Micron 43 (2012) 85103

Contents lists available at ScienceDirect

Micron
journal homepage: www.elsevier.com/locate/micron

Review

Electron microscopy of nanoemulsions: An essential tool for characterisation and

stability assessment
Victoria Klang a,,1 , Nadejda B. Matsko b,,1 , Claudia Valenta a , Ferdinand Hofer b
a

University of Vienna, Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Life Sciences, Althanstrasse 14, 1090 Vienna, Austria
Graz University of Technology and Centre for Electron Microscopy Graz, Institute for Electron Microscopy and Fine Structure Research (FELMI-ZFE), Steyrergasse 17, 8010 Graz,
Austria
b

a r t i c l e

i n f o

Article history:
Received 30 May 2011
Received in revised form 18 July 2011
Accepted 19 July 2011
Keywords:
Nanoemulsion
Submicron emulsion
Electron microscopy
Scanning electron microscopy
Transmission electron microscopy
Cryo ATEM

a b s t r a c t
The characterisation of pharmaceutical formulations by microscopic techniques is essential to obtain
reliable data about the actual morphology of the system. Since the size range of colloidal drug delivery systems has long ago reached the lower end of the nanometer scale, classical light microscopy has
been replaced by electron microscopy techniques which provide sufcient resolution for the visualisation of nano-sized structures. Indeed, the superior resolution and methodological versatility of electron
microscopy has rendered this technique an indispensable tool for the analysis of nanoemulsions. Microscopic analysis of these lipid-based drug delivery systems with particle sizes in the lower submicron
range provides critical information about the size, shape and internal structure of the emulsion droplets.
Moreover, surfactant aggregates such as liposomes or multilamellar structures which remain unnoticed
during particle size measurements can be detected in this fashion. This review provides a brief overview
about both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques
which have been employed to characterise nanoemulsions. Of special interest are sophisticated cryo techniques of sample preparation for both TEM and SEM which deliver high-quality images of nanoemulsions
in their natural state. An overview about the instrumentation and sample preparation for all presented
methods is given. Important practical aspects, sources of error and common artefacts as well as recent
methodological advances are discussed. Selected examples of electron microscopic studies of nanoemulsions are presented to illustrate the potential of this technique to reveal detailed and specic information.
2011 Elsevier Ltd. All rights reserved.

Contents
1.
2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nano-sized emulsion systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Properties and characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Laser light scattering versus electron microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Development of microscopic techniques for visualisation of nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Scanning electron microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Experimental setup, sample preparation and potential artefacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
SEM-based techniques for characterisation of nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transmission electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Experimental setup, sample preparation and potential artefacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

86
86
86
87
88
89
89
89
89
89

Abbreviations: TEM, transmission electron microscopy; SEM, scanning electron microscopy; EELS, electron energy-loss spectroscopy; EDX, energy dispersive X-ray
spectroscopy; DLS, dynamic light scattering; PDI, polydispersity index; AFM, atomic force microscopy; ESEM, environmental scanning electron microscopy; STEM, scanning
transmission electron microscopy; FF-TEM, freeze-fracture transmission electron microscopy; EFTEM, energy-ltered transmission electron microscopy; MDM, minimum
detectable mass; MMF, minimum detectable mass fraction; ELNES, energy-loss near edge structure.
Corresponding author. Tel.: +43 316 873 8335; fax: +43 316 811 596.
Corresponding author. Tel.: +43 4277 55404; fax: +43 4277 9554.
E-mail addresses: victoria.klang@univie.ac.at (V. Klang), nadejda.matsko@felmizfe.at (N.B. Matsko).
1
These authors contributed equally to this work.
0968-4328/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2011.07.014

86

5.

6.

7.
8.

V. Klang et al. / Micron 43 (2012) 85103

4.2.
Conventional TEM for characterisation of nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Cryo-preparation techniques for SEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.1.
FF-SEM and cryo SEM: experimental setup, sample preparation and potential artefacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.2.
FF-SEM and cryo SEM for characterisation of nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Cryo-preparation techniques for TEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.1.
Freeze-etching and freeze-fracturing for TEM: experimental setup, sample preparation and potential artefacts . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.2.
Freeze-fracture TEM for characterisation of nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
6.3.
Cryo TEM: experimental setup, sample preparation and potential artefacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
6.4.
Cryo TEM for characterisation of nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Advanced techniques: cryo analytical TEM (cryo ATEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

1. Introduction

2. Nano-sized emulsion systems

Colloidal systems which consist of nely dispersed structures

in the lower submicron range are constantly being developed
for pharmaceutical or technological applications. Among those,
nanoemulsions have a long history of practical application as parenteral nutrition systems (Baspinar, 2009; Calder et al., 2010;
Pinnamaneni et al., 2003; Wabel, 1998). Later on, they were investigated as drug delivery vehicles for different routes of application,
such as the parenteral, oral, dermal or ocular route (Abdulrazik
et al., 2001; Baker and Naguib, 2005; Ilan et al., 1996; Klang et
al., 1996; Piemi et al., 1999; Schwarz et al., 1995; Tamilvanan and
Benita, 2004; Yilmaz and Borchert, 2005). The benet of these
systems lies within their composition, which usually comprises
highly biocompatible and biodegradable compounds. This renders
them useful for sensitive routes of drug delivery as well as for
cosmetic applications (Klang and Valenta, 2011c; Patravale and
Mandawgade, 2008; Sonneville-Aubrun et al., 2004). However, the
large variety of employed excipients may result in a variable structure of the different formulations. Thus, a thorough characterisation
of novel nanoemulsion systems is essential to obtain reliable information about their physicochemical properties and their long-term
stability.
This review provides an insight into the nature of nanoemulsions and their characterisation to highlight the value and the
necessity of electron microscopy for an exact visualisation of such
systems. The development of common microscopic techniques for
analysis of nanoemulsions is briey summarised. Subsequently,
the most frequently employed electron microscopic techniques are
presented. Advantages of the different methods as well as limitations are discussed. Numerous examples and images are included
to demonstrate the richness of information that can be obtained by
the discussed techniques.
The nanoemulsion structure can be viewed in an electron
microscope without further preparation and certain conclusions
can be drawn from the obtained images if the circumstances
are favourable. However, far more representative information
can be gained upon observation of the sample in its native
state after cryo-preparation in thin vitried layers or at a fracture plane using both transmission electron microscopy (TEM)
and scanning electron microscopy (SEM) (Mason et al., 2006).
Although some of the described techniques are rarely employed
for nanoemulsion characterisation due to the time-consuming
and complex sample preparation and high costs, all methods
reported in the literature are briey described. Since TEM in
combination with cryogenic techniques of sample preparation is
among the most appropriate methods for the investigation of
nanoemulsions in their original state, a focus is laid on these
and related techniques. Advanced techniques such as cryogenic
electron energy-loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDX) are likewise presented in this
context.

2.1. Properties and characterisation

In literature, the term nanoemulsion can be found for an
astonishing variety of formulations. From a strictly technical viewpoint, nanoemulsions are conventional oil-in-water or, more rarely,
water-in-oil emulsions with mean droplet sizes below 100 nm
(Mason et al., 2006). However, classical nanoemulsions which are
produced by high-pressure homogenisation seldom reach droplet
sizes in this range. Originally, lecithin-based submicron emulsions
were employed in parenteral nutrition (Baspinar, 2009; Calder
et al., 2010; Wabel, 1998). Along with their adaptation for other
pharmaceutical or cosmetic applications their composition and
production methods were varied. Likewise, the terminology was
adapted, presumably to serve marketing purposes. As a result,
the vast majority of literature dealing with nanoemulsions actually describes systems in the submicron range slightly or well
above 100 nm. Since this trend already dominates in the respective literature, the term nanoemulsion will be maintained in this
review as well. However, the reader should be aware of the slightly
misleading terminology. Importantly, they should not be confused with microemulsions, which are thermodynamically stable
systems that usually contain large amounts of surfactants and
solvents and may exhibit various different structures (Anton and
Vandamme, 2011). Further details can be found in recent reviews
on this topic (Anton and Vandamme, 2011; Klang and Valenta,
2011c).
In general, nanoemulsions can thus be described as oil-in-water
emulsions with particle sizes below 1000 nm, most frequently
between 100 and 500 nm (Daniels, 2001). The employed excipients such as the oils and surfactants are mostly of a biocompatible,
mild nature (Klang and Valenta, 2011c; Patravale and Mandawgade,
2008; Sonneville-Aubrun et al., 2004). Unlike microemulsions,
nanoemulsions are not thermodynamically stable. Their good physical long-term stability stems from the fact that the conventional
destabilisation phenomena such as creaming and coalescence are
largely prevented or decelerated due to the small particle size of
the droplets which are not affected by gravity, but rather subjected
to Brownian motion. The main source of instability is generally
reported to be Ostwald ripening, which is the tendency of small
droplets to merge with larger droplets due to differences in solubility (Klang and Valenta, 2011c; Mason et al., 2006). This slow
increase in droplet size will eventually lead to a break-down of the
system and is particularly problematic if the dispersed phase has
signicant water solubility (Welin-Berger and Bergenstahl, 2000).
In addition, reversible destabilisation phenomena such as occulation, creaming (Baker and Naguib, 2005; Wabel, 1998) or separation
of a clear aqueous phase from the concentrated emulsion may occur
(Klang and Valenta, 2011c).
Nanoemulsions can be produced by high-energy emulsication
methods using high pressure-homogenisation or ultrasonication

