to use pulsed laser
 A.S. Elkady / Micron 38 (2007) 848–853 851

Fig. 4. Monitoring crystal growth in black cumin seed suspension. Shown are different images obtained for the same object using different modes of the newly
developed microscope: (a) image taken in reflection mode; (b) polarized light mode; (c) scanning transmitted laser mode; (d) multicolored 3D image using the
FemtoScan software. Scale bar = 1000 mm.

beam for visualizing faster spatiotemporal changes. This
possibility will allow for studying the impact of external
physical conditions (e.g. temperature, magnetic field and
radiation) on the microscopic structure and phase transition. It
would also allow for studying fast dynamics and real-time
structural studies of different organic and inorganic materials,
time-resolved self-assembly and interfacial interactions at
semi-macroscopic length scale; thus bridging the gap between
the nanoscopic and semi-macroscopic structure features.
Various nanomaterials are being produced nowadays for
technological and biomedical applications, which include drug
and gene nanocarriers used in the field of nanomedicine
(Elkady and Zhdanov, 2006), as well as raw materials used in
pharmaceutical industry. Usually, nanoparticles do not exist in
individual particles but rather as larger colloidal aggregates and
matrix composites (Dutta and Hofmann, 2003; Tartaj et al.,
2003). The new microscopy is well suited for studying the
micro- and macro-structure of such nanoaggregates. It allows

Fig. 5. Macroscopic (a) and nanoscopic (b) characterization of a suspension of black cumin seed, the first image is obtained using STLM in fluorescent mode, while
the image in (b) was obtained using AFM in friction mode. Some suspension particles are aggregating forming a lamellar phase and a typical nanostructure of such
aggregates is given in (b). Scale bar = 1000 mm in (a).
852 A.S. Elkady / Micron 38 (2007) 848–853
for assessment of morphological and bulk structure functional
properties of giant supramolecular nanoaggregates and
nanoclusters (Elkady, manuscript in preparation). Thus, the
self-assembled nanomaterials that reached to hundreds of
microns in their size via a bottom-up approach could be
efficiently studied using the new facility. Besides, the
precipitation behavior and macroscopic phase separation that
take place in soft condensed matter, drugs and gene delivery
systems represent one important usage area of the new
microscopy.
Taken all together, the approach applied here for enhancing
interference within sample volume would also be useful if
applied to another 3D radiation imaging techniques, e.g. X-ray,
g-rays and neutron microscopy, radiography and tomography.
Making use of the recent advances in neutron optics, the
quantum-mechanical phase shifts of neutron de Broglie wave
packets induced by the influence of macroscopic objects
(Beguiristain et al., 2002; Pfeiffer et al., 2006) combined with
our approach would result in high quality neutron imaging.
We propose to implement a neutron density recognition unit
and suitable software that correlates the reflected/transmitted
neutron intensity to different elements and isotopes neutron
cross-sections in neutron scanners and neutron microscopy.
This would significantly enhance construction of images for
quantitative elemental analysis. The capability of such
techniques in studying, e.g. the hydration effects in biological
samples and hydrogenous materials (Cremer et al., 2005) as
well as the structure of magnetic particles that are of both
technological and medical importance will become then a
relevant research field using neutron imaging. We propose also
to use neutron mirrors (e.g. Ni58 plasma coated mirrors and
supermirrors) that would provide a high reflectivity over a wide
range of neutron wavelengths (Maayouf et al., 1996,
1997a,b,c), thus securing a high luminosity at sample position
and increasing the current available resolution (0.1 mm), which
is mainly limited by the available beam intensity (Herrmann
et al., 1985). Due to their high penetration power, neutrons will
be advantageous for non-destructive testing of thick specimens
that have poor contrast when imaged using conventional
techniques. However, neutron detectors do not have the
resolution to image the interference fringes (effects) directly
or to detect tiny shifts in them. This could be overcome by
enhancing neutron (radiation) detection capability by using
nanomaterials-based sensitive detectors. The fabrication of
three-dimensional nanomaterial composites for radiation
detection has great potential benefits over conventional
semiconductor- and scintillation-based technologies, because
of the precise control of material–radiation interaction and
modulation of signal output (Wang et al., 2006; Létant and
Wang, 2006a,b; Cholewa et al., 2007).
