Download - Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
1/19
http://localhost/var/www/apps/conversion/tmp/scratch_7/dx.doi.org/10.1016/j.micron.2011.07.014mailto:[email protected]:[email protected]://www.elsevier.com/locate/micronhttp://www.sciencedirect.com/science/journal/09684328http://localhost/var/www/apps/conversion/tmp/scratch_7/dx.doi.org/10.1016/j.micron.2011.07.014 -
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
2/19
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
3/19
V. Klang et al. / Micron 43 (2012) 85103 87
Fig. 1. (A) Visual appearance and cryo TEM image of a nanoemulsion. Images reprinted from Sonneville-Aubrun et al. (2004) with permission from Elsevier. (B) Cryo TEM
image of a nanoemulsion with nano-sized oil droplets (homogeneously filled circles) and vesicles (unfilled circles). The black scale bar represents 200nm. Image reprinted
from Norden et al. (2001) with permission from Elsevier. (C) TEM micrograph of an amphotericin B nanoemulsion with particle sizes between 100 and 400nm, prepared
by the freeze-fracturing technique. Image reprinted from Benita and Levy (1993) with permission from Wiley. (D) Visualisation of the size distribution and morphology of
nanoemulsions by atomic force microscopy, reprinted from Marxeret al. (2011) with permission from Elsevier.
(Klang et al., 2011a; Takegami et al., 2008; Yilmaz and Borchert,
2005). In addition, different methods of low-energy emulsification
have been described which employ the chemical properties of the
system to create nano-sized emulsion droplets from a microemul-
sion matrix (Anton et al., 2007; Bouchemal et al., 2004; Sole et al.,
2010; Tadros et al., 2004). In both cases, highly fluid emulsion
systems emerge. The optical appearance may range from trans-
parent systems with droplet sizes well below 100 nm to opaque ormilky systems with droplet sizes slightly above 100 nm. A bluish
tone points to the occurrence of Rayleigh Scattering for small
droplet sizes. Emulsions with larger droplet sizes appear as plain
white fluids due to multiple scattering of light (Klang and Valenta,
2011c; Mason et al., 2006). Representative examples of a translu-
cent nanoemulsion and a corresponding cryo TEM image are given
in Fig. 1A.
The characterisation of nano-sized colloidal systems requires
diverse techniques and a certain experience. Basic formulation
characteristics such as visual appearance, pH, mean particle size,
particle surface charge, chemical stability of employed excipients
and the localisation of incorporated drugs provide useful infor-
mation (Klang and Valenta, 2011c). For an exhaustive overview of
the different physicochemical techniques for nanoemulsion char-acterisation the reader is referred to recent reviews on this topic
(Benita and Levy, 1993; Klang and Valenta, 2011c; Solans et al.,
2005; Tadros et al., 2004).
Especially the mean particle size and the particle size dis-
tribution are frequently employed to characterise the long-term
stability of novel formulations. These parameters can be deter-
mined by optical light scattering techniques. More specifically,
dynamic light scattering (DLS, photon correlation spectroscopy) is
frequently employed for determination of nano-sized oil droplets
within emulsions. Among the obtained results, the mean particle
size as intensity-weighted mean of the hydrodynamic diameter
and the polydispersity index (PDI) are most frequently presented
(Hoeller et al., 2009; Klang et al., 2011a; Preetz et al., 2010; Yilmaz
and Borchert, 2005). The latter characterises the width of the
particle size distribution and thus the homogeneity of the for-
mulation. A small PDI below 0.2 indicates a narrow droplet size
distribution and thus better stability against destabilisation phe-
nomena such as Ostwald ripening (Klang and Valenta, 2011c).
The characterisation and stability assessment of nanoemulsions is
strongly associated with their droplet size and PDI. If both param-
eters remain largely unchanged during a prolonged observation
period, a formulation is usually considered physically stable. Ini-tial and regular subsequent measurements are required for an
exact stability assessment. Apart from light scattering techniques,
microscopic methods can be employed to monitor changes in
droplet size. Thus, electron microscopy is not only a valuable
tool for formulation characterisation, but also for the stability
assessment of nanoemulsions since certain changes in formula-
tion properties remain undetected during DLS analysis, as detailed
below.
2.2. Laser light scattering versus electron microscopy
In context with nanoemulsion characterisation and stability
assessment, DLS exhibits certain limitations and may provide
incomplete information. Firstly, it may fail to recognise thepresence of a small population of large droplets present in
nanoemulsions (Benita and Levy, 1993). Likewise, other surfac-
tant aggregates such as liposomal vesicles or lamellar structures
are not detected; the exact composition of the colloidal system
thus remains unknown. However, such structures are frequent
by-products of high-pressure homogenisation and should be
accounted for (Klang et al.,2011a). Fig.1B demonstratesthe compo-
sition of sucha mixedcolloidal dispersionof nano-sized oil droplets
and liposomes. Moreover, the shape of the analysed oil droplets is
usually assumed to be a perfect sphere for calculation of the DLS
results, which is not always the case. Thus, determined particle
sizes for droplets of variable shape may not be entirely repre-
sentative. Furthermore, most samples have to be diluted prior to
DLS measurements to ensure sufficient transparency for accurate
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
4/19
88 V. Klang et al. / Micron 43 (2012) 85103
droplet size determination (Klang and Valenta, 2011c). As a conse-
quence, reversible destabilisation phenomena such as flocculation
or the appearance of larger aggregates may remain unnoticed.
In order to account for these issues, additional techniques of
analysis are highly recommendable. Methods such as sedimen-
tation field flow fractionation (Benita and Levy, 1993), nuclear
magnetic resonance spectroscopy (Norden et al., 2001; Takegami
et al., 2008) or Fourier transform infrared spectroscopy (Scheuing,
1990; Whittinghill et al., 1999) have been proposed in this con-
text. However, the microscopic visualisation of the investigated
nanoemulsions might represent the most reliable and informative
method for formulation characterisation.
When employing microscopic techniques for nanoemulsion
characterisation, the presence of larger droplets is not an entirely
uncommon observation (Hatanaka et al., 2010; Preetz et al., 2010),
albeit a rarely reported one. Experience has shown that it is possi-
ble to obtain excellent DLS data for nanoemulsions over months of
stability monitoring while a microscopic analysis of the same sam-
ple reveals a definite change of the internal structure. Recently,
Preetz et al. (2010) demonstrated the importance of microscopic
analysis for the characterisation of nanoemulsions and nanocap-
sules. It was found that the mean droplet size determined by
DLS was around 150 nm for all investigated systems. In contrast,
freeze-fracture TEM revealed variable droplet sizes between 50and 500nm with the highest frequency around100 nm, which was
additionally confirmed by atomic force microscopy (Preetz et al.,
2010).
Thus, the importance of electron microscopic techniques for
the analysis of nanoemulsion droplet size and overall morphology
needs to be emphasized. Surprisingly, hardly any review articles
can be found on this specific topic. An excellent overview of cryo
TEM analysis of colloidalsystems in general was recently published
(Kuntscheet al.,2011). Cryo TEMis certainlyamong the most useful
techniques for the investigation of nanoemulsions since it delivers
detailed information about the internal structure of the observed
colloidal systems in their native state. The present review aims to
give an overview of the electron microscopy techniques that have
been employed for the analysis of nanoemulsions so far and possi-ble future developments. It is important to note that all mentioned
techniques have theirbenefits anddrawbacks. In anycase, thestud-
ied images have to be representative of the whole sample. Image
analysis software shouldbe employed only for systems with a suit-
ablecontrast andcomposition.Several rounds of analysis are highly
recommendable. A good overview of the investigated systems can
be obtained by combination of DLS or static laser diffraction and
cryo TEM (Kuntsche et al., 2009).
2.3. Development of microscopic techniques for visualisation of
nanoemulsions
Optical light microscopy can be considered the simplest method
to examine the microstructure of nanoemulsions. However, it wassoon realised that this technique is futile for the exact visualisa-
tion of nano-sized droplets below 500 nm (Benita and Levy, 1993;
Drzymala and Krajczyk, 1985). In context with nanoemulsions, the
applications of light microscopy are limited to the detection of
pronounced destabilisation phenomena such as coalescence and
Ostwald ripening (Jumaa and Mueller, 1998; Welin-Berger and
Bergenstahl, 2000) or monitoring of phase transitions (Jahanzad
et al., 2010; Rao and McClements, 2010). Likewise, the presence of
larger aggregates, droplets (Baker and Naguib, 2005; Burapapadh
et al., 2010; Corts-Munoz et al., 2009; Wang et al., 2008) or
undissolved drug crystals (Akkar and Mueller, 2003; Araujo et al.,
2011) can be determined and the background movement within
the images indicates the presence of nano-sized droplets in Brow-
nian motion (Schalbart et al., 2010). For an exact visualisation of
the nanoemulsion structure and determination of the particle size
a higher resolution is necessary. Thus, electron microscopy is an
essential tool for these tasks.
Early methods for visualisation of parenteral fat emulsionswere
presented by Du Plessis et al. (1986) who analysed thin sections of
emulsions enclosed in agar capsules after fixation with osmium
tetroxide. Well-defined droplets with particle sizes around 250nm
could be visualised. This methodproved to be time-consuming and
expensive; first results could only be obtained after 30h. Faster
and less expensive methods were subsequently developed which
involved fixation of the diluted sample and placing a thin layer on
a formvar-coated copper grid for TEM examination after nega-
tive staining (Benita and Levy, 1993; Du Plessis et al., 1987). As
for all hydrated systems investigated by conventional electron
microscopy, artefacts induced by fixation, inclusion in agar, nega-
tive staining and drying have to be expected. Structural alterations
such as distortion or collapse of the colloidal system caused by
investigation under vacuum and beam damage always need to be
taken into consideration wheninterpreting such images (Kuntsche
et al., 2011).