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 lled circles) and vesicles (unlled circles). The black scale bar represents 200 nm. 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 400 nm, 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 Marxer et 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 emulsication
have been described which employ the chemical properties of the
system to create nano-sized emulsion droplets from a microemulsion matrix (Anton et al., 2007; Bouchemal et al., 2004; Sole et al.,
2010; Tadros et al., 2004). In both cases, highly uid emulsion
systems emerge. The optical appearance may range from transparent systems with droplet sizes well below 100 nm to opaque or
milky 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 uids due to multiple scattering of light (Klang and Valenta,
2011c; Mason et al., 2006). Representative examples of a translucent 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 information (Klang and Valenta, 2011c). For an exhaustive overview of
the different physicochemical techniques for nanoemulsion characterisation 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 distribution are frequently employed to characterise the long-term
stability of novel formulations. These parameters can be determined by optical light scattering techniques. More specically,
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 formulation. A small PDI below 0.2 indicates a narrow droplet size
distribution and thus better stability against destabilisation phenomena 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 parameters remain largely unchanged during a prolonged observation
period, a formulation is usually considered physically stable. Initial 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 formulation 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 the
presence of a small population of large droplets present in
nanoemulsions (Benita and Levy, 1993). Likewise, other surfactant 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 demonstrates the composition of such a mixed colloidal dispersion of 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 representative. Furthermore, most samples have to be diluted prior to
DLS measurements to ensure sufcient transparency for accurate

88

V. Klang et al. / Micron 43 (2012) 85103

droplet size determination (Klang and Valenta, 2011c). As a consequence, reversible destabilisation phenomena such as occulation
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 sedimentation eld ow 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 context. 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 possible to obtain excellent DLS data for nanoemulsions over months of
stability monitoring while a microscopic analysis of the same sample reveals a denite change of the internal structure. Recently,
Preetz et al. (2010) demonstrated the importance of microscopic
analysis for the characterisation of nanoemulsions and nanocapsules. 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 50
and 500 nm with the highest frequency around 100 nm, which was
additionally conrmed 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 specic topic. An excellent overview of cryo
TEM analysis of colloidal systems in general was recently published
(Kuntsche et al., 2011). Cryo TEM is certainly among 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 possible future developments. It is important to note that all mentioned
techniques have their benets and drawbacks. In any case, the studied images have to be representative of the whole sample. Image
analysis software should be employed only for systems with a suitable contrast and composition. 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 was
soon realised that this technique is futile for the exact visualisation 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 Brownian 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 emulsions were
presented by Du Plessis et al. (1986) who analysed thin sections of
emulsions enclosed in agar capsules after xation with osmium
tetroxide. Well-dened droplets with particle sizes around 250 nm
could be visualised. This method proved to be time-consuming and
expensive; rst results could only be obtained after 30 h. Faster
and less expensive methods were subsequently developed which
involved xation of the diluted sample and placing a thin layer on
a formvar -coated copper grid for TEM examination after negative staining (Benita and Levy, 1993; Du Plessis et al., 1987). As
for all hydrated systems investigated by conventional electron
microscopy, artefacts induced by xation, inclusion in agar, negative 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 when interpreting such images (Kuntsche
et al., 2011).
At roughly the same time, it was realised that SEM likewise represents a valuable tool to determine the droplet size of parenteral
fat emulsions (Benita and Levy, 1993; Bullock, 1984; HamiltonAttwell et al., 1987). A denite benet of the SEM technique is
the topography dependent nature of the obtained images, i.e. additional topographical information at a considerable depth of focus
on a 2D-image can be obtained. However, the xation of lipids is
a challenging task and specic xation protocols for SEM are necessary 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 xation
of a nanoemulsion with osmium trioxide and deposition of the xed
droplets on a lter 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 accuracy
of 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
identication of nely 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 cryoxation techniques (Groves et al., 1985; Rotenberg et al., 1991b)
that the well-established presence of liposomes in nanoemulsions
was rst observed. The development of cryo-preparation methods
for liquid specimens (Bachmann and Talmon, 1984) offered new
possibilities for nanoemulsion characterisation by cryo-TEM
(Rotenberg et al., 1991a; Teixeira et al., 2000) and cryo-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; Takegami 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.

V. Klang et al. / Micron 43 (2012) 85103

3. Scanning electron microscopy

3.1. Experimental setup, sample preparation and potential
artefacts
In SEM (Ardenne, 1938a,b; Knoll, 1935; Reimer, 1993), the image
is formed step by step by scanning a focused electron beam across
the specimen. The primary electrons penetrate the solid specimen
and are deected by a large number of elastic scattering processes.
Various signals are generated as a result of the impact of the incident electrons, which are collected to form an image or to analyse
the sample surface (Bogner et al., 2007). The electrons that are collected by the detector system give specic 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 50 eV. Secondary electrons can emerge from the specimen only from within a thin surface layer of a few nanometers.
The image contrast depends on the selected angular range of the
electrons collected (Goldstein et al., 1981).
II. Material contrast can be obtained by back-scattered electrons
which possess energies between 50 eV 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 ultrastructural investigation of different kinds of hydrated materials,
including nanoemulsions. Firstly, topography dependent information 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 magnications 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 resolution and the risk of electron beam damage (Reimer, 1993).
3.2. SEM-based techniques for characterisation of nanoemulsions
Calderilla et al. investigated different colloidal systems based on
sucrose esters. While SEM was employed for the characterisation
of nanocapsules by a xation 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 critical point drier. The samples were then sputter coated with gold
and examined. The authors stated that SEM was a suitable technique for the investigation of 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 propofol nanoemulsions was performed by Masaki et al. (2003) to
analyse notable changes in droplet size. A one-step xation technique for the oil droplets on lter 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 lipid dissolved in aqueous glutaraldehyde. Thus, this
specic xation technique permitted visualisation of awless oil

89

droplets in the SEM. A lter paper immersed in the saline dilution

medium alone was used as control. The samples were dehydrated
by different alcohols and after 2 h of freeze-drying they were coated
with gold vapour and observed under a SEM to detect the maximum
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
and those 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 the long-term stability of the
nanoemulsion and may render it unsuitable for intravenous application. This underlines the need for additional methods of analysis
such as electron microscopic techniques.
A SEM-based technique that can be used for dynamic experiments of hydrated nanocarriers is environmental scanning electron
microscopy (ESEM) (do Amaral et al., 2005; Ruozi et al., 2011). This
technique is based on the use of a multiple aperture and a graduated vacuum system that allows the chamber to be maintained at
pressures around 5000 Pa. 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 procedure and by controlling the temperature using a Peltier stage
(Ruozi et al., 2011). However, the resolution is not sufcient 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 electron microscopy (wet STEM in ESEM) or SEM of uids in ow cells
(Bogner et al., 2005, 2007). Nevertheless, accurate results regarding
droplet size and size distribution of nano-sized emulsions can be
obtained by wet STEM in ESEM (Bogner et al., 2005; do Amaral et al.,
2005). Thus, further developments in this eld may be anticipated
with interest.

4. Transmission electron microscopy

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 specimen is not illuminated by light, but
by an electron beam. This requires operation in a vacuum since gas
molecules would deect the electrons. At the top of the microscope
column is an electron gun and a system of electromagnetic lenses
focuses the electron beam on the sample. The image contrast in TEM
is obtained by the interactions of the electrons with the material,
i.e. electron scattering. The resolution in TEM is directly proportional 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 120 kV and microscopes in the range
of 200400 kV 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
0.31.0 nm (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 eld mode and thus

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V. Klang et al. / Micron 43 (2012) 85103

appear to be absorbed. In the case of ordered or crystalline sample 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 brightness depends on the local mass thickness. Thus, darker regions
in the bright eld image mode are regions of higher scattering
(Sawyer and Grubb, 1996). For more detailed information on electron 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 without 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).
The most common staining agents are salts of heavy metals such as
molybdenum, tungsten or uranium which possess atomic numbers
between 42 and 92. These agents must be benign to the wet specimens, form a thin glassy layer upon drying and 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 uranyl
acetate (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 solution of a heavy metal salt is applied for staining. The sample is
then left to dry and nally observed by TEM at room temperature. This technique allows for the identication of the dehydrated
shells of the nanoemulsion droplets which are stabilised by surfactant. The strongly scattering metal ions form an amorphous
shield enveloping the weakly scattering oil droplets to enhance the
electron microscopy contrast (Brenner and Horne, 1959). A high
reverse contrast is thus seen in bright eld TEM images, with light
droplets against a darker background. Negative staining can be used
to visualise the size, shape and internal structure of the sample.
The negative stain not only provides contrast for weakly scattering
specimens, but also physical support against collapse of the sample
structure during drying and protection against electron beam damage (Massover, 2008). The sample may also be coated with a carbon
lm 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 preservation of the structural integrity are identical in both TEM and SEM
(Mueller, 1991). Both drying and staining techniques can affect the
structure and morphology of the sample; thus, great care should
be taken during interpretation of the obtained images (Friedrich
et al., 2010). Severe shrinkage or even complete collapse, selective dimensional modication and aggregation of the constituents
of colloidal systems due to complete dehydration and drying usually cause strong structural changes. In addition, the use of heavy
metal salts leads to a selective sample appearance since only structures 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 react with the staining agents are clearly observed while
many compounds of the sample remain practically invisible. As a
result, the nal EM images may describe completely modied structures which have nothing in common with the original formulation
morphology. Moreover, conventional negative staining on continuous carbon support lms bears the risk of sample distortion due

to adsorption and attening during the drying of the thin aqueous lm of negative stain or evaporation in the TEM (Harris, 2008).
Apart from adsorption artefacts, variable spreading and incomplete
specimen coverage by the stain solution can lead to non-uniform
staining results. Specimen distortion from surface tension forces
during evaporative drying or the formation of a saturated salt solution before the nal drying may occur as well (Massover, 2008).
Techniques based on cryoxation 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 justied (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 experimental data can be found in the respective literature. Image 2
gives an overview of images obtained by TEM at room temperature after negative staining. Although these images might provide
some 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 preliminary 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 limitations 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 observation. As can be seen in Fig. 2A, a pronounced difference in mean
droplet diameters was demonstrated for the two systems. Likewise, Kelmann et al. (2007) investigated the microstructure of
a 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 nanoemulsions 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 afnity of the staining
agent to interfacial components.
In another approach, nanoemulsions stabilised by semisolid
polymer interphases were analysed by TEM without specied sample preparation (Nam et al., 2010). Signicant variations were found
in size distribution and morphology of nanoemulsions in dependence of different oil phases (Fig. 2C). The surface morphology of the
systems might have been affected by the drying process. Ganta et al.
(2008) employed TEM after negative staining with phosphotungstic
acid. The peculiar arrangement of the nano-sized droplets (Fig. 2D)
was most likely caused by the sample preparation. A similar technique 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 submicron emulsions (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 observed for emulsions with soybean oil (Fig. 2F). It remained

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 et al. (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 parafn. Scale bars
represent 150 nm. Images reprinted from Nam et al. (2010) with permission from the American Chemical Society (ACS). (D) TEM photomicrograph of a parenteral emulsion
after negative staining using phosphotungstic acid. The scale bar represents 500 nm. Image reprinted from Ganta et al. (2008) with permission from Elsevier. (E), (F) TEM
photomicrographs of nano-sized emulsions with castor oil (E) or soybean oil (F). The scale bars represent 500 nm. For the latter system, coalescence phenomena are visible.
Images reprinted from Ibrahim et al. (2009) with permission from Elsevier. (G) TEM photomicrographs of thalidomid-loaded and blank nanoemulsions with polysorbate. The
scale bars represent 100 nm.
Image reprinted from Araujo et al. (2011) with permission from Elsevier.

unclear whether the latter was inherent to the formulation or

merely an artefact caused by sample preparation.
Several authors employed specic diffraction modes during
conventional TEM bright eld imaging to visualise the form and
size of nanoemulsions and to determine the amorphous or crystalline character of their components (Bouchemal et al., 2004;
Shaq et al., 2007). However, the contrast of the obtained images of
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 conventional TEM are comparatively rare. An appropriate staining
technique appears to be essential to obtain sufcient 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 conditions 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 remain intact and can be presented
(Fig. 2G). In this case, no information is obtained about the polydispersity of the droplet size distribution or 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
lm. After evaporation of the water the sample was observed at

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V. Klang et al. / Micron 43 (2012) 85103

Fig. 3. Comparison of cryo TEM and conventional TEM after negative staining with uranyl acetate: nanoemulsions stabilised by either 5% (A and B) or 2.5% (w/w) of sucrose
stearate (C and D) were investigated with both methods. On the left hand side (A, C), the cryo TEM images are given. On the right hand side (B, D), the corresponding 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
droplets that had been concentrated and deformed during the evaporation process could be observed. Apparently, the silicone oil was
of sufciently large molecular weight so as not to signicantly
evaporate under the experimental conditions. The specic reasons
why 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 and D). A direct comparison with images of the same
samples obtained by cryo TEM (Fig. 3A and C) suggests that the morphology of the systems is comparatively well preserved despite the
unfavourable surroundings within the TEM. Surprisingly intact, if
deformed and aggregated droplet shells were obtained. Nevertheless, care has to be taken to avoid artefacts caused by electron beam
damage (Fig. 3D, right-hand corner), which are more imminent in
TEM at room temperature.
Summarising our experiences with TEM analysis of nanoemulsions 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 efcacy 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 emulsied using different amounts of a sucrose ester surfactant. Perfectly clear phase
boundaries of the droplet shells after evaporation in the TEM were
found for systems of an ideal surfactant concentration of 2.5% (w/w)
and high physical stability (Fig. 3D). A surplus of surfactant did not
improve the formulations general properties (Fig. 3B), but rather
destabilised the system by introducing aggregates and leading to
droplet deformation. The obtained images of both formulations corresponded 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 certain information that might be useful in nanoemulsion studies, e.g.
about sample stability against external stress. The stability of the
emulsied 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 conned to cryo TEM. Interpretation
of TEM images of nanoemulsions should therefore be performed
with caution.

V. Klang et al. / Micron 43 (2012) 85103

5. Cryo-preparation techniques for SEM

5.1. FF-SEM and 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 freezefracture 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 eld.
In contrast to freeze-fracture electron microscopy, the frozen
specimens prepared for cryo electron microscopy are not replicated, 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 most
frequently 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 benets 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 solutions (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 rmly onto the carbon coated grid and are not readily removed by lter paper. The
resulting layer may not be thin enough to be transparent for the
electron beam. On the other hand, the dimensions of the holes in
the carbon support lm 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 insufcient for a comprehensive understanding of the sample organisation. In contrast,
cryo SEM delivers information about the distribution of specic
structures such as aggregates on the surface of the sample irrespective 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 sample areas can be investigated by freeze-fracture SEM, which also
delivers a good overview of the sample morphology and homogeneity.
When working with either freeze-fracture or cryo electron
microscopy, some general aspects on the benets and limitations
of the different methods of sample preparation should be considered to identify the most appropriate technique for a specic
nanoemulsion sample. Vitrication of samples for TEM is typically achieved by plunging the thin liquid specimens into liquid
ethane. In case of oil-rich samples, apolar compounds such as hydrcarbons, 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 usually do not contain large amounts of apolar compounds or solvents,
plunge-freezing is usually a suitable approach. However, in case
of water-in-oil nanoemulsions or microemulsions dissolution of

93

sample compounds may occur. Therefore, high pressure-freezing

represents the optimal cryoxation method in such cases. It allows
the vitrication of samples with a thickness of up to 200 m.
Moreover, during high pressure freezing the sample is encapsulated in a metal shell, which helps to avoid dissolution problems.
Conventional freeze-fracture techniques allow for the direct visualisation of oil-rich samples which would dissolve in the liquid
ethane used for vitrication of the samples during cryo-techniques
(Belkoura et al., 2004). In addition, non-blotting methods for freezefracture preparation and cryo-energy ltered transmission electron
microscopy are suitable alternatives (Belkoura et al., 2004; Burauer
et al., 2003; Schmidtgen et al., 1998).
5.2. FF-SEM and 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 carriers such as nanoemulsions and solid lipid nanoparticles by cryo
SEM (Saupe et al., 2006). The liquid colloidal systems were plungefrozen and viewed in their natural state after fracturing and coating
with platinum. Deviating nano-sized structures which may emerge
during production could be detected by this technique since both
shape 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 (BilbaoSainz et al., 2010). A freeze-fracture SEM technique was employed
to characterise the interfacial and bulk structure of silica-coated
nano-sized emulsions as well as conventional nanoemulsions without additional coating (Eskandar et al. 2009). The coated emulsions
were stabilised by mixed interfacial layers of either lecithin or oleylamine and hydrophilic silica nanoparticles. The reported method
included cryoxation of the emulsion, fracturing, etching, platinum
coating and subsequent imaging. The samples were injected into a
split brass tube and were cryoxed by plunging the latter into a
liquid nitrogen-solid nitrogen slush together with the cryo transfer specimen holder. The specimen was then transferred to the
exchange chamber under vacuum and the split brass tube was broken 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 conductivity prior to SEM imaging. The obtained images revealed that
the adsorption of the silica nanoparticles to the oil droplet interface was inuenced by the nature of the surfactant and the charge
it conveyed to the interfacial lm (Fig. 4B). The distribution of the
silica nanoparticles between the interfacial lm and 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 and potential artefacts
Freeze-etching, freeze-fracturing and cryo electron microscopy
of frozen uids are complementary techniques mainly because the
respectively obtained information is based on different mechanisms (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 freezefracture 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

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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 about the internal structure of

nano-sized colloidal carriers is obtained (Kuntsche et al., 2011).
The key steps involved in this procedure are rapid freezing of
the sample, fracturing, replication and 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. cryoxation, of the nano-sized suspension 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
xation with glutaraldehyde beforehand serves to avoid artefacts induced by the cryoprotectant. In many cases, successful
freezing of hydrated samples requires ultrarapid freezing tech-

niques, such as optimised plunge freezing, jet freezing, spray

freezing, 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 microtome blade. If deemed necessary, an additional etching step may
be performed which consists of vacuum sublimation of ice after
fracturing. In other words, the ice can be removed from the surface of the fractured specimen by freeze-drying by increasing
the temperature to about 100 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

V. Klang et al. / Micron 43 (2012) 85103

95

the investigated sample (Benita and Levy, 1993; Buchheim, 1982).

As for all microscopic techniques, care must be taken to avoid
misinterpretation due to artefacts. Artefacts may occur due to insufcient freezing rates or re-deposition of solvent molecules onto the
sample plane after fracturing (Kuntsche et al., 2011). Therefore,
considerate specimen preparation is essential to ensure reproducible and reliable data.
6.2. Freeze-fracture TEM for characterisation of nanoemulsions

Fig. 5. Schematic illustration of the sample preparation during the freeze-fracture

process followed by the freeze-etching technique. The obtained specimens can subsequently be investigated by TEM or SEM.

then covered with a strengthening layer of electron-lucent carbon to stabilise the ultra-thin metal lm (Preetz et al., 2010).
More specically, 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 carbon, 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 underlying biological material by careful chemical digestion with acid
solutions or detergents. The still-oating replica is thoroughly
washed free from residual chemicals, carefully placed on ne
grids, dried and then investigated in the TEM (Preetz et al., 2010;
Severs and Robenek, 2008).
Further details and practical advice on freeze-fracture electron 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 different 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 signal
to 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 important feature 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 xation,
cryoprotective pre-treatment, cryoxation, freeze-fracturing, etching and replication, may signicantly inuence the appearance of

The morphology of a lecithin-based nanoemulsion for topical

application was investigated by FF-TEM following a standard protocol of sample cryoxation, freeze-fracturing, freeze-etching and
covering with platinum/carbon (Zhou et al., 2010). The inuence
of increasing amounts of glycerol within the systems was clearly
shown (Fig. 6). The droplet size, shape and size distribution could
be monitored very well by this technique. Likewise, instability phenomena such as the agglomeration of droplets as observed in 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 conrming 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
vitried frozen-hydrated state. As with freeze-fracture TEM, information 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 displayed in its original state and 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 an
electron microscope. Therefore, the development of cryo-electron
microscopy of vitried specimens represented a major progress in
this eld (Bouchet-Marquis and Hoenger, 2011; Steinbrecht and
Zierold, 1987). During vitrication, a thin lm containing the specimen is plunged into a suitable cryogen such as liquid ethane. Liquid
nitrogen is used to maintain the temperature of the ethane near its
melting point of 183 C. With a freezing rate around 1,000,000 K/s,
the uid surrounding the specimen does not have time to form crystalline ice, which would damage the fragile sample; it is vitried
instead. The nature of the liquid is preserved since the rst-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 vitried layer of
aqueous specimens exist, such as plunge-, impact-, spray- or high
pressure-freezing. For investigation of nanoemulsions, plungefreezing and high pressure-freezing are the most commonly used
techniques. Detailed information on these cryoxation procedures
can be found in the literature (Dubochet et al., 1982; Lepault et al.,
1983; Sitte et al., 1987). Various kinds of cryo specimen holders and
plunge-freezing devices are available.
One of the basic requirements for TEM is that the specimen
must be very thin. To produce lms of adequate thickness, three
main techniques can be employed. Firstly, blotting of the sample
is performed followed by vitrication (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

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 inuence 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 the arrows in image A can be detected.

of less than 200 nm. Secondly, cryo-sectioning using high pressure freezing devices after vitrication of the bulk sample can be
performed (Moor, 1987; Mueller and Moor, 1984). In this context, different cutting techniques to obtain vitried sections thin
enough for high resolution observation are available (Al-Amoudi
et al., 2003; Dubochet et al., 1983; McDowall et al., 1983). Thirdly,
a lm of the liquid sample can be prepared that is subsequently
vitried as a thin layer. The thickness of the lms can be controlled
interferometrically before vitrication (Denkov et al., 1996). The
latter two methods can be applied for various tasks, but are rather
complex and demanding in nature. Thus, vitrication 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 3 l) of a uid suspension containing the sample is applied to the surface of a supporting
substrate such as a holey or continuous carbon lm that is
attached to the surface of a standard TEM specimen grid (Fukami
and Adachi, 1965; Reichelt et al., 1977).
II. Subsequently, the droplet is carefully blotted with lter 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 lms, the thin sample
lm is left stretched over the holes (Fig. 7A).
III. The thin uid layer is rapidly immersed into a suitable cryogen of high heat capacity, which leads to instantaneous and
contaminant-free freezing. This vitrication or shock-freezing
by propelling the grids into a cryogen is ideally performed by
means of a plunging device and a humidity- and temperaturecontrolled environmental vitrication system (Dubochet et al.,
1987; Norden et al., 2001). The vitried samples then need to be
transferred into the TEM cryo holder of the microscope under

liquid nitrogen and are examined at 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 that
biochemical integrity of the specimen is given: the proper preparation of the support substrate and the considerate blotting of the
sample droplet to a thin uid layer on the substrate prior to freezing. The rapid drop in temperature during vitrication 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 comparatively poor contrast in cryo TEM. The inherently low contrast of
unstained vitried specimens can be compensated to a large extent
by an optimal use of phase contrast (Adrian et al., 1984). A variation in focus can serve to reveal different parts of a structure while a
total defocus creates optical artefacts (Kuntsche et al., 2011). Additional staining techniques can be employed to produce images with
improved contrast (Adrian et al., 1998).
As in all electron microscopic techniques, different artefacts may
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
modications due to humidity or temperature changes may hinder
the structural analysis. Inadequate cooling within the microscope
leads to damage of the vitried 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 observation of frozen materials in cryo TEM although a certain protection
of the sample is obtained after the cryo-xation procedure. Electron beam damage is especially severe in the presence of water
(Belkoura et al., 2004). Since most colloidal systems are sensitive to

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 lm is shown. The letters
indicate the carbon layer (C), the holes in the carbon lm (H) and the frozen hydrated sample (S). The scheme on the right hand side depicts the formation of the thin liquid
sample layer which remains after blotting with a lter paper just before the freezing procedure. (B) Segregation of nanoemulsion droplets: smaller droplets remain in the
thinner parts of the sample lm while larger ones are only found in the thicker parts of the sample layer.

radiation damages, the time period for sample viewing is limited

and low dose conditions are recommended. Such minimal dose
microscopy can be performed on all modern electron microscopes;
thus, the strength of the electron beam can be handled (Belkoura
et al., 2004). Severe electron beam damage may result in bubbling
of the sample and out-of-focus images (Kuntsche et al., 2011). The
modied technique of cryo negative 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 attening of fragile structures (Harris, 2008).
The potential presence of cryogen residues that remain from 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 vitried ice into cubic or hexagonal ice (Kuntsche et al., 2011). Likewise, ice contamination may
occur due to a high content of evaporated water in the TEM column (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 limitations to the cryo TEM technique for investigation of nanoemulsions.
To obtain adequate lm thickness is currently the major difculty in
preparing samples for direct imaging with cryo TEM (Belkoura et al.,
2004). The maximum specimen thickness that can be observed is
limited to a few hundred nanometers, i.e. the lm has to be thin
enough to allow investigation by cryo TEM. However, at the same
time the lm must be thick enough not to inuence the microstructure of the sample (Belkoura et al., 2004).
As already mentioned, blotting of the sample followed by vitrication 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 sample 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 polydisperse systems may be biased towards small particles due to the
preparation technique. The specimen preparation involves application of the liquid sample on the microscopic grid and removal
of the surplus liquid with lter paper until an ultra-thin sample
lm remains in the holes of the grid, particularly in their centre (Fig. 7B) (Jores, 2004a; Jores et al., 2004b). Structures which

exceed the thickness of this lm are either removed or relocated

to thicker areas of the lm 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 measurements 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 specic programmes.
Nevertheless, cryo TEM of frozen-hydrated unstained specimens is presently among the preferred approaches for highresolution studies because it provides data on the fully native
structure and some protection of the specimens against electron radiation damage (Massover, 2008). Technical improvements
of all steps of sample preparation, such as environmental control during vitrication and improved data processing through
CCD cameras and digitalisation, promote the further use of this
technique (Kuntsche et al., 2011). Three-dimensional reconstitution (Orlova et al., 1999) or cryoelectron tomography (Koning
and Koster, 2009) are interesting developments to obtain information about the three-dimensional structure of the investigated
systems. Thus, the projection limitation of TEM can be partially 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
vitried, but also from negatively stained or freeze-fractured
samples (Baumeister, 2002; Frank, 1992; Hoppe, 1981). Furthermore, freeze-fracture direct imaging offers interesting prospects for
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.

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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 vesicles among nanoemulsion droplets (Fig. 1B) (Norden et al., 2001).
Overall, nanoemulsion droplets are comparatively simple to distinguish 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 systems (Klang et al., 2010, 2011a,b; Teixeira et al., 2000). Teixeira
et al. reported unusual bicompartmental structures with associated oil and water phases among regular nanoemulsion droplets
(Teixeira et al., 2000). These handbag-shaped structures were
slightly 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 morphology of lecithin-based nanoemulsions both in the presence (Klang
et al., 2010) and absence (Klang et al., 2011a) of additional emulsiers or stabilisers. It was found that the lecithin mixture alone led to
the formation of a random mnagerie of rather irregularly shaped
droplets and vesicles (Fig. 9A). This corresponds well with the
respective literature. Phosphatidylcholine alone is rather unsuitable for the formation of curved surfaces (Shchipunov, 1997; Trotta
et al., 2002), and the performance of phospholipids mixtures which
largely consist of phosphatidylcholine can be improved by additional surfactants (Hoeller et al., 2009; Trotta et al., 2002; Yilmaz
and Borchert, 2005). Interestingly, a remarkable change in formulation microstructure was also achieved by addition of -cyclodextrin
as stabilising agent (Fig. 9C). The number of additional vesicular
structures decreased notably while the droplet shape appeared
more homogeneous and spherical. These ndings 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, namely
sucrose stearate S-970, revealed that this mixture of eudermic surfactants was more suitable for the formation of nano-sized droplets
than the lecithin mixture (Fig. 9B). In this case, further addition
of -cyclodextrin had no notable impact on the composition of
the 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 properties (Klang et al., 2010). Similar observations were recently made
for a nanoemulsion stabilised by 5% of sucrose stearate (Klang et al.,
2011b). As can be seen in Fig. 10B, a highly crowded microstructure
was observed despite dilution of the sample. The emulsion droplets
appeared slightly deformed, which may have been related either to
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 (cryo ATEM)

The technique of electron energy-loss spectroscopy is of great
interest for the analysis of lipid nanocarriers, especially in combination 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 specic energy loss. Thus, the elemental composition of the
particle or droplet can be determined by analysis of this specic
energy by means of a spectroscope attached to the electron microscope (Egerton, 1986). Based on this phenomenon, EELS can be
used for chemical analysis of various liquid dispersions, including
systems containing polymers.
Another technique which is widely used for the elemental analysis in combination with electron microscopy is energy dispersive
X-ray spectroscopy. When a high-energy electron beam impinges
upon a specimen, X-ray photons are produced. Characteristic Xrays have well dened 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 broadening 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-ltered transmission electron microscopy
(EFTEM) imaging are usually applied to the detection of light elements such as C, N or O, where EDX is less sensitive (Egerton, 1986).
Isaacson and Johnson (1975) provided the theoretical basis for the
prediction of detection limits in EELS by introducing the concepts of
minimum detectable mass (MDM) and minimum detectable mass
fraction (MMF). The MDM describes the smallest amount of material that can be detected in a given matrix. A small beam diameter is
desirable, thus the use of a scanning transmission electron microscope equipped with a eld emission source is preferable (Krivanek
et al., 1991; Leapman, 2003). Alternatively, the MFF represents the
smallest concentration of elements that can be measured in a given
matrix. This parameter depends primarily on the total current available in the probe. Thermal emission tips that provide large beam
currents and conventional transmission electron microscopy mode
may therefore be preferable (Kothleitner and Hofer, 1993; Zhu et 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 200 kV instrument (Riegler and Kothleitner,
2010). However, the cryo EELS detection limit is much lower, thus
rendering the analysis of nanoemulsion compositions a challenging task (Yakovlev et al., 2010). Frozen hydrated nanoemulsions
may contain only low amounts of specic 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 200 kV (in contrast to the conventionally used 80120 kV for
cryo TEM imaging) and high beam currents, which lead to complete
beam damage of the specimen structure before the adequate spectra can be recorded (Egerton, 1986). Currently there are two general
types of energy lters (post-column(GIF) and column-integrated
(omega)) which are used for EELS/EFTEM analysis (Egerton, 1986,
2005).

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 latter image appears to suffer from severe out-of-focus problems, perhaps due to insufcient freezing of the sample. The large structures indicated by the arrows cannot
be clearly identied 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: 186.41 11.06, n = 3) or (C) 2.5% of lecithin and -cyclodextrin (mean particle size: 175.82 00.47, n = 3), (B) 2.5% of sucrose stearate alone (mean particle size:
141.21 08.73, n = 3) or (D) 2.5% of sucrose stearate and -cyclodextrin (mean particle size: 144.77 10.61, n = 3). The addition of the cyclodextrin apparently promotes the
formation of spherical droplets in case of lecithin-based systems (Fig. 7A versus 7C). The arrowheads included in Image 7A indicate the presence of vesicular structures, such
as liposomes and 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.

100

V. Klang et al. / Micron 43 (2012) 85103

Fig. 10. (A) Cryo TEM micrograph of a nanoemulsion stabilised by lecithin, sucrose stearate and -cyclodextrin. The excess of surfactant induces the formation of 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 the deformation of the droplets was caused
during production or during sample preparation for cryo TEM.
Image reprinted from Klang et al. (2011b).

Cryo EELS has not yet found broader application for nanoemulsion characterisation. Firstly, frozen hydrated nanoemulsions
usually contain specic atoms like N, P, Cl or F in such low concentrations that the detection limit of cryo EELS does not allow to

obtain ne ELNES details of these elements in the spectra, which

is not the case for conventional biological samples or water mapping. 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 100 nm. The corresponding chemical composition of the analysed compound is depicted on the right hand side (C, D). Vitried specimens were examined in
a eld emission Tecnay F20 TEM operating at an accelerating voltage of 200 kV using an Oxford CT3500 cryo holder (Oxford Instruments, UK) that maintained the vitried
specimens at 160 C during sample observation. Images were recorded digitally on a cooled UltraScan CCD (Gatan, USA).

V. Klang et al. / Micron 43 (2012) 85103

of the specimen structure before adequate spectra can be record.

However, cryo EELS can be extremely useful for nanoemulsions
containing additives such as pharmaceutical substances or functionalised mineral particles (Kim et al., 2006; Oleshko et al., 1998).
The localisation of the additives can be determined without damaging the native structure of the system. Work is in progress in this
area. Fig. 11 elucidates the importance of cryo EELS for the identication of mineral particles within a lipid nanocarrier solution. In
this context, the ice particles which may appear on the sample surface during the cryo-transfer of the sample to the chamber of the
electron microscope are a particular source of misinterpretations.
Ice crystals may exhibit various different forms, including spherical,
cubic or hexagonal shapes. Unfortunately, drug crystals or mineral
particles which may be present in the investigated systems exhibit
similar shapes and cannot be clearly distinguished from ice crystals. In the presented case, cryo EELS spectra of two structures with
almost identical appearance clearly show that the former is composed of calcium carbonate (Fig. 11A, C) while the latter is an ice
(Fig. 11B, D).
8. Conclusion
Electron microscopy techniques are an essential tool to obtain
precise information about basic structural properties of colloidal
drug delivery systems such as nanoemulsions. Although both conventional SEM and TEM can be employed for this task, more
representative and artefact-free images can be obtained with cryogenic methods of sample preparation. Among those, cryo TEM is
most frequently used for nanoemulsion visualisation. Although
the availability of this technique is increasing, the comparatively
high nancial effort and the complexity of the time-consuming
sample preparation are limitations to its widespread use for quality control purposes or other everyday applications. Nevertheless,
the progress of cryo analytical techniques such as cryo EELS and
advanced techniques such as freeze-fracture direct imaging might
offer highly interesting possibilities for nanoemulsion characterisation in the future.
References
Abdulrazik, M., Tamilvanan, S., Khoury, K., Benita, S., 2001. Ocular delivery of
cyclosporin A II. Effect of submicron emulsions surface charge on ocular distribution of topical cyclosporin A. STP Pharma. Sci. 11, 427432.
Adrian, M., Dubochet, J., Fuller, S.D., Harris, J.R., 1998. Cryo-negative staining. Micron
29, 145160.
Adrian, M., Dubochet, J., Lepault, J., McDowall, A.W., 1984. Cryo Electron microscopy
of viruses. Nature (London) 308, 3236.
Akkar, A., Mueller, R.H., 2003. Formulation of intravenous carbamazepine emulsions
by SolEmuls technology. Eur. J. Pharm. Biopharm. 55, 305312.
Al-Amoudi, A., Dubochet, J., Gnaegi, H., Luthi, W., Studer, D., 2003. An oscillating
cryoknife reduces cutting-induced deformation of vitreous ultrathin sections. J.
Microsc. 212, 2633.
Anton, N., Gayet, P., Benoit, J.P., Saulnier, P., 2007. Nano-emulsions and nanocapsules
by the PIT method: an investigation on the role of the temperature cycling on
the emulsion phase inversion. Int. J. Pharm. 344, 4452.
Anton, N., Vandamme, T.F., 2011. Nano-emulsions and micro-emulsions: clarications of the critical differences. Pharm. Res. 28, 978985.
Araujo, F.A., Kelmann, R.G., Araujo, B.V., Finatto, R.B., Teixeira, H.F., Koester, L.S.,
2011. Development and characterization of parenteral nanoemulsions containing thalidomide. Eur. J. Pharm. Sci. 42, 238245.
Ardenne, M., 1938a. Das Elektronen-Rastermikroskop.Theoretische Grundlagen. Z.
Phys. 109, 553572.
Ardenne, M., 1938b. Das Elektronen-Rastermikroskop. Praktische Ausfuehrung. Z.
Tech. Phys. 19, 407416.
Bachmann, L., Mayer, E., 1987. Physics of water and Ice: implications for cryoxation. In: Steinbrecht, R., Zierold, K. (Eds.), Cryotechniques in Biological Electron
Microscopy. Springer Verlag, Berlin, pp. 331.
Bachmann, L., Schmitt-Fumian, W.W., 1973. Spray-freeze-etching of dissolved
macromolecules emulsions and subcellular components. In: Benetti, E.L., Favard,
P. (Eds.), Freeze-etching: Techniques and Applications. Soc. Franc. Microsc. Electron, Paris, pp. 6372.
Bachmann, L., Talmon, Y., 1984. Cryomicroscopy of liquid and semiliquid specimens:
direct imaging versus replication. Ultramicroscopy 14, 211218.

101

Baker, M.T., Naguib, M., 2005. Propofol: the challenges of formulation. Anesthesiology 103, 860876.
Bali, V., Ali, M., Ali, J., 2010. Study of surfactant combinations and development of
a novel nanoemulsion for minimising variations in bioavailability of ezetimibe.
Colloids Surf. B Biointerfaces 76, 410420.
Baspinar, Y., 2009. Nano- and microemulsions for topical application of poorly soluble immunosuppressives. PhD Thesis. Free University Berlin, Germany.
Baumeister, W., 2002. Electron tomography: towards visualizing the molecular
organization of the cytoplasm. Curr. Opin. Struct. Biol. 12, 679684.
Belkoura, L., Stubenrauch, C., Strey, R., 2004. Freeze fracture direct imaging: a new
freeze fracture method for specimen preparation in cryo-transmission electron
microscopy. Langmuir 20, 43914399.
Benita, S., Levy, M.Y., 1993. Submicron emulsions as colloidal drug carriers for
intravenous administration: comprehensive physicochemical characterization.
J. Pharm. Sci. 82, 10691079.
Benita, S., Magalhaes, N.S., Cave, G., Seiller, M., 1991. The stability and in vitro release
kinetics of a clobride emulsion. Int. J. Pharm. 76, 225237.
Bilbao-Sainz, C., Avena-Bustillos, R.J., Wood, D.F., Williams, T.G., McHugh, T.H., 2010.
Nanoemulsions prepared by a low-energy emulsication method applied to
edible lms. J. Agric. Food Chem. 58, 1193211938.
Bogner, A., Jouneau, P.H., Thollet, G., Basset, D., Gauthier, C., 2007. A history of scanning electron microscopy developments: towards wet-STEM imaging. Micron
38, 390401.
Bogner, A., Thollet, G., Basset, D., Jouneau, P.H., Gauthier, C., 2005. Wet STEM: a
new development in environmental SEM for imaging nano-objects included in
a liquid phase. Ultramicroscopy 104, 290301.
Bouchemal, K., Briancon, S., Perrier, E., Fessi, H., 2004. Nano-emulsion formulation
using spontaneous emulsication: solvent, oil and surfactant optimisation. Int.
J. Pharm. 280, 241251.
Bouchet-Marquis, C., Hoenger, A., 2011. Cryo-electron tomography on vitried sections: a critical analysis of benets and limitations for structural cell biology.
Micron 42, 152162.
Bozzola, J.J., Russell, L.D., 1999. Electron Microscopy. Jones & Bartlett Learning Publishers, Sudbury, Massachusetts.
Brandl, M., Drechsler, M., Bachmann, D., Bauer, K.H., 1997. Morphology of semisolid
aqueous phosphatidylcholine dispersions, a freeze fracture electron microscopy
study. Chem. Phys. Lipids 87, 6572.
Brenner, S., Horne, R.W., 1959. A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 103110.
Buchheim, W., 1982. Aspects of sample preparation for freeze-fracture/freeze-etch
studies of proteins and lipids in food systems. Food Microstruct. 1, 189208.
Bullock, G.R., 1984. The current status of xation for electron microscopy: a review.
J. Microsc. 133, 115.
Burapapadh, K., Kumpugdee-Vollrath, M., Chantasart, D., Sriamornsak, P., 2010.
Fabrication of pectin-based nanoemulsions loaded with itraconazole for pharmaceutical application. Carb. Polym. 82, 384393.
Burauer, S., Belkoura, L., Stubenrauch, C., Strey, R., 2003. Bicontinuous microemulsions revisited: a new approach to freeze fracture electron microscopy (FFEM).
Colloids Surf. A: Physicochem. Eng. Aspects 228, 159170.
Calder, P.C., Jensen, G.L., Koletzko, B.V., Singer, P., Wanten, G.J., 2010. Lipid emulsions
in parenteral nutrition of intensive care patients: current thinking and future
directions. Intensive Care Med. 36, 735749.
Calderilla-Fajardo, S.B., Cazares-Delgadillo, J., Villalobos-Garcia, R., QuintanarGuerrero, D., Ganem-Quintanar, A., Robles, R., 2006. Inuence of sucrose esters
on the in vivo percutaneous penetration of octyl methoxycinnamate formulated in nanocapsules, nanoemulsion, and emulsion. Drug Dev. Ind. Pharm. 32,
107113.

Corts-Munoz,
M., Chevalier-Lucia, D., Dumay, E., 2009. Characteristics of submicron
emulsions prepared by ultra-high pressure homogenisation: effect of chilled or
frozen storage. Food Hydrocolloids 23, 640654.
Daniels, R., 2001. Galenic Principles of Modern Skin Care Products, Skin Care Forum.
Society for Dermopharmacy.
Danino, D., Talmon, Y., 2000. Cryo-transmission electron microscopy. In: Baskin,
A., Norde, W. (Eds.), Physical Chemistry of Biological Interfaces. Marcel Dekker,
New York, pp. 799821.
Denkov, N.D., Yoshimura, H., Nagayama, K., Kouyama, T., 1996. Nanoparticle arrays
in freely suspended vitried lms. Phys. Rev. Lett. 76, 23542357.
Desai, A., Vyas, T., Amiji, M., 2008. Cytotoxicity and apoptosis enhancement in brain
tumor cells upon coadministration of paclitaxel and ceramide in nanoemulsion
formulations. J. Pharm. Sci. 97, 27452756.
do Amaral, M., Bogner, A., Gauthier, C., Thollet, G., Jouneau, P.-H., Cavaille, J.Y., Asua, J.M., 2005. Novel experimental technique for the determination of
monomer droplet size distribution in miniemulsion. Macromol. Rapid Commun.
26, 365368.
Drzymala, J., Krajczyk, L., 1985. Electron microscopic observation of ne polydispersed emulsion. J. Colloids Surf. 15, 249253.
Du Plessis, J., Tiedt, L.R., Kotze, A.F., Van Wyk, C.J., Ackermann, C., 1987. A transmission electron microscope method for determination of droplet size in parenteral
fat emulsions using negative staining. Int. J. Pharm. 46, 177178.
Du Plessis, J., Tiedt, L.R., Van Wyk, C.J., Ackermann, C., 1986. A new transmission
electron microscope method for the determination of particle size in parenteral
fat emulsions. Int. J. Pharm. 34, 173174.
Dubochet, J., Adrian, M., Chang, J.J., Lepault, J., McDowall, A.W., 1987. Cryoelectron microscopy of vitried specimens. In: Steinbrecht, R., Zierold, K. (Eds.),
Cryotechniques in Biological Electron Microscopy. Springer Verlag, Berlin, pp.
114131.

102

V. Klang et al. / Micron 43 (2012) 85103

Dubochet, J., Chang, J.-J., Freeman, R., Lepault, J., McDowall, A.W., 1982. Frozen aqueous suspensions. Ultramicroscopy 10, 5562.
Dubochet, J., McDowall, A., Menge, B., Schmid, E.N., Lickfeld, K.G., 1983. Electron
microscopy of frozen-hydrated bacteria. J. Bacteriol. 155, 381390.
Egelhaaf, S.U., Schurtenberger, P., Mueller, M., 2000. New controlled environment
vitrication system for cryo-transmission electron microscopy: design and
application to surfactant solutions. J. Microsc. 200, 128139.
Egerton, R.F., 1986. Electron Energy-loss Spectroscopy in the Electron microscope.
Plenum Press, New York, USA.
Egerton, R.F., 2005. Physical Principles of Electron Microscopy: An Introduction to
TEM, SEM and AEM. Springer, New York, USA.
Eskandar, N.G., Simovic, S., Prestidge, C.A., 2009. Nanoparticle coated submicron
emulsions: sustained in-vitro release and improved dermal delivery of all-transretinol. Pharm. Res. 26, 17641775.
Fang, J.Y., Leu, Y.L., Chang, C.C., Lin, C.H., Tsai, Y.H., 2004. Lipid nano/submicron
emulsions as vehicles for topical urbiprofen delivery. Drug Deliv. 11,
97105.
Fox, C.B., 2009. Squalene emulsions for parenteral vaccine and drug delivery.
Molecules 14, 32863312.
Frank, W., 1992. Electron Tomography: Three-Dimensional Imaging with the Transmission Electron Microscope. Plenum Press, New York, USA.
Friedrich, H., Frederik, P.M., de With, G., Sommerdijk, N.A., 2010. Imaging of selfassembled structures: interpretation of TEM and cryo-TEM images. Angew.
Chem. Int. Ed. Engl. 49, 78507858.
Fukami, A., Adachi, K., 1965. A new method of preparation of a self-perforated micro
plastic grid and its application. J. Electron Microsc. (Tokyo) 14, 112118.
Ganta, S., Amiji, M., 2009. Coadministration of Paclitaxel and curcumin in nanoemulsion formulations to overcome multidrug resistance in tumor cells. Mol. Pharm.
6, 928939.
Ganta, S., Paxton, J.W., Baguley, B.C., Garg, S., 2008. Pharmacokinetics and pharmacodynamics of chlorambucil delivered in parenteral emulsion. Int. J. Pharm. 360,
115121.
Goldstein, G.I., Newbury, D.E., Echlin, P., Joy, D.C., Fiori, C., Lifshin, E., 1981. Scanning
Electron Microscopy and X-ray Microanalysis. Plenum Press, New York, USA.
Groves, M.J., Wineberg, M., Brian, A.P.R., 1985. The presence of liposomal
material in phosphatide stabilized emulsions. J. Dispersion Sci. Technol. 2,
237243.
Gulik-Krzywicki, T., 1997. Freeze-fracture transmission electron microscopy. Curr.
Opin. Colloid. Interface Sci. 2, 137144.
Hamilton-Attwell, V.L., Du Plessis, J., Van Wyk, C.J., 1987. A new scanning electron
microscope (SEM) method for the determination of particle size in parenteral
fat emulsions. J. Microsc. 145, 347349.
Harris, J.R., 2008. Negative staining across holes: application to bril and tubular
structures. Micron 39, 168176.
Hatanaka, J., Chikamori, H., Sato, H., Uchida, S., Debari, K., Onoue, S., Yamada, S., 2010.
Physicochemical and pharmacological characterization of alpha-tocopherolloaded nano-emulsion system. Int. J. Pharm. 396, 188193.
Hatziantoniou, S., Deli, G., Nikas, Y., Demetzos, C., Papaioannou, G.T., 2007. Scanning electron microscopy study on nanoemulsions and solid lipid nanoparticles
containing high amounts of ceramides. Micron 38, 819823.
Hayat, M.A., 2000. Principles and Techniques of Electron Microscopy: Biological
Applications. Cambridge University Press, Cambridge, UK.
Hoeller, S., Sperger, A., Valenta, C., 2009. Lecithin based nanoemulsions: a comparative study of the inuence of non-ionic surfactants and the cationic
phytosphingosine on physicochemical behaviour and skin permeation. Int. J.
Pharm. 370, 181186.
Hoppe, W., 1981. Three-dimensional electron microscopy. Annu. Rev. Biophys. Bioeng. 10, 563592.
Ibrahim, S.S., Awad, G.A., Geneidi, A., Mortada, N.D., 2009. Comparative effects of different cosurfactants on sterile prednisolone acetate ocular submicron emulsions
stability and release. Colloids Surf. B Biointerfaces 69, 225231.
Ilan, E., Amselem, S., Weisspapir, M., Schwarz, J., Yogev, A., Zawoznik, E., Friedman, D.,
1996. Improved oral delivery of desmopressin via a novel vehicle: mucoadhesive
submicron emulsion. Pharm. Res. 13, 10831087.
Isaacson, M., Johnson, D., 1975. The microanalysis of light elements using transmitted energy loss electrons. Ultramicroscopy 1, 3352.
Jahanzad, F., Josephides, J., Mansourian, A., Sajjadi, S., 2010. Dynamics of transitional
phase inversion emulsication: effect of addition time on the type of inversion
and drop size. Ind. Eng. Chem. Res. 49, 76317637.
Jores, K., 2004a. Lipid nanodispersions as drug carrier systemsa physicochemical characterization. PhD Thesis. Martin-Luther-University, Halle-Wittenberg,
Germany.
Jores, K., Mehnert, W., Drechsler, M., Bunjes, H., Johann, C., Maeder, K., 2004b.
Investigations on the structure of solid lipid nanoparticles (SLN) and oil-loaded
solid lipid nanoparticles by photon correlation spectroscopy, eld-ow fractionation and transmission electron microscopy. J. Control. Release 95, 217
227.
Jumaa, M., Mueller, B.W., 1998. The effect of oil components and homogenization
conditions on the physicochemical properties and stability of parenteral fat
emulsions. Int. J. Pharm. 163, 8189.
Kelmann, R.G., Kuminek, G., Teixeira, H.F., Koester, L.S., 2007. Carbamazepine parenteral nanoemulsions prepared by spontaneous emulsication process. Int. J.
Pharm. 342, 231239.
Kesselman, E., Talmon, Y., Bang, B., Abbas, S., Li, Z., Lodge, T.P., 2005. Cryogenic
transmission electron microscopy imaging of vesicles formed by a polystyrenepolyisoprene diblock copolymer. Maromolecules 38, 67796781.

Kim, G., Sousa, A., Meyers, D., Shope, M., Libera, M., 2006. Diffuse polymer interfaces
in lobed nanoemulsions preserved in aqueous media. J. Am. Chem. Soc. 128,
65706571.
Klang, S.H., Baszkinb, A., Benita, S., 1996. The stability of piroxicam incorporated in a
positively-charged submicron emulsion for ocular administration. Int. J. Pharm.
132, 3344.
Klang, V., Matsko, N., Raupach, K., El-Hagin, N., Valenta, C., 2011a. Development
of sucrose stearate-based nanoemulsions and optimisation through gammacyclodextrin. Eur. J. Pharm. Biopharm..
Klang, V., Matsko, N., Zimmermann, A.M., Vojnikovic, E., Valenta, C., 2010. Enhancement of stability and skin permeation by sucrose stearate and cyclodextrins in
progesterone nanoemulsions. Int. J. Pharm. 393, 152160.
Klang, V., Schwarz, J.C., Matsko, N., Rezvani, E., El-Hagin, N., Wirth, M., Valenta, C.,
2011b. Semi-solid sucrose stearate-based emulsions as dermal drug delivery
systems. Pharmaceutics 3, 275306.
Klang, V., Valenta, C., 2011c. Lecithin-based nanoemulsions. J. Drug Del. Sci. Tech.
21, 5576.
Knoll, M., 1935. Auadepotentiel und Sekundremission elektronenbestrahlter Krper. Z. Tech. Phys. 16, 467475.
Koning, R.I., Koster, A.J., 2009. Cryo-electron tomography in biology and medicine.
Ann. Anat. 191, 427445.
Kothleitner, G., Hofer, F., 1993. Quantitative microanalysis using electron-loss
spectrometry I. Li and Be in oxides. Microsc. Microanal. Microstruct. 4,
539560.
Krivanek, O.L., Mory, C., Tence, M., Colliex, C., 1991. EELS quantication near the
single-atom detection level. Microsc. Microanal. Microstruct. 2, 257267.
Kuntsche, J., Horst, J.C., Bunjes, H., 2011. Cryogenic transmission electron microscopy
(cryo-TEM) for studying the morphology of colloidal drug delivery systems. Int.
J. Pharm., doi:10.1016/j.ijpharm.2011.02.001.
Kuntsche, J., Klaus, K., Steiniger, F., 2009. Size determinations of colloidal fat emulsions: a comparative study. J. Biomed. Nanotechnol. 5, 384395.
Leapman, R.D., 2003. Detecting single atoms of calcium and iron in biological structures by electron energy-loss spectrum-imaging. J. Microsc. 210, 515.
Lepault, J., Booy, F.P., Dubochet, J., 1983. Electron microscopy of frozen biological
suspensions. J. Microsc. (Oxford) 129, 89102.
Liu, C.H., Yu, S.Y., 2010. Cationic nanoemulsions as non-viral vectors for plasmid
DNA delivery. Colloids Surf. B Biointerfaces 79, 509515.
Marxer, E.E., Brussler, J., Becker, A., Schummelfeder, J., Schubert, R., Nimsky, C.,
Bakowsky, U., 2011. Development and characterization of new nanoscaled ultrasound active lipid dispersions as contrast agents. Eur. J. Pharm. Biopharm. 77,
430437.
Masaki, Y., Tanaka, M., Nishikawa, T., 2003. Physicochemical compatibility of
propofol-lidocaine mixture. Anesth. Analg. 97, 16461651.
Mason, T.G., Wilking, J.N., Meleson, K., Chang, C.B., Graves, S.M., 2006. Nanoemulsions: formation, structure, and physical properties. J. Phys. Condens. Matter 18,
R635R666.
Massover, W.H., 2008. On the experimental use of light metal salts for negative
staining. Microsc. Microanal. 14, 126137.
Massover, W.H., 2011. New and unconventional approaches for advancing
resolution in biological transmission electron microscopy by improving macromolecular specimen preparation and preservation. Micron 42, 141151.
McDowall, A.W., Chang, J.-J., Freeman, R., Lepault, J., Walter, C.A., Dubochet, J., 1983.
Electron microscopy of frozen-hydrated sections of vitreous ice and vitried
biological samples. J. Microsc. (Oxford) 131, 19.
Menold, R., Luttge, B., Kaiser, W., Schmidt, A., 1972. Gefrierbruchtechnik zur
Untersuchung von Suspensionen und Emulsionen. Chem. Ing. Tech. 44, 1226
1232.
Mohammed, A.R., Weston, N., Coombes, A.G.A., Fitzgerald, M., Perrie, Y., 2004. Liposome formulation of poorly water soluble drugs: optimisation of drug loading
and ESEM analysis of stability. Int. J. Pharm. 285, 2334.
Moor, H., 1987. Theory and practice of high-pressure freezing. In: Steinbrecht, R.A.,
Zierold, K. (Eds.), Cryo-techniques in Biological Electron Microscopy. SpringerVerlag, Berlin, Heidelberg, pp. 175191.
Mou, D., Chen, H., Du, D., Mao, C., Wan, J., Xu, H., Yang, X., 2008. Hydrogel-thickened
nanoemulsion system for topical delivery of lipophilic drugs. Int. J. Pharm. 353,
270276.
Mueller, M., 1991. Cryotechniques in biologycal electron microscopy. Encyclopedia
Hum. Biol. 2, 721730.
Mueller, M., Moor, H., 1984. Cryoxation of thick specimens by high pressure freezing. In: Mueller, M., Becker, R.P., Boyde, A., Wolosewick, J.J. (Eds.), The Science
of Biological Specimen Preparation. SEM, AMF OHare, Chicago, Illinois, pp.
131138.
Nam, Y.S., Kim, J.W., Shim, J., Han, S.H., Kim, H.K., 2010. Nanosized emulsions stabilized by semisolid polymer interphase. Langmuir 26, 1303813043.
Norden, T.P., Siekmann, B., Lundquist, S., Malmsten, M., 2001. Physicochemical
characterisation of a drug-containing phospholipid-stabilised o/w emulsion for
intravenous administration. Eur. J. Pharm. Sci. 13, 393401.
Oleshko, V.P., Gijbels, R.H., Van Daele, A.J., Jacob, W.A., Xu, Y.E., Wang, S.E., Park, I.Y.,
Kang, T.S., 1998. Combined characterization of composite tabular silver halide
microcrystals by cryo-EFTEM/EELS and cryo-STEM/EDX techniques. Microsc.
Res. Tech. 42, 108122.
Orlova, E.V., Sherman, M.B., Chiu, W., Mowri, H., Smith, L.C., Gotto Jr., A.M., 1999.
Three-dimensional structure of low density lipoproteins by electron cryomicroscopy. Proc. Natl. Acad. Sci. U.S.A. 96, 84208425.
Pathan, I.B., Setty, C.M., 2011. Enhancement of transdermal delivery of tamoxifen
citrate using nanoemulsion vehicle. Int. J. Pharm. Tech. Res. 3, 287297.

V. Klang et al. / Micron 43 (2012) 85103

Patravale, V.B., Mandawgade, S.D., 2008. Novel cosmetic delivery systems: an application update. Int. J. Cosmet. Sci. 30, 1933.
Piemi, M.P., Korner, D., Benita, S., Marty, J.-P., 1999. Positively and negatively charged
submicron emulsions for enhanced topical delivery of antifungal drugs. J. Control. Release 58, 177187.
Pinnamaneni, S., Das, N.G., Das, S.K., 2003. Comparison of oil-in-water emulsions manufactured by microuidization and homogenization. Pharmazie 58,
554558.
Preetz, C., Hauser, A., Hause, G., Kramer, A., Mader, K., 2010. Application of atomic
force microscopy and ultrasonic resonator technology on nanoscale: distinction
of nanoemulsions from nanocapsules. Eur. J. Pharm. Sci. 39, 141151.
Rao, J., McClements, D.J., 2010. Stabilization of phase inversion temperature
nanoemulsions by surfactant displacement. J. Agric. Food Chem. 58, 70597066.
Reichelt, R., Koenig, T., Wangermann, G., 1977. Preparation of microgrids as specimen supports for high resolution electron microscopy. Micron 8, 2931.
Reimer, L., 1993. Image Formation in Low-Voltage Scanning Electron Microscopy.
SPIE Optical Engineering Press, Bellingham & Washington.
Riegler, K., Kothleitner, G., 2010. EELS detection limits revisited: Rubya case study.
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, and CLSM 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. Structural investigations on nanoemulsions,
solid lipid nanoparticles and nanostructured lipid carriers by cryo-eld emission 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 nanoemulsion
by low-energy emulsication methods. Int. J. Refrigeration 33, 16121624.
Scheuing, D.R., 1990. Fourier Transform Infrared Spectroscopy in Colloid and Interface Science. American Chemical Society, Washington, DC, pp. 121.
Schmidtgen, M.C., Drechsler, M., Lasch, J., Schubert, R., 1998. Energy-ltered cryotransmission electron microscopy of liposomes prepared from human stratum
corneum lipids. J. Microsc. 191, 177186.
Schwarz, J.S., Weisspapir, M.R., Friedman, D.I., 1995. Enhanced transdermal delivery of diazepam by submicron emulsion (SME) creams. Pharm. Res. 12, 687
692.
Seki, J., Sonoke, S., Saheki, A., Fukui, H., Sasaki, H., Mayumi, T., 2004. A nanometer
lipid emulsion, lipid nano-sphere (LNS), as a parenteral drug carrier for passive
drug targeting. Int. J. Pharm. 273, 7583.
Severs, N., Robenek, H., 2008. Freeze-fracture cytochemistry in cell biology. Methods
Cell Biol. 88, 181204.
Severs, N.J., 2007. Freeze-fracture electron microscopy. Nat. Protocols 2, 547576.
Shaq, S., Shakeel, F., Talegaonkar, S., Ahmad, F.J., Khar, R.K., Ali, M., 2007. Design
and development of oral oil in water ramipril nanoemulsion formulation: in vitro
and in vivo assessment. J. Biomed. Nanotech. 3, 2844.
Shakeel, F., Baboota, S., Ahuja, A., Ali, J., Aqil, M., Shaq, S., 2007. Nanoemulsions as
vehicles for transdermal delivery of aceclofenac. AAPS Pharm. 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 the
isoavone genistein. Pharmazie 64, 3235.
Singh, K.K., Vingkar, S.K., 2008. Formulation, antimalarial activity and biodistribution
of oral lipid nanoemulsion of primaquine. Int. J. Pharm. 347, 136143.

103

Sitte, H., Edelmann, L., Neumann, K., 1987. Cryoxation without pretreatment at
ambient pressure. In: Steinbrecht, R., Zierold, K. (Eds.), Cryotechniques in Biological Electron Microscopy. Springer, Berlin, Heidelberg, Germany, pp. p.87p.113.
Sjoeblom, E., Friberg, S., 1978. Light-scattering and electron microscopy determinations 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. Nanoemulsions. 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 variables and scale up. J. Colloid Interface Sci. 344, 417423.
Sonneville-Aubrun, O., Simonnet, J.T., LAlloret, F., 2004. Nanoemulsions: a new vehicle for skincare products. Adv. Colloid Interface Sci. 108109, 145149.
Steinbrecht, R.A., Zierold, K., 1987. Cryo Techniques in Biological Electron
Microscopy. Springer, Berlin, Heidelberg, Germany.
Sutton, S.R., Newville, M., Rivers, M.L., 2003. Synchrotron X-ray microscope analysis.
Geophys. Res. Abstracts 5, 13260.
Tadros, T., Izquierdo, P., Esquena, J., Solans, C., 2004. Formation and stability of nanoemulsions. Adv. Colloid Interface Sci. 108109, 303318.
Takegami, S., Kitamura, K., Kawada, H., Matsumoto, Y., Kitade, T., Ishida, H., Nagata,
C., 2008. Preparation and characterization of a new lipid nano-emulsion containing two cosurfactants, sodium palmitate for droplet size reduction and sucrose
palmitate for stability enhancement. Chem. Pharm. Bull. (Tokyo) 56, 10971102.
Talmon, Y., 1999. Cryogenic temperature transmission electron microscopy in the
study of surfactant systems. In: Binks, B.P.E. (Ed.), Modern Characterization
Methods of Surfactant Systems. Marcel Dekker, New York, p. p147.
Tamilvanan, S., Benita, S., 2004. The potential of lipid emulsion for ocular delivery
of lipophilic drugs. Eur. J. Pharm. Biopharm. 58, 357368.
Teixeira, H., Dubernet, C., Rosilio, V., Benita, S., Lepault, J., Erk, I., Couvreur, P., 2000.
New bicompartmental structures are observed when stearylamine is mixed with
triglyceride emulsions. Pharm. Res. 17, 13291332.
Trotta, M., Pattarino, F., Ignoni, T., 2002. Stability of drug-carrier emulsions containing phosphatidylcholine mixtures. Eur. J. Pharm. Biopharm. 53, 203208.
Wabel, C., 1998. Inuence of lecithin on structure and stability of parenteral
fat emulsions. PhD Thesis. Friedrich-Alexander-University, Erlangen-Nrnberg,
Germany.
Wang, X., Jiang, Y., Wang, Y., Huang, M., Ho, C., Huang, Q., 2008. Enhancing antiinammatory activity of curcumin through O/W nanoemulsions. Food Chem.,
108.
Welin-Berger, K., Bergenstahl