Acknowledgements
The author is grateful for Igor V. Yaminsky and Alexander S.
Filonov, Moscow State University for providing the package of
software ‘‘FemtoScan’’, and for El-Nasr Pharmaceuticals
Chemicals Co., Cairo, for providing the neomycin drug.
References
Beguiristain, H.R., Anderson, I.S., Dewhurst, C.D., Piestrup, M.A., Cremer,
J.T., Pantell, R.H., 2002. A simple neutron microscope using a compound
refractive lens. Appl. Phys. Lett. 81 (22), 4290–4292.
Boncheva, M., Bruzewicz, D.A., Whitesides, G.M., 2003. Millimeter-scale self-
assembly and its applications. Pure Appl. Chem. 75 (5), 621–630.
Boncheva, M., Gracias, D.H., Jacobs, H.O., Whitesides, G.M., 2002. Biomi-
metic self-assembly of a functional asymmetrical electronic device. Proc.
Natl. Acad. Sci. U.S.A. 99 (8), 4937–4940.
Cholewa, M., Moser, H.O., Huang, L., Lau, S.P., Yoo, J., An, S.J., Yi, G.C.,
Xingyu, G., Wee, A.T.S., Bettiol, A., Watt, F., Fischer, B., 2007. Secondary
electron emission properties of III-nitride/ZnO coaxial heterostructures
under ion and X-ray bombardment. J. Nucl. Inst. Meth. Phys. Res. Section
B: Beam Interact. Mater. Atoms 254 (1), 55–58.
Clark, T.D., Boncheva, M., German, J.M., Weck, M., Whitesides, G.M., 2002.
Design of three-dimensional, millimeter-scale models for molecular fold-
ing. J. Am. Chem. Soc. 124 (1), 18–19.
Cremer, J.T., Piestrup, M.A., Park, H., Gary, C.K., Pantell, R.H., Glinka, C.J.,
Barker, J.G., 2005. Imaging hydrogenous materials with a neutron micro-
scope. Appl. Phys. Lett. 87, 161–913.
Demir, H., Kanter, M., Coskun, O., Uz, Y.H., Koc, A., Yildiz, A., 2006. Effect of
black cumin (Nigella sativa) on heart rate, some hematological values, and
pancreatic beta-cell damage in cadmium-treated rats. Biol. Trace Elem. Res.
110 (2), 151–162.
Dubois, M., Zemb, T., 1997. Swelling limits for bilayer microstructures: the
implosion of lamellar structure versus disordered lamellae. Curr. Opin.
Colloid Interface Sci. 5, 27–37.
Dutta, J., Hofmann, H., 2003. Self-organization of colloidal nanoparticles. In:
Nalwa, H.S. (Ed.), Encyclopedia of Nanoscience and Nanotechnology, vol.
X. American Scientific Publishers, pp. 1–23.
Elkady, A.S., Zhdanov, R.I., 2006. Dicationic DEGA-based lipid systems for
gene transfer and delivery: supramolecular structure and activity. In:
Mozafari, M.R. (Ed.), Nanocarrier Technologies: Frontiers of Nanotherapy.
Springer, Netherlands, pp. 175–190.
Filonov, A.S., Yaminsky, I.V., 2006. FemtoScan SPM Image Processing Soft-
ware Manual, Advanced Technologies Center, Moscow, 91 pp., http://
www.nanoscopy.net.
Forrest, A.K., 2007. A scanner with multi-angle response. J. Opt. A: Pure Appl.
Opt. 9, 335–340.
Herrmann, P., Steinhauser, K., Gahler, R., Steyerl, A., Mampe, W., 1985.
Neutron microscope. Phys. Rev. Lett. 54, 1969–1972.
Kaler, E.W., Murthy, A.K., Rodriguez, B.E., Zasadzinski, J.A.N., 1989. Spon-
taneous vesicle formation in aqueous mixtures of single-tailed surfactants.
Science 245, 1371–1374.
Létant, S.E., Wang, T.F., 2006a. Semiconductor quantum dot scintillation under
gamma-ray irradiation. Nano Letters 6, 2877–2880.
Létant, S.E., Wang, T.F., 2006b. Study of porous glass doped with quantum
dots or laser dyes under alpha irradiation. Appl. Phys. Lett. 88, 103110–
103113.