At roughly the same time, it wasrealised that SEM likewise rep-
resents a valuable tool to determine the droplet size of parenteral
fat emulsions (Benita and Levy, 1993; Bullock, 1984; Hamilton-
Attwell et al., 1987). A definite benefit of the SEM technique isthe topography dependentnature of the obtained images, i.e. addi-
tional topographical information at a considerable depth of focus
on a 2D-image can be obtained. However, the fixation of lipids is
a challenging task and specific fixation protocols for SEM are nec-
essary that preserve the shape and size of nanoemulsion droplets
(Benita and Levy, 1993). One of the earliest techniques in this
respect was developed by Hamilton et al. which combines fixation
of a nanoemulsionwith osmium trioxideand deposition of thefixed
droplets on a filter support (Benita et al., 1991; Hamilton-Attwell
et al., 1987).
In addition, freeze-fracturing techniques (Bachmann and
Schmitt-Fumian, 1973) emerged in context with electron
microscopy of nanoemulsions (Benita and Levy, 1993; Menold
et al., 1972; Sjoeblom and Friberg, 1978), thus increasing accuracyof the obtained images. The technique of rapid freezing and
fracturing of the sample to prepare carbon replicas was found
time-consuming since it involved a two-stage Triafol-carbon
replica technique and required the use of a special vacuum coater
(Drzymala and Krajczyk, 1985). However, it allowed for the
identification of finely structured molecules such as lipids and
various formations composed of surfactant. Early research already
produced images of high quality (Fig. 1C) (Benita and Levy, 1993).
Thus, the detection of vesicles, micelles, liquid crystals and other
structures together with fat droplets became feasible (Buchheim,
1982; Groves et al., 1985; Norden et al., 2001; Rotenberg et al.,
1991b). It was mainly by means of freeze-fracturing and cryofix-
ation techniques (Groves et al., 1985; Rotenberg et al., 1991b)
that the well-established presence of liposomes in nanoemulsionswas first observed. The development of cryo-preparation methods
for liquid specimens (Bachmann and Talmon, 1984) offered new
possibilities for nanoemulsion characterisation by cryo-TEM
(Rotenberget al., 1991a; Teixeira et al., 2000) andcryo-SEM (Saupe
et al., 2006).
Other microscopic techniques such as atomic force microscopy
(AFM) have been successfully employed for the investigation of
nanoemulsions (Fig. 1D) (Corts-Munoz et al., 2009; Fang et al.,
2004; Marxer et al., 2011; Preetz et al., 2010; T akegami et al.,
2008). Since the focus of this review lies with electron microscopy,
AFM will not be further discussed in this context. The electron
microscopic techniques that are most frequently employed for the
analysis of nanoemulsions today as well as recent methodological
advances will be elucidated in the following chapters.
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
5/19
V. Klang et al. / Micron 43 (2012) 85103 89
3. Scanning electronmicroscopy
3.1. Experimental setup, sample preparation and potential
artefacts
InSEM(Ardenne,1938a,b; Knoll, 1935; Reimer, 1993), theimage
is formed step by step by scanning a focused electron beam across
the specimen. The primary electrons penetrate the solid specimen
and are deflected by a large number of elastic scattering processes.
Various signals are generated as a result of the impact of the inci-
dent electrons, which are collected to form an image or to analyse
the sample surface (Bogner et al., 2007). The electrons that are col-
lected by the detector system give specific information and types
of contrast as detailed below:
I. The surface topography of the sample is primarily registered by
secondary electrons, i.e. all emitted electrons with exit energies
below 50eV. Secondary electrons can emerge from the speci-
men only from within a thin surface layer of a few nanometers.
The image contrastdepends on theselectedangular range of the
electrons collected (Goldstein et al., 1981).
II. Material contrast can be obtained by back-scattered electrons
which possess energies between 50eV and the primary energy
at the point when they pass through the surface of the specimen.This contrast results in an increase in intensity with increasing
mean atomic number (Reimer, 1993).
The SEM technique has a few important characteristics which
make it a highly popular microscopic technique for the ultra-
structural investigation of different kinds of hydrated materials,
including nanoemulsions. Firstly, topography dependent informa-
tion of the sample surface can be obtained in this fashion, as
opposed to the two-dimensional projections of the section volume
which can be obtained by TEM. Secondly, a great depth of focus is
achieved by SEM. At low magnifications it can be in the range of
a few millimeters which is especially important for samples with
high surface corrugation. However, there are certain limitations.
These include a lack of internal details, a somewhat limited resolu-tion and the risk of electron beam damage (Reimer, 1993).
3.2. SEM-based techniques for characterisation of nanoemulsions
Calderillaet al. investigated differentcolloidal systems basedon
sucrose esters. While SEM was employed for the characterisation
of nanocapsules by a fixation procedure with osmium tetroxide
and covering of the dried samples with gold particles, no such
approach was reported for the nanoemulsions presented in this
study (Calderilla-Fajardo et al., 2006). Hatziantoniou et al. (2007)
investigated both nanoemulsions and solid lipid nanoparticles by
SEM. The samples were placed on a polycarbon substrate and
left to dry at room temperature, followed by drying in a criti-
cal point drier. The samples were then sputter coated with goldand examined. The authors stated that SEM was a suitable tech-
nique for the investigationof nanoemulsions and advantages when
compared to FF-TEM were found, such as lower costs and the
opportunity to examine large areas of dispersed particles in a short
time.
Another variation of a SEM analysis for visualisation of propo-
fol nanoemulsions was performed by Masaki et al. (2003) to
analyse notable changes in droplet size. A one-step fixation tech-
nique for the oil droplets on filter papers with a combination
of glutaraldehyde-malachite green and osmium tetroxide was
employed. These compounds are otherwise used to stabilise i.v.
fat emulsions. Malachite green is useful as a dye and at the same
time stabilises lipiddissolvedin aqueous glutaraldehyde. Thus,this
specific fixation technique permitted visualisation of flawless oil
droplets in the SEM. A filter paper immersed in the saline dilution
medium alone was used as control. The samples were dehydrated
bydifferent alcohols and after 2 h of freeze-drying they were coated
with gold vapour and observedundera SEMto detectthemaximum
size of the observed droplets. The resulting images clearly showed
the coalescence process of the emulsion droplets with propofol
after addition of lidocaine. Not surprisingly, the authors reported a
certain discrepancy between the observed maximum droplet sizes
andthose previously determined by DLS. As already discussed, DLS
only determines an average diameter of all droplets and thus may
fail to acknowledge the presence of a few large droplets among
a large population of small droplets. However, the presence of
larger droplets may be detrimental to thelong-term stabilityof the
nanoemulsion and may render it unsuitable for intravenous appli-
cation. This underlines the need for additional methods of analysis
such as electron microscopic techniques.
A SEM-based technique that can be used for dynamic experi-
ments of hydratednanocarriersis environmental scanningelectron
microscopy (ESEM) (do Amaral et al., 2005; Ruozi et al., 2011). This
technique is based on the use of a multiple aperture and a gradu-
ated vacuum system that allows the chamber to be maintained at
pressures around 5000Pa. Specimens can be viewed under water
vapour or other auxiliary gases. Moreover, dehydration of hydrated
samples can be partially inhibited by using a pump-down pro-cedure and by controlling the temperature using a Peltier stage
(Ruozi et al., 2011). However, the resolution is not sufficient to
obtain detailed information regarding the surface properties and
architecture of nanoscale structures (Mohammed et al., 2004). This
lack of resolution is likewise the current main limitation for other
SEM-based techniques such as wet scanning transmission elec-
tron microscopy (wet STEM in ESEM) or SEM of fluids in flow cells
(Bogneret al., 2005,2007). Nevertheless, accurate results regarding
droplet size and size distribution of nano-sized emulsions can be
obtained bywet STEM inESEM (Bogneret al., 2005; do Amaral et al.,
2005). Thus, further developments in this field may be anticipated
with interest.
4. Transmission electronmicroscopy
4.1. Experimental setup, sample preparation and potential
artefacts
Conventional transmission electron microscopes (Ruska and
Knoll, 1932) are electron optical instruments analogous to light
microscopes.However,the specimenis notilluminated by light, but
by an electron beam. This requires operation in a vacuum since gas
moleculeswoulddeflect theelectrons. At the topof the microscope
column is an electron gun and a system of electromagnetic lenses
focuses theelectron beam onthe sample. Theimage contrastin TEM
is obtained by the interactions of the electrons with the material,
i.e. electron scattering. The resolution in TEM is directly propor-tional to the acceleration voltage of the electrons. High resolution
is obtained due to the short wavelength of the electrons when the
voltage increases. However, increasing acceleration voltage leads
to poorer contrast since the scattering of the electrons is decreased
at higher velocity (Kuntsche et al., 2011). Typical instruments are
capable of voltages from 40 to 120kV andmicroscopes in the range
of 200400kV are becoming more common. In TEM investigations
of colloidal systems, voltages between 80 and 200 kV are usually
employed (Kuntsche et al., 2011).
The resolution in TEM is rather limited by the properties of the
specimens than by those of instrumentation. Its present level is at
0.31.0nm (Massover, 2011). The absorption of electrons into the
specimen is unusual, but electrons scattered to large angles do not
contribute to the image in the usual bright field mode and thus
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
6/19
90 V. Klang et al. / Micron 43 (2012) 85103
appear to be absorbed. In the case of ordered or crystalline sam-
ple material, this results in diffraction contrast, which is strongly
dependent on the crystal orientation. In amorphous materials,
a mass thickness contrast is formed, where the image bright-
ness depends on the local mass thickness. Thus, darker regions
in the bright field image mode are regions of higher scattering
(Sawyer and Grubb, 1996). For more detailed information on elec-
tron microscopy, general literature is recommended (Bozzola and
Russell, 1999; Egerton, 2005).
In case of nanoemulsions, cryo preparation methods are of
course the most suitable methods of analysis. If cryo TEM is not
available, a conventional negative staining analysis with or with-
out dilution can be performed on nanoemulsions to obtain certain
basic informations. Staining techniques are frequently employed
for imaging of colloidal systems with TEM since they are easy, fast
and universally applicable (Kuntsche et al., 2011; Massover, 2008).
Themost commonstaining agents are salts of heavy metals such as
molybdenum, tungsten or uranium which possess atomic numbers
between42 and 92. These agents must be benign to the wet speci-
mens, form a thin glassy layer upon dryingand must resist electron
beam radiation damage to a satisfying extent (Massover, 2008).
In context with nanoemulsion analysis, phosphotungstic acid or
its salt solutions (Desai et al., 2008; Ganta and Amiji, 2009; Liu
and Yu, 2010; Pathan and Setty, 2011; Seki et al., 2004) or uranylacetate (Araujo et al., 2011; Hatanaka et al., 2010; Schalbart et al.,
2010) are most frequently employed. During sample preparation,
a droplet of the nanoemulsion is placed on a carbon coated grid
onto which it is rapidly adsorbed. Subsequently, an aqueous solu-
tion of a heavy metal salt is applied for staining. The sample is
then left to dry and finally observed by TEM at room tempera-
ture. This technique allows for the identification of the dehydrated
shells of the nanoemulsion droplets which are stabilised by sur-
factant. The strongly scattering metal ions form an amorphous
shieldenveloping the weakly scattering oil droplets to enhance the
electron microscopy contrast (Brenner and Horne, 1959). A high
reverse contrast is thus seen in bright field TEM images, with light
dropletsagainsta darker background.Negative stainingcan be used
to visualise the size, shape and internal structure of the sample.The negative stain not only provides contrast for weakly scattering
specimens, butalso physical support against collapse of the sample
structure during drying and protection against electron beam dam-
age (Massover, 2008). Thesample may also be coatedwith a carbon
film under ambient conditions before investigation (Burapapadh
et al., 2010).
However, it should be kept in mind that conventional electron
microscopy is prone to artefacts in case of surfactant solutions, i.e.
hydrated colloidal dispersions (Egelhaaf et al., 2000). For hydrated
samples such as nanoemulsions, the factors affecting the preserva-
tion of the structural integrity are identical in both TEM and SEM
(Mueller, 1991). Both drying and staining techniques canaffect the
structure and morphology of the sample; thus, great care should
be taken during interpretation of the obtained images (Friedrichet al., 2010). Severe shrinkage or even complete collapse, selec-
tive dimensional modification and aggregation of the constituents
of colloidal systems due to complete dehydration and drying usu-
ally cause strong structural changes. In addition, the use of heavy
metal salts leads to a selectivesample appearance since only struc-
tures that can be reached by or react with the staining agents can
be detected (Hayat, 2000). Consequently, the uppermost surface
of the sample as well as the compounds of the system that can
chemically reactwith thestainingagentsare clearly observedwhile
many compounds of the sample remain practically invisible. As a
result,thefinalEMimagesmay describecompletelymodifiedstruc-
tureswhich have nothing in common withthe original formulation
morphology. Moreover, conventional negative staining on contin-
uous carbon support films bears the risk of sample distortion due
to adsorption and flattening during the drying of the thin aque-
ous film of negative stain or evaporation in the TEM (Harris, 2008).
Apartfrom adsorptionartefacts, variable spreadingand incomplete
specimen coverage by the stain solution can lead to non-uniform
staining results. Specimen distortion from surface tension forces
duringevaporative dryingor the formationof a saturated salt solu-
tion before the final drying may occur as well (Massover, 2008).
Techniques basedon cryofixation and low temperature electron
microscopy help to overcome these major problems. However, not
all TEM laboratories are equipped with cryo electron microscopy
facilities. Therefore, the application of air-dried negative staining
techniques for biological specimens or other aqueous systems such
as colloidal dispersions remains justified (Harris, 2008).
4.2. Conventional TEM for characterisation of nanoemulsions
Conventional TEM is frequently employed for the analysis of
colloidal systems since it can visualise coexisting structures and
microstructure transitions (Bali et al., 2010; Silva et al., 2009).
Despite the obvious risk of artefacts, a large body of experi-
mental data can be found in the respective literature. Image 2
gives an overview of images obtained by TEM at room tempera-
ture after negative staining. Although these images might providesome basic information in regard to droplet stability or general
size range, they are of course far from a successful microscopic
analysis. Conventional TEM may have its place as a useful pre-
liminary technique to obtain a rapid impression of a colloidal
system. However, this approach should not be confused with
a substantiated structural analysis. We present some images of
variable quality to show the potential as well as the obvious lim-
itations of this technique. Seki et al. (2004) employed TEM for
visualisation of two different emulsion systems. The samples were
placed on a collodion-coated specimen mesh and negative staining
with sodium phosphotungstate was performed before observa-
tion. As can be seen in Fig. 2A, a pronounced difference in mean
droplet diameters was demonstrated for the two systems. Like-
wise, Kelmann et al. (2007) investigated the microstructure ofa carbamazepine-loaded nanoemulsion by TEM. A droplet of the
sample was placed on a carbon-coated copper grid, stained with
uranyl acetate and covered with formvar. As demonstrated in
Fig. 2B, a clear image of the undiluted nanoemulsion was obtained.
A similar staining technique using uranyl acetate was employed for
visualisation of a nanoemulsion for the administration of paclitaxel
and curcumin(Ganta and Amiji, 2009). Likewise, topical nanoemul-
sions with genistein were visualised in this fashion (Silva et al.,
2009). The increased contrast observed at the interface of the oil
droplets was assumed to be related to an affinity of the staining
agent to interfacial components.
In another approach, nanoemulsions stabilised by semisolid
polymer interphases were analysed by TEM without specified sam-
ple preparation (Namet al., 2010). Significant variations werefoundin size distribution and morphology of nanoemulsions in depen-
denceof differentoilphases (Fig.2C). The surface morphology of the
systems might havebeen affected by thedrying process. Gantaetal.
(2008) employedTEM afternegative stainingwithphosphotungstic
acid. The peculiar arrangement of the nano-sized droplets (Fig. 2D)
was most likely caused by the sample preparation. A similar tech-
nique was employed for visualisation of gelated nanoemulsions
(Mou et al., 2008); however, the contrast of the obtained images
was poorer, possibly due to the altered formulation properties. In
another work, the same negative staining method was employed
for ocular submicronemulsions(Ibrahim et al., 2009). The obtained
images revealed a crowded droplet structure and poor contrast
for emulsions with castor oil (Fig. 2E). Coalescence phenomena
were observedfor emulsionswith soybean oil(Fig.2F). It remained
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
7/19
V. Klang et al. / Micron 43 (2012) 85103 91
Fig. 2. (A) Electron micrographs obtained after negative staining of a lipid nanoemulsion system (lipid nano-spheres, LNS,left-hand side) and a conventional fat emulsion
for parenteral nutrition (LM, right-hand side). Images reprinted from Seki etal. (2004) with permission from Elsevier. (B) TEM photomicrograph of a carbamazepine-loaded
nanoemulsion. Image reprinted from Kelmann et al. (2007) with permission from Elsevier. (C) TEM micrographs of nanoemulsions prepared with different types of oil:
(1) phenyl trimethicone, (2) poly-dimethylsiloxane, (3) cetyl ethylhexanoate, (4) dioctanoyl-decanoyl-glycerol, (5) isopropyl myristate and (6) liquid paraffin. Scale bars
represent 150nm. Images reprinted from Nam etal. (2010) with permission from theAmerican Chemical Society (ACS).(D) TEMphotomicrograph of a parenteral emulsion
after negative staining using phosphotungstic acid. The scale bar represents 500nm. Image reprinted from Ganta et al. (2008) with permission from Elsevier. (E), (F) TEM
photomicrographs of nano-sized emulsionswith castoroil (E)or soybean oil (F). Thescale bars represent500 nm. For the latter system, coalescence phenomena are visible.
Images reprintedfrom Ibrahim et al.(2009) with permission fromElsevier.(G) TEM photomicrographsof thalidomid-loaded and blanknanoemulsionswith polysorbate. The
scale bars represent100 nm.
Image reprinted from Araujo et al. (2011) with permission from Elsevier.
unclear whether the latter was inherent to the formulation ormerely an artefact caused by sample preparation.
Several authors employed specific diffraction modes during
conventional TEM bright field imaging to visualise the form and
size of nanoemulsions and to determine the amorphous or crys-
talline character of their components (Bouchemal et al., 2004;
Shafiq et al., 2007). However, the contrast of the obtained imagesof
dark droplets against a bright background was comparatively poor
in these as well as in similar cases (Shakeel et al., 2007; Singh and
Vingkar, 2008).
Overall, nanoemulsion images of good quality obtained by con-
ventional TEM are comparatively rare. An appropriate staining
technique appears to be essential to obtain sufficient contrast;
both type and amount of the staining solution should be optimised
before the respective task. Nevertheless, oil droplet aggregation
or destruction has to be expected under the dehydration condi-tions of electron microscopy (Liu and Yu, 2010). Even if images
of more or less intact oil droplets are obtained, the information
that is conveyed is frequently limited since a very restricted area of
the investigated specimen may remainintact and canbe presented
(Fig. 2G). In this case, no information is obtained about the polydis-
persity of the droplet size distributionor the presence of additional
structures within the sample.
Apparently, conventional TEM may be most successfully
employed for the real-space determination of nanoemulsion
droplets after continuous phase-evaporation if the employed oil
has a large molecular weight, as observed by Mason et al. (2006). A
silicon oil-in-water nanoemulsion was stained with uranyl acetate
and placed on a TEM grid coated with a monolayer polymer
film. After evaporation of the water the sample was observed at
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
8/19
92 V. Klang et al. / Micron 43 (2012) 85103
Fig. 3. Comparison of cryo TEMand conventional TEM after negative staining with uranyl acetate: nanoemulsions stabilised by either5% (A and B) or 2.5% (w/w) of sucrose
stearate (C andD) were investigated with both methods. On theleft hand side (A,C), thecryo TEMimages aregiven.On therighthand side (B,D), thecorresponding images
obtained by conventional TEM at room temperature are given. For conventional TEM, the risk of beam damage is imminent as indicated in the lower right hand corner of
image D.
Image A and B are reprinted from Klang et al. (2011b), image C is reprinted from Klang et al. (2011a) with permission from Elsevier.
room temperature. At low oil volume fractions, coalescence of the
droplets during evaporation was apparently avoided since they
did not completely cover the grid. This effect was most likely
enhanced by the staining agent. However, even nanoemulsion
dropletsthat hadbeen concentratedand deformedduringthe evap-
oration process could be observed. Apparently, the silicone oil was
of sufficiently large molecular weight so as not to significantly
evaporate under the experimental conditions. The specific reasonswhy this simple method provided such comparatively good results
remained unclear. In recent experiments (Klang et al., 2011a,b),
conventional nano-sized oil-in-water emulsions were investigated
by TEM at room temperature after negative staining with uranyl
acetate (Fig.3B andD).A directcomparison with imagesof thesame
samples obtainedby cryo TEM(Fig.3A and C)suggeststhatthemor-
phology of thesystems is comparatively well preserveddespite the
unfavourable surroundings within the TEM. Surprisingly intact, if
deformed and aggregated droplet shells were obtained. Neverthe-
less, care has to be taken to avoid artefactscaused by electronbeam
damage (Fig. 3D, right-hand corner), which are more imminent in
TEM at room temperature.
Summarising our experiences with TEM analysis of nanoemul-
sions at room temperature, it may be assumed that it is not
primarily the nature of the oil which is decisive for the quality of
the obtained images, but the efficacy of the employed surfactant
to stabilise the oil droplets. In Fig. 3, a conventional cosmetic oil
of a molecular weight around 300 g/mol was emulsified using dif-
ferent amounts of a sucrose ester surfactant. Perfectly clear phase
boundaries of the droplet shells after evaporation in the TEM were
foundfor systems of an idealsurfactantconcentration of 2.5%(w/w)
and high physical stability (Fig. 3D). A surplus of surfactant did notimprove the formulations general properties (Fig. 3B), but rather
destabilised the system by introducing aggregates and leading to
droplet deformation.The obtainedimagesof bothformulations cor-
responded comparatively well to the native structures as observed
by cryo TEM. Although the obtained TEM images are of course far
from describing the real state of the emulsion, they provide cer-
tain information that might be useful in nanoemulsion studies, e.g.
about sample stability against external stress. The stability of the
emulsified oil droplets is after all an important quality parameter.
However, it is again emphasised that the potential of TEM at room
temperature is limited to such basic aspects; an exact visualisation
of the droplet shape remains confined to cryo TEM. Interpretation
of TEM images of nanoemulsions should therefore be performed
with caution.
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
9/19
V. Klang et al. / Micron 43 (2012) 85103 93
5. Cryo-preparation techniques for SEM
5.1. FF-SEMand cryo SEM: experimental setup, sample
preparation and potential artefacts
Freeze-fracture techniques and direct imaging by cryo electron
microscopy are clearly more appropriate methods for analysis of
nanoemulsions than conventional electron microscopy. In freeze-
fracture electron microscopy the specimens are rapidly frozen,
fractured, shadowed with metal and replicated (Belkoura et al.,
2004). The metal replica of the fracture surface are then viewed
either by SEM or TEM, the former of which may include additional
steps sample preparation. Further details on the freeze-fracture
technique for analysis of nanoemulsions are given in context
with freeze-fracture TEM in section 6.1., which is more frequently
employed in this field.
In contrast to freeze-fracture electron microscopy, the frozen
specimens prepared for cryo electron microscopy are not repli-
cated, but immediately transferred to a low temperature stage
within the microscope and viewed directly (Belkoura et al., 2004).
In cryo SEM, additional steps such as coating with metal for
enhanced conductance of the electrons are usually performed
(Preetz et al., 2010). Details on cryo preparation methods are given
in context with cryo TEM in Section 6.3, which is by far the mostfrequently employed technique for nanoemulsion analysis.
Images of nanoemulsions in their natural hydrated state can be
obtained by these techniques of sample preparation before SEM
analysis. The benefits of cryo SEM are particularly useful for the
analysis of certain types of colloidal nanoemulsion systems which
are not entirely suitable for the commonly employed cryo TEM
investigation. For samples which are highly viscous, have a strong
tendency to aggregation or contain large amounts of oil, cryo SEM
of the freeze fracture-freeze dried samples is among the best solu-
tions (Severs and Robenek, 2008). On the one hand, an ultrathin
layer of such a solution for cryo TEM investigation can hardly be
obtained. Viscous nanoemulsions are adsorbed firmly onto the car-
bon coated grid and are not readily removed by filter paper. The
resulting layer may not be thin enough to be transparent for theelectron beam. On the other hand, the dimensions of the holes in
the carbon support film are usually much smaller than the size
of large aggregates or multilamellar structures potentially present
in nanoemulsions. Thus, such structures may remain undetected.
In cryo TEM micrographs only a small part of an entire aggregate
structure can be observed, which may be insufficient for a com-
prehensive understanding of the sample organisation. In contrast,
cryo SEM delivers information about the distribution of specific
structures such as aggregates on the surface of the sampleirrespec-
tive of their location on the grid. Thus, cryo SEM provides a more
adequate impression of the overall morphology of inhomogeneous
and/or viscous nanoemulsions. Likewise, comparatively large sam-
ple areas can be investigated by freeze-fracture SEM, which also
delivers a good overview of the sample morphology and homo-geneity.
When working with either freeze-fracture or cryo electron
microscopy, some general aspects on the benefits and limitations
of the different methods of sample preparation should be con-
sidered to identify the most appropriate technique for a specific
nanoemulsion sample. Vitrification of samples for TEM is typi-
cally achieved by plunging the thin liquid specimens into liquid
ethane. In case of oil-rich samples, apolarcompounds such as hydr-
carbons, alkales or aromatics will dissolve in the cryo-medium
ethane, thus rendering analysis of the intact system impossible
(Kesselman et al., 2005). Since oil-in-water nanoemulsions usu-
ally do notcontainlargeamounts of apolarcompounds or solvents,
plunge-freezing is usually a suitable approach. However, in case
of water-in-oil nanoemulsions or microemulsions dissolution of
sample compounds may occur. Therefore, high pressure-freezing
represents the optimal cryofixation methodin such cases. It allows
the vitrification of samples with a thickness of up to 200m.Moreover, during high pressure freezing the sample is encapsu-
lated in a metal shell, which helps to avoid dissolution problems.
Conventional freeze-fracture techniques allow for the direct visu-
alisation of oil-rich samples which would dissolve in the liquid
ethane used for vitrification of the samples during cryo-techniques
(Belkouraet al.,2004). In addition, non-blottingmethods for freeze-
fracture preparation andcryo-energyfilteredtransmissionelectron
microscopy are suitable alternatives (Belkoura et al., 2004; Burauer
et al., 2003; Schmidtgen et al., 1998).
5.2. FF-SEMand cryo SEM for characterisation of nanoemulsions
Cryogenic treatment of samples prior to SEM allows for the
investigation of soft carriers such as nanoemulsions in their native
state. Saupe et al. investigated different nano-structured lipid car-
riers such as nanoemulsions and solid lipid nanoparticles by cryo
SEM (Saupe et al., 2006). The liquid colloidal systems were plunge-
frozenand viewedin theirnatural state after fracturing andcoating
withplatinum. Deviating nano-sized structures which may emerge
during production could be detected by this technique since bothshape and size of the lipid structures were visualised (Fig. 4A).
The technique of freeze-fracture SEM is among the more rarely
employed techniques for the analysis of nanoemulsions (Mason
et al., 2006). Few reports can be found in the literature (Bilbao-
Sainz et al., 2010). A freeze-fracture SEM technique was employed
to characterise the interfacial and bulk structure of silica-coated
nano-sized emulsionsas wellas conventionalnanoemulsions with-
out additional coating (Eskandaret al. 2009). The coated emulsions
were stabilisedby mixedinterfacial layers of either lecithin or oley-
lamine and hydrophilic silica nanoparticles. The reported method
includedcryofixation of the emulsion,fracturing, etching, platinum
coating and subsequent imaging. The samples were injected into a
split brass tube and were cryofixed by plunging the latter into a
liquid nitrogen-solid nitrogen slush together with the cryo trans-fer specimen holder. The specimen was then transferred to the
exchange chamber under vacuumand the split brass tube was bro-
ken with a precooled scalpel. The surface ice was removed during
careful sublimation to avoid droplet disintegration. The fractured
and etched sample was then coated with platinum to provide con-
ductivity prior to SEM imaging. The obtained images revealed that
the adsorption of the silica nanoparticles to the oil droplet inter-
face was influenced by the nature of the surfactant and the charge
it conveyed to the interfacial film (Fig. 4B). The distribution of the
silicananoparticles between the interfacial filmand the bulk phase
could be determined in this fashion.
6. Cryo-preparation techniques for TEM
6.1. Freeze-etching and freeze-fracturing for TEM: experimental
setup, sample preparation andpotential artefacts
Freeze-etching, freeze-fracturing and cryo electron microscopy
of frozen fluids are complementary techniques mainly because the
respectively obtained information is based on different mecha-
nisms (Severs and Robenek, 2008; Steinbrecht and Zierold, 1987).
Freeze-fracture electron microscopy techniques emerged during
the 1950s to 1960s and have been successfully employed for the
analysis of hydrated specimens for well over 30 years. The freeze-
fracture technique physically fractures a frozen biological sample;
the structural details exposed by the fracture plane are visualised
by vacuum-deposition of platinum-carbon to make the replica for
examination in a TEM (Severs, 2007). Thus, no drying process is
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
10/19
94 V. Klang et al. / Micron 43 (2012) 85103
Fig. 4. (A) Cryo SEM images of nanoemulsions. Image reprinted from Saupe et al. (2006) with permission from Elsevier. (B) Freeze-fracture SEM images of conventional
nanoemulsions stabilised by lecithin (L) or oleylamine (O) and corresponding silica-coated lecithin (LSA) or oleylamin (OSA) nanoemulsions.
Images reprinted from Eskandar et al. (2009) with kind permission from Springer Science and Business Media.
required and additional information aboutthe internal structure ofnano-sized colloidal carriers is obtained (Kuntsche et al., 2011).
The key steps involved in this procedure are rapid freezing of
thesample, fracturing,replicationand replica cleaning.A schematic
overview of the freeze-fracturing process for TEM as well as SEM
analysis is given in Fig. 5.
I. The rapid freezing, i.e. cryofixation, of the nano-sized suspen-
sion is usually performed by swiftly immersing the sample
into a liquid coolant such as subcooled liquid nitrogen. In this
context, pre-treatment with cryoprotectants such as glycerol
is sometimes necessary to avoid ice crystal damage. Chemical
fixation with glutaraldehyde beforehand serves to avoid arte-
facts induced by the cryoprotectant. In many cases, successful
freezing of hydrated samples requires ultrarapid freezing tech-
niques, such as optimised plunge freezing, jet freezing, sprayfreezing, high-pressure freezing or freezing by impact against a
cold metal block (Severs, 2007).
II. Subsequently, the fracturing of the sample is carried out under
vacuum at liquid nitrogen temperature by breaking the sample
in a hinged device or by using a liquid nitrogen-cooled micro-
tomeblade.If deemed necessary, an additional etchingstep may
be performedwhich consists of vacuumsublimationof iceafter
fracturing. In other words, theice can be removed from the sur-
face of the fractured specimen by freeze-drying by increasing
the temperature to about100 C for several minutes to let ice
sublime.
III. The replicas are then prepared by shadowing and backing of
the specimen. The surface of the sample is usually shadowed
with platinum to achieve a good topographic contrast and
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
11/19
V. Klang et al. / Micron 43 (2012) 85103 95
Fig. 5. Schematic illustration of the sample preparation during the freeze-fracture
process followed by thefreeze-etching technique.The obtained specimenscan sub-
sequently be investigated by TEM or SEM.
then covered with a strengthening layer of electron-lucent car-
bon to stabilise the ultra-thin metal film (Preetz et al., 2010).
More specifically, the cold fractured surface, possibly etched,
is shadowed with evaporated platinum or gold at an average
angle of 45 in a high vacuum evaporator. A second coat of car-
bon, evaporated perpendicular to the average surface plane, is
often performed to improve stability of the replica coating. The
topographical features of the frozen, fractured surface are thus
transformed into variations in the thickness of the deposited
platinum layer of the replica (Severs, 2007).
IV. The specimen is then returned to ambient temperature and
pressure and the extremely fragile pre-shadowed metal
replica of the fracture surface is released from the underly-ing biological material by careful chemical digestion with acid
solutions or detergents. The still-floating replica is thoroughly
washed free from residual chemicals, carefully placed on fine
grids,dried andtheninvestigatedin the TEM(Preetzetal.,2010;
Severs and Robenek, 2008).
Further details and practical advice on freeze-fracture elec-
tron microscopy can be found in the literature (Gulik-Krzywicki,
1997; Severs, 2007). Overall, freeze-fracture electron microscopy
can be employed for the analysis of a large spectrum of differ-
ent materials, including liquids and dispersions, at intermediate
to low resolution. Freeze-fracture TEM (FF-TEM) is well adapted
to study lipid-containing colloidal suspensions, such as liposomes,
nanoemulsions and nanoparticles despite the relatively low signalto noise ratio of the replicas. Polymer solutions, microemulsions
and biological systems can be investigated as well (Brandl et al.,
1997; Gulik-Krzywicki, 1997; Yan et al., 2005; Zhou et al., 2010).
The most importantfeature of this technique is the tendency of the
fracture plane to follow a plane through the central hydrophobic
core of frozen membranes, thus splitting them in half. As a result,
planar views of the internal structure of the samples are obtained
(Severs, 2007).
The main drawback of FF-TEM is that the obtained information
strongly depends on the quality of the fracture (Belkoura et al.,
2004). Freeze-fracturing techniques are complex in nature and the
different steps of sample preparation, such as chemical fixation,
cryoprotective pre-treatment,cryofixation, freeze-fracturing, etch-
ing and replication, may significantly influence the appearance of
the investigated sample (Benita and Levy, 1993; Buchheim, 1982).
As for all microscopic techniques, care must be taken to avoid
misinterpretationdue to artefacts. Artefacts mayoccurduetoinsuf-
ficient freezing rates or re-deposition of solvent molecules ontothe
sample plane after fracturing (Kuntsche et al., 2011). Therefore,
considerate specimen preparation is essential to ensure repro-
ducible and reliable data.
6.2. Freeze-fracture TEM for characterisation of nanoemulsions
The morphology of a lecithin-based nanoemulsion for topical
application was investigated by FF-TEM following a standard pro-
tocol of sample cryofixation, freeze-fracturing, freeze-etching and
covering with platinum/carbon (Zhou et al., 2010). The influence
of increasing amounts of glycerol within the systems was clearly
shown (Fig. 6). The droplet size, shape and size distribution could
be monitoredvery well by thistechnique. Likewise, instability phe-
nomena suchas the agglomeration of dropletsas observedin Fig.6A
could be detected. In the same fashion, Zeevi et al. (1994) analysed
positively charged submicron emulsions using a freeze-fracture
and etching technique, thus confirming a spherical particle shape
and random particle distribution within the fracture plane.
6.3. Cryo TEM: experimental setup, sample preparation and
potential artefacts
Cryo TEM allows for the direct investigation of colloids in the
vitrified frozen-hydrated state. As with freeze-fracture TEM, infor-
mation about the internal structure of the colloidal system is
obtained (Kuntsche et al., 2011). By means of a complex sample
preparation the formulation microstructure is displayedin its origi-
nal stateand a clear differentiation between nano-sized oil droplets
and other structures can be obtained (Fox, 2009). It is therefore
the electron microscopic technique of choice for an artefact-free
visualisation of nanoemulsions.
Conventional TEM analysis of hydrated systems only provides a
limited amount of information since the aqueous compounds of
the system will evaporate rapidly under the vacuum within anelectron microscope. Therefore, the development of cryo-electron
microscopy of vitrified specimens represented a major progress in
this field (Bouchet-Marquis and Hoenger, 2011; Steinbrecht and
Zierold, 1987). During vitrification, a thin film containing the spec-
imen is plunged into a suitable cryogen such as liquidethane.Liquid
nitrogen is used to maintain the temperature of the ethane near its
melting point of183 C. Witha freezingrate around 1,000,000 K/s,
thefluidsurrounding the specimendoesnothavetimeto form crys-
talline ice, which would damage the fragile sample; it is vitrified
instead. The nature of the liquid is preserved since the first-order
exothermic phase transition between the liquid and the solid does
not take place (Bachmann and Mayer, 1987). Thus, the specimen
that is embedded within the layer of vitreous ice is essentially
preserved in its native state at high spatial resolution.Different methods for preparation of a thin vitrified layer of
aqueous specimens exist, such as plunge-, impact-, spray- or high
pressure-freezing. For investigation of nanoemulsions, plunge-
freezing and high pressure-freezing are the most commonly used
techniques. Detailed information on these cryofixation procedures
can be found in the literature (Dubochet et al., 1982; Lepault et al.,
1983; Sitte et al., 1987). Various kinds of cryo specimenholdersand
plunge-freezing devices are available.
One of the basic requirements for TEM is that the specimen
must be very thin. To produce films of adequate thickness, three
main techniques can be employed. Firstly, blotting of the sample
is performed followed by vitrification (Danino and Talmon, 2000).
This method is simple and rapid, but is limited to the analysis of
liquid suspensions of moderate viscosity with particle diameters
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
12/19
96 V. Klang et al. / Micron 43 (2012) 85103
Fig. 6. Freeze-fracture TEM micrographs of different lecithin-based nanoemulsions. Images reprinted from Zhou et al. (2010) with kind permission from Springer Science
and Business Media. The influence of increasing contents of glycerol within the formulation is demonstrated by decreasing particle sizes and a more homogeneous droplet
size distribution. Coalescence phenomena as indicated by thearrows in image A can be detected.
of less than 200n m. Secondly, cryo-sectioning using high pres-
sure freezing devices after vitrification of the bulk sample can be
performed (Moor, 1987; Mueller and Moor, 1984). In this con-
text, different cutting techniques to obtain vitrified sections thin
enough for high resolution observation are available (Al-Amoudi
et al., 2003; Dubochet et al., 1983; McDowall et al., 1983). Thirdly,a film of the liquid sample can be prepared that is subsequently
vitrified as a thin layer. The thicknessof the films can be controlled
interferometrically before vitrification (Denkov et al., 1996). The
latter two methods can be applied for various tasks, but are rather
complex and demanding in nature. Thus, vitrification after blotting
of the sample is the most widely employed technique.
The sample preparation for cryo TEM involves three main
steps:
I. A small aliquot (approximately 3l) of a fluid suspension con-taining the sample is applied to the surface of a supporting
substrate such as a holey or continuous carbon film that is
attachedto the surface ofa standardTEM specimengrid(Fukamiand Adachi, 1965; Reichelt et al., 1977).
II. Subsequently, the droplet is carefully blotted with filter paper
until most of the supernatant liquid is removed and only a thin
layer of approximately 100 nm thickness is left on the support
substrate. On perforated carbon support films, the thin sample
film is left stretched over the holes (Fig. 7A).
III. The thin fluid layer is rapidly immersed into a suitable cryo-
gen of high heat capacity, which leads to instantaneous and
contaminant-free freezing. This vitrification or shock-freezing
by propelling the grids into a cryogen is ideally performed by
means of a plunging device and a humidity- and temperature-
controlled environmental vitrification system (Dubochet et al.,
1987;Norden et al., 2001). Thevitrifiedsamples then need to be
transferred into the TEM cryo holder of the microscope under
liquidnitrogen and areexaminedat temperatures around 100 K
(Marxer et al., 2011; Yilmaz and Borchert, 2005).
There are two key factors regarding specimen preparation that
are critical to obtaining high quality cryo TEM data, assuming thatbiochemical integrity of the specimen is given: the proper prepa-
ration of the support substrate and the considerate blotting of the
sample droplet to a thin fluid layer on the substrate prior to freez-
ing. The rapid drop in temperature during vitrification provides
the possibility to capture hydrated specimens in their momentary
movement without changing their structure and helps to reduce
the effect of electron beam damage.
Most colloidal systems such as nanoemulsions give compara-
tively poor contrast in cryo TEM. The inherently low contrast of
unstained vitrified specimens can be compensated to a large extent
by an optimal use of phase contrast (Adrian et al., 1984). A varia-
tion in focus canserve to revealdifferent parts of a structurewhile a
total defocus creates optical artefacts (Kuntsche et al., 2011). Addi-
tional staining techniques canbe employed to produce images withimproved contrast (Adrian et al., 1998).
As in allelectronmicroscopictechniques,different artefactsmay
hinder cryo TEM analysis. Adequate cooling of the sample at all
stages of preparation and analysis is essential. Otherwise, artefacts
may emerge during the freezing process. Ice crystal formation or
modifications due to humidity or temperature changes may hinder
the structural analysis. Inadequate cooling within the microscope
leads to damage of the vitrified sample within the vacuum of the
TEM by a kind of freeze-drying effect (Kuntsche et al., 2011).
Moreover, electron beam damage may occur during observa-
tion of frozen materials in cryo TEM although a certain protection
of the sample is obtained after the cryo-fixation procedure. Elec-
tron beam damage is especially severe in the presence of water
(Belkoura et al., 2004). Since most colloidalsystems are sensitiveto
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
13/19
V. Klang et al. / Micron 43 (2012) 85103 97
Fig. 7. Images of a typical nanoemulsion analysed by cryo TEM after plunge freezing. (A) A conventional TEM grid coated with a holey carbon film is shown. The letters
indicate thecarbon layer (C), theholesin thecarbonfilm (H)and thefrozen hydrated sample (S). Thescheme on theright hand side depicts theformation of thethin liquid
sample layer which remains after blotting with a filter paper just before the freezing procedure. (B) Segregation of nanoemulsion droplets: smaller droplets remain in the
thinner parts of the sample filmwhile larger ones areonly found in thethicker parts of thesample layer.
radiation damages, the time period for sample viewing is limited
and low dose conditions are recommended. Such minimal dose
microscopy canbe performedon all modern electronmicroscopes;
thus, the strength of the electron beam can be handled (Belkoura
et al., 2004). Severe electron beamdamage may result in bubbling
of the sample and out-of-focus images (Kuntsche et al., 2011). The
modified techniqueof cryonegative staining,which should provide
both maintained sample hydration and protection against electron
beam damage, bears the risk of selective particle orientation due to
interfacial forces and flattening of fragile structures (Harris, 2008).
Thepotential presence of cryogen residues that remainfrom the
plunge-freezing process likewise needs to be taken into account.
An increased energy input may remove these remnants, but may
induce a phase transition of the vitrified ice into cubic or hexag-onal ice (Kuntsche et al., 2011). Likewise, ice contamination may
occur due to a high content of evaporated water in the TEM col-
umn (Friedrich et al., 2010). For examples of the potential artefacts
in cryo TEM analysis of colloidal systems the reader is referred to a
recent article by Kuntsche et al. (2011).
Apart from the risk of artefacts, there are certain general limita-
tions to the cryoTEM technique for investigationof nanoemulsions.
To obtain adequatefilm thicknessis currently themajor difficulty in
preparingsamplesfor direct imagingwith cryoTEM (Belkoura et al.,
2004). The maximum specimen thickness that can be observed is
limited to a few hundred nanometers, i.e. the film has to be thin
enough to allow investigation by cryo TEM. However, at the same
time the filmmust be thickenough notto influence themicrostruc-
ture of the sample (Belkoura et al., 2004).As already mentioned, blotting of the sample followed by vitri-
fication is the most commonly employed technique in cryo TEM of
nanoemulsions. However, the blotting procedure bears the risk of
preparation artefacts. Concentration changes of the thinned sam-
ple drop, size segregation and changes of internal structure due
to shearing effects may occur (Belkoura et al., 2004). Thus, the
blotting procedure has to be adjusted for each particular sample
(Talmon, 1999). Nevertheless, cryo electron micrographs of poly-
disperse systems may be biased towards small particles due to the
preparation technique. The specimen preparation involves appli-
cation of the liquid sample on the microscopic grid and removal
of the surplus liquid with filter paper until an ultra-thin sample
film remains in the holes of the grid, particularly in their cen-
tre (Fig. 7B) (Jores, 2004a; Jores et al., 2004b). Structures which
exceed the thickness of this film are either removed or relocated
to thicker areas of the film during this procedure. Unfortunately,
areas of increased thickness are often too sensitive towards the
electron beam to deliver reliable results upon investigation. As a
consequence, aggregates or droplets with large dimensions may
remain undetected. Thus, a direct comparison of droplet sizes as
observed in cryo TEM with the results of particle size measure-
ments by DLS or laser diffraction should be performed with great
caution (Jores, 2004a,b). The data obtained by cryo TEM should
be regarded as complementary qualitative information about the
shape and size of the observed particles. A quantitative evaluation
of cryo electron micrographs of a certain formulation aiming at an
accurate size distribution of the observed droplets would require
evaluation of large amounts of individual images and specific pro-grammes.
Nevertheless, cryo TEM of frozen-hydrated unstained spec-
imens is presently among the preferred approaches for high-
resolution studies because it provides data on the fully native
structure and some protection of the specimens against elec-
tron radiation damage (Massover, 2008). Technical improvements
of all steps of sample preparation, such as environmental con-
trol during vitrification and improved data processing through
CCD cameras and digitalisation, promote the further use of this
technique (Kuntsche et al., 2011). Three-dimensional reconstitu-
tion (Orlova et al., 1999) or cryoelectron tomography (Koning
and Koster, 2009) are interesting developments to obtain infor-
mation about the three-dimensional structure of the investigated
systems. Thus, the projection limitation of TEM can be par-tially solved. Three-dimensional tilt-series-based tomography or
even simple stereo pairviews obtained with tilting capable stages
and CCD camera image acquisition give a rapid insight into the
three-dimensional structure of nanocarrier systems not only from
vitrified, but also from negatively stained or freeze-fractured
samples (Baumeister, 2002; Frank, 1992; Hoppe, 1981). Further-
more,freeze-fracture direct imagingoffers interesting prospectsfor
investigation of hydrated colloidal samples (Belkoura et al., 2004).
This method combines elements of the freeze-fracture technique
with direct imaging by cryo TEM. Many experimental artefacts
caused by the blotting procedure in cryo TEM can be avoided;
thus, this technique, although not yet applied to nanoemulsion
samples, represents a promising approach to obtain artefact-free
images.
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
14/19
98 V. Klang et al. / Micron 43 (2012) 85103
Cryo TEM is particularly useful to investigate structural details
of colloidal nano-sized systems, e.g. to detect the presence of vesi-
cles among nanoemulsion droplets (Fig. 1B) (Norden et al., 2001).
Overall, nanoemulsion droplets are comparatively simple to dis-
tinguish in cryo TEM images. They always appear as spherical dark
droplets while solid lipid nanoparticles, liposomes or other related
lipid structures may appear as needle- or rod-like structures when
viewed edge-on (Jores, 2004a,b). The systems can be investigated
with (Hatanaka et al., 2010) or without dilution (Marxer et al.,
2011). In general, dilution of the investigated nanoemulsions is
advisable since examination of undiluted samples frequently leads
to crowded images where individual structures cannot be clearly
characterised.
6.4. Cryo TEM for characterisation of nanoemulsions
A number of recent cryo-microscopic investigations of
nanoemulsions based on eudermic surfactants have allowed for
detailed insights into the versatile nature of such colloidal sys-
tems (Klang et al., 2010, 2011a,b; Teixeira et al., 2000). Teixeira
et al. reported unusual bicompartmental structures with associ-
ated oil and water phases among regular nanoemulsion droplets
(Teixeira et al., 2000). These handbag-shaped structures wereslightly deformed (Fig. 8A), which was ascribed to molecular
rearrangements at the interface due to an interaction between
the employed lecithin and the positively charged stearylamine.
Similar conclusions were reported for nanoemulsions with the
positively charged phytosphingosine (Yilmaz and Borchert, 2005)
although the latter image (Fig. 8B) is of questionable quality. This
emphasises the need for careful interpretation of the obtained
results so as to avoid misinterpretations which are still frequently
observed.
In more recent approaches, we have investigated the morphol-
ogy of lecithin-based nanoemulsions both in the presence (Klang
et al., 2010) and absence (Klang et al., 2011a) of additional emulsi-
fiers orstabilisers. Itwas foundthatthe lecithinmixture aloneled to
the formation of a random mnagerie of rather irregularly shapeddroplets and vesicles (Fig. 9A). This corresponds well with the
respective literature. Phosphatidylcholine alone is rather unsuit-
able forthe formationof curvedsurfaces(Shchipunov, 1997; Trotta
et al., 2002), and the performance of phospholipids mixtures which
largely consist of phosphatidylcholine can be improved by addi-
tional surfactants (Hoeller et al., 2009; Trotta et al., 2002; Yilmaz
andBorchert, 2005). Interestingly, a remarkable change in formula-
tionmicrostructurewas alsoachieved by additionof-cyclodextrinas stabilising agent (Fig. 9C). The number of additional vesicular
structures decreased notably while the droplet shape appeared
more homogeneous and spherical. These findings highlight the
importance of a considerate choice of excipients during formulation
development and the role of electron microscopy to monitor the
results. Comparative studies with a sucrose ester mixture, namelysucrose stearate S-970, revealed that this mixture of eudermic sur-
factantswas moresuitable for the formationof nano-sized droplets
than the lecithin mixture (Fig. 9B). In this case, further addition
of -cyclodextrin had no notable impact on the composition ofthe colloidal system or the droplet shape (Fig. 9D). In fact, any
surplus of surfactant might lead to the formation of additional
aggregates such as multilamellar structures (Fig. 10A) which may
or may not be favourable for the respective formulation proper-
ties (Klang et al., 2010). Similar observations were recently made
fora nanoemulsionstabilised by 5% of sucrose stearate (Klang et al.,
2011b). Ascanbeseenin Fig. 10B, a highly crowded microstructure
was observed despite dilution of the sample. The emulsion droplets
appeared slightly deformed, which may have been related eitherto
altered formulation viscosity and behaviour during high-pressure
homogenisation or to droplet repulsion due to the high negative
zeta potential values (Klang et al., 2011b).
7. Advanced techniques: cryo analytical TEM (cryoATEM)
The technique of electron energy-loss spectroscopy is of great
interest for the analysis of lipid nanocarriers, especially in com-
bination with cryo-preparation techniques (Cryo EELS). Cryo EELS
represents a valuable approach to analyse both the morphology
of a formulation and its chemical composition since the energy
spectrum of electrons passing through the specimens also contains
information about the element composition. When the electron
beam impacts the particle, some of the electrons are inelastically
scattered and lose a part of their energy. Every element possesses
its own specific energyloss.Thus, the elemental composition of the
particle or droplet can be determined by analysis of this specific
energy by means of a spectroscope attached to the electron micro-
scope (Egerton, 1986). Based on this phenomenon, EELS can be
used for chemical analysis of various liquid dispersions, including
systems containing polymers.
Another technique whichis widely used for the elemental anal-
ysis in combination with electron microscopy is energy dispersive
X-ray spectroscopy. When a high-energy electron beam impingesupon a specimen, X-ray photons are produced. Characteristic X-
rays have well defined energies which are characteristic of the
atoms in the specimen (Sawyer and Grubb, 1996; Sutton et al.,
2003). Thus, the elemental composition of the investigated sample
can be determined. In most cases EELS is expected to offer higher
spatial resolution than EDX because the effect of beam broaden-
ing and aberrations of the probe-forming lens can be controlled by
means of an angle-limiting collection aperture. The technique of
EELS as well as energy-filtered transmission electron microscopy
(EFTEM) imaging are usually applied to the detection of light ele-
ments suchas C,N orO, where EDX islesssensitive (Egerton, 1986).
Isaacson and Johnson (1975) provided the theoretical basis for the
prediction of detectionlimits in EELSby introducingthe concepts of
minimum detectable mass (MDM) and minimum detectable massfraction (MMF). The MDMdescribes the smallest amount of mate-
rial that can be detected in a given matrix. A small beam diameter is
desirable, thus the use of a scanning transmission electron micro-
scope equipped with a field emission sourceis preferable (Krivanek
et al., 1991; Leapman, 2003). Alternatively, the MFF represents the
smallest concentration of elements that canbe measured in a given
matrix. Thisparameter depends primarilyon thetotal currentavail-
able in the probe. Thermal emission tips that provide large beam
currents and conventional transmission electron microscopy mode
may thereforebe preferable(Kothleitner andHofer, 1993; Zhuet al.,
2001). In the ideal case of a sample that is not susceptible to beam
damage, traces of chemical elements as low as 0.030.01% can be
detected when using a 200kV instrument (Riegler and Kothleitner,
2010). However, the cryo EELS detection limit is much lower, thusrendering the analysis of nanoemulsion compositions a challeng-
ing task (Yakovlev et al., 2010). Frozen hydrated nanoemulsions
may contain only low amounts of specific atoms like N, P, Cl or F.
Thus, the statistical noise obscures the weak energy-loss near edge
structure (ELNES) signals leading to increased errors in background
extrapolation and further deterioration of the detection limits.
Moreover, cryo EELS usually requires the use of primary energies
of 200kV (in contrast to the conventionally used 80120kV for
cryo TEMimaging) andhighbeamcurrents, whichleadto complete
beam damage of the specimen structure before the adequate spec-
tracanbe recorded (Egerton,1986). Currentlythere are twogeneral
types of energy filters (post-column(GIF) and column-integrated
(omega)) which are used for EELS/EFTEM analysis (Egerton, 1986,
2005).
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
15/19
V. Klang et al. / Micron 43 (2012) 85103 99
Fig. 8. (A) Cryo TEM micrograph of a positively charged nanoemulsion containing deformed bicompartmental structures. Image reprinted from Teixeira et al. (2000) with
kind permission from Springer Science and Business Media. (B) Cryo TEM image which presumably shows similar structures containing phytosphingosine and ceramides.
The latterimageappears to sufferfrom severeout-of-focus problems,perhapsdue to insufficient freezing of thesample. Thelargestructures indicatedby thearrows cannot
be clearly identified as emulsion droplets and might be ice crystals.
Image reprinted with permission from Elsevier (Yilmaz and Borchert, 2005).
Fig. 9. Visualisation of differences in formulation morphology: cryo TEM micrographs of diluted nanoemulsions (1:10, v/v) stabilised by (A) 2.5% of lecithin alone (mean
particle size: 186.4111.06, n=3) or(C) 2.5% oflecithinand-cyclodextrin (meanparticle size:175.8200.47, n =3), (B)2.5% of sucrose stearate alone (mean particle size:141.2108.73, n=3) or (D) 2.5% ofsucrose stearate and -cyclodextrin (meanparticle size:144.7710.61, n=3). Theaddition of thecyclodextrin apparently promotes the
formationof spherical droplets in case of lecithin-based systems (Fig.7A versus7C). Thearrowheads included in Image 7A indicate thepresence of vesicular structures, such
as liposomesand multilamellar layers. In case of sucrose-stearate based systems, hardly any differences were observed after addition of-CD.
All images reprinted from Klang et al. (2011a) with permission from Elsevier.
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
16/19
100 V. Klang et al. / Micron 43 (2012) 85103
Fig.10. (A)Cryo TEMmicrograph of a nanoemulsionstabilised by lecithin,sucrose stearate and-cyclodextrin. The excess of surfactant induces the formationof additional
multilamellar structures. Image reprinted from Klang et al. (2010) with permission from Elsevier. (B) Cryo TEM micrograph of a nanoemulsion stabilised by comparatively
high amounts of sucrose stearate (5%, w/w) after dilution with distilled water (1:10, v/v). It remains to be investigated whether thedeformation of thedroplets was caused
during production or during sample preparation forcryo TEM.
Image reprinted from Klang et al. (2011b).
Cryo EELS has not yet found broader application for nanoemul-
sion characterisation. Firstly, frozen hydrated nanoemulsions
usually contain specific atoms like N, P, Cl or F in such low con-
centrations that the detection limit of cryo EELS does not allow to
obtain fine ELNES details of these elements in the spectra, which
is not the case for conventional biological samples or water map-
ping. Secondly, cryo EELS usually requires usage of an electron
beam voltage up to 200 kV, which leads to complete beam damage
Fig. 11. Application of cryo EELS for the analysis of frozen hydrated samples: differentiation of calcium carbonate particles (A) from ice crystal formations (B). The scale
bars represent 100nm. The corresponding chemical composition of the analysed compound is depicted on the right hand side (C, D). Vitrified specimens were examined in
a field emission Tecnay F20 TEM operating at an accelerating voltage of 200kV using an Oxford CT3500 cryo holder (Oxford Instruments, UK) that maintained the vitrified
specimens at 160
C during sample observation.Images were recorded digitally on a cooled UltraScan CCD (Gatan, USA).
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
17/19
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
18/19
-
7/23/2019 Electron Microscopy of Nanoemulsions_An Essential Tool for Characterisation and Stability Assessment
19/19
V. Klang et al. / Micron 43 (2012) 85103 103
Patravale,V.B., Mandawgade,S.D., 2008.Novel cosmetic delivery systems:an appli-cation update. Int. J. Cosmet. Sci. 30, 1933.
Piemi, M.P.,Korner, D.,Benita,S., Marty, J.-P., 1999.Positively andnegatively chargedsubmicron emulsions for enhanced topical delivery of antifungal drugs. J. Con-trol. Release 58, 177187.
Pinnamaneni, S., Das, N.G., Das, S.K., 2003. Comparison of oil-in-water emul-sions manufactured by microfluidization and homogenization. Pharmazie 58,554558.
Preetz, C., Hauser, A., Hause, G., Kramer, A., Mader, K., 2010. Application of atomicforcemicroscopy and ultrasonic resonatortechnology on nanoscale: distinctionofnanoemulsions from nanocapsules. Eur. J. Pharm. Sci. 39, 141151.
Rao, J., McClements, D.J., 2010. Stabilization of phase inversion temperaturenanoemulsionsby surfactantdisplacement.J. Agric.Food Chem.58, 70597066.
Reichelt, R., Koenig, T., Wangermann, G., 1977. Preparation of microgrids as speci-mensupports forhigh resolution electron microscopy. Micron8, 2931.
Reimer, L., 1993. Image Formation in Low-Voltage Scanning Electron Microscopy.SPIE Optical Engineering Press, Bellingham & Washington.
Riegler, K., Kothleitner, G., 2010.EELS detectionlimits revisited:Rubya casestudy.Ultramicroscopy 110, 10041013.
Rotenberg, M., Rubin, M., Bor, A., Meyuhas, D., Talmon, Y., Lichtenberg, D., 1991a.Physico-chemical characterization of Intralipid emulsions. Biochim. Biophys.Acta 1086, 265272.
Rotenberg, M., Rubin, M., Bor, A., Meyuhas, D., Talmon, Y., Lichtenberg, D., 1991b.Physico-chemical characterization of IntralipidTM emulsions. Biochim. Biophys.Acta 1086, 265272.
Ruozi, B.,Belletti, D.,Tombesi,A., Tosi, G.,Bondioli, L.,Forni, F.,Vandelli, M.A., 2011.AFM, ESEM TEM, andCLSM in liposomal characterization: a comparative study.Int.J. Nanomed. 6, 557563.
Ruska, E.,Knoll, M., 1932. Das Elektronenmikroskop. Z. Phys. 78, 318339.Saupe, A., Gordon, K.C.,Rades, T., 2006. Structuralinvestigationson nanoemulsions,
solid lipid nanoparticles and nanostructured lipid carriers by cryo-field emis-sion scanning electron microscopy and Raman spectroscopy. Int. J. Pharm.314,5662.
Sawyer, L.C., Grubb, D.T., 1996. Polymer Microscopy, 2nd ed. Alden Press, Oxford,England.
Schalbart, P.,Kawaji, M., Fumoto, K., 2010. Formation of tetradecane nanoemulsionby low-energy emulsification methods. Int. J. Refrigeration 33, 16121624.
Scheuing, D.R., 1990. Fourier TransformInfrared Spectroscopy in Colloid and Inter-face Science. American Chemical Society, Washington, DC, pp. 121.
Schmidtgen, M.C., Drechsler, M., Lasch, J., Schubert, R., 1998. Energy-filtered cry-otransmission electron microscopyof liposomespreparedfrom humanstratumcorneum lipids.J. Microsc. 191,177186.
Schwarz, J.S., Weisspapir, M.R., Friedman, D.I., 1995. Enhanced transdermal deliv-ery of diazepam by submicron emulsion (SME) creams. Pharm. Res. 12, 687692.
Seki, J., Sonoke, S., Saheki, A., Fukui, H., Sasaki, H., Mayumi, T., 2004. A nanometerlipid emulsion, lipid nano-sphere (LNS),as a parenteral drug carrier for passivedrug targeting. Int. J. Pharm. 273, 7583.
Severs,N., Robenek,H., 2008.Freeze-fracturecytochemistryin cellbiology.MethodsCell Biol. 88, 181204.Severs, N.J., 2007. Freeze-fracture electron microscopy. Nat. Protocols 2, 547576.Shafiq, S., Shakeel, F., Talegaonkar, S., Ahmad, F.J., Khar, R.K., Ali, M., 2007. Design
anddevelopmentof oraloil inwater ramiprilnanoemulsionformulation: invitroand in vivoassessment. J. Biomed. Nanotech.3, 2844.
Shakeel, F., Baboota, S., Ahuja, A., Ali, J., Aqil, M., Shafiq, S., 2007. Nanoemulsions asvehicles for transdermal delivery of aceclofenac. AAPSPharm. Sci.Tech. 8, E104.
Shchipunov, Y.A., 1997. Self-organising structures of lecithin. Russ. Chem. Rev. 66,301322.
Silva, A.P., Nunes, B.R., De Oliveira, M.C., Koester, L.S., Mayorga, P., Bassani, V.L.,Teixeira, H.F., 2009. Development of topical nanoemulsions containing theisoflavone genistein. Pharmazie 64, 3235.
Singh, K.K.,Vingkar,S.K., 2008.Formulation, antimalarial activityand biodistributionoforal lipid nanoemulsion of primaquine. Int. J. Pharm.347, 136143.
Sitte, H., Edelmann, L., Neumann, K., 1987. Cryofixation without pretreatment atambient pressure.In: Steinbrecht,R., Zierold,K. (Eds.),Cryotechniques in Biolog-ical ElectronMicroscopy. Springer,Berlin,Heidelberg, Germany,pp. p.87p.113.
Sjoeblom, E., Friberg, S., 1978. Light-scattering and electron microscopydetermina-tions of association structures in W/O microemulsions. J. Colloid Interface Sci.67, 1630.
Solans, C., Izquierdo, P., Nolla, J ., Azemar, N., Garcia-Celma, M.J., 2005. Nano-emulsions. Curr. Opin. Coll. Int. Sci. 10, 102110.
Sole, I., Pey, C.M., Maestro, A., Gonzalez, C., Porras, M., Solans, C., Gutierrez, J.M.,2010. Nano-emulsions prepared by the phase inversion composition method:preparation variablesand scale up. J. Colloid Interface Sci. 344, 417423.
Sonneville-Aubrun,O., Simonnet, J.T.,LAlloret,F., 2004.Nanoemulsions:a newvehi-cle forskincare products. Adv. Colloid Interface Sci. 108109, 145149.
S te inbrecht , R.A., Ziero ld, K. , 1987. Cry o Techniques in Biological Electro nMicroscopy. Springer, Berlin, Heidelberg, Germany.
Sutton, S.R.,Newville,M., Rivers, M.L.,2003. Synchrotron X-raymicroscopeanalysis.Geophys. Res. Abstracts 5, 13260.
Tadros, T., Izquierdo, P., Esquena,J., Solans, C., 2004.Formation andstabilityof nano-emulsions. Adv. Colloid Interface Sci. 108109, 303318.
Takegami, S.,Kitamura, K., Kawada, H., Matsumoto, Y., Kitade, T., Ishida, H.,Nagata,C., 2008.Preparationand characterization of a newlipid nano-emulsioncontain-ing two cosurfactants, sodium palmitate fordroplet size reductionand sucrosepalmitatefor stabilityenhancement.Chem.Pharm.Bull. (Tokyo) 56, 10971102.
Talmon, Y., 1999. Cryogenic temperature transmission electron microscopy in thestudy of surfactant systems. In: Binks, B.P.E. (Ed.), Modern CharacterizationMethods of Surfactant Systems. Marcel Dekker, New York, p. p147.
Tamilvanan, S., Benita, S., 2004. The potential of lipid emulsion for ocular deliveryoflipophilic drugs. Eur. J. Pharm. Biopharm. 58, 357368.
Teixeira, H., Dubernet, C., Rosilio, V.,Benita, S.,Lepault, J., Erk, I., Couvreur, P.,2000.Newbicompartmental structuresareobservedwhenstearylamineismixed with
trigly