Maayouf, R.M., Elkady, A.S., Abdel-Latif, I., 1997a. CFDF—a new Fourier
RTOF-diffractometer for stress and powder diffraction investigations. J.
Neutron Res. 5, 1–6.
Maayouf, R.M., Abdel-Latif, I., Elkady, A., El-Shafey, A., Khalil, M., El-
Shaer, Y., 1997b. Assessment and main characteristic parameters of the
Cairo Fourier diffractometer facility. J. Nucl. Inst. Meth. Phys. Res. A
398, 295–302.
Maayouf, R.M., Elkady, A.S., Abdel-Latif, I., Elkady, A., El-Shafey, A., Khalil,
M., El-Shaer, Y., 1997c. Performance and main characteristics of the Cairo
Fourier diffractometer facility at the ET-RR-1 reactor. International Atomic
Energy Agency Report, Vienna, INDC (EGY)-007.
Maayouf, R.M., Abdel-Latif, I., Elkady, A.S., 1996. Neutron Guide Facility at
the ET-RR-1 Reactor. Int. Symp. Neutron Optics and Related research
Facilities NOK96, March 19–21, 1996, Kyoto University, Kumatori,
Japan.
Michalet, X., Pinaud, F.F., Bentolila, L.A., Tsay, J.M., Doose, S., Li, J.J.,
Sundaresan, G., Wu, A.M., Gambhir, S.S., Weiss, S., 2005. Quantum dots
for live cells, in vivo imaging, and diagnostics. Science 307, 538–544.
 A.S. Elkady / Micron 38 (2007) 848–853
Pfeiffer, F., Grünzweig, C., Bunk, O., Frei, G., Lehmann, E., David, C., 2006.
Neutron phase imaging and tomography. Phys. Rev. Lett. 96, 215–505.
Sabanayagam, C.R., Lakowicz, J.R., 2007. Increasing the sensitivity of DNA
microarrays by metal-enhanced fluorescence using surface-bound silver
nanoparticles. Nucleic Acids Res. 35 (2), e13.
Sackmann, E., Lipowsky, R., 1995. In: Hoff, A.J. (Ed.), Handbook of
Biological Physics, Vol. IB. North-Holland, Amsterdam.
Salomi, N.J., Nair, S.C., Jayawardhanan, K.K., Varghese, C.D., Panikkar, K.R.,
1992. Antitumour principles from Nigella sativa seeds. Cancer Lett. 63 (1),
41–46.
Smith, A.M., Dave, S., Nie, S., True, L., Gao, X., 2006. Multicolor quantum dots
for molecular diagnostics of cancer. Exp. Rev. Mol. Diagn. 6 (2), 231–244.
Stanley, S.K., Coffee, S.S., Ekerdt, J.G., 2006. Directed self assembly of
nanocrystals within macroscopic to nanoscopic features in assembly at
the nanoscale—toward functional nanostructured materials. In: Ozkan, C.,
853
Rosei, F., Lopinski, G., Wang, Z. (Eds.), Materials Research Society
Symposium Proceedings 901E, Paper 0901-Ra19-03.1.
Tartaj, P., Morales, M., Verdaguer, S.V., Carreno, T.G., Serna, C.J., 2003. The
preparation of magnetic nanoparticles for applications in biomedicine. J.
Phys. D: Appl. Phys. 36, R182–R197.
Wang, T.F., Letant, S.E., Nikolic, R.J., Chueng, C.L., 2006. FY05 LDRD Final
Report Nanomaterials for Radiation Detection. Report number UCRL-TR-
218736, 6 pp.
Whitesides, G.M., Boncheva, M., 2002. Beyond molecules: self-assembly of
mesoscopic and macroscopic components. PNAS 99 (8), 4769–4774.
Winfree, E., Furong, L., Wenzler, L., Seeman, N.C., 1998. Design and self-
assembly of two-dimensional DNA crystals. Nature 394, 539–544.
Wong, G.C.L., Tang, J.X., Lin, A., Li, Y., Janmey, P.A., Safinya, C.R., 2000.
Hierarchical self-assembly of F-Actin cationic lipid complexes: stacked
three-layer tubule networks. Science 288, 2035–2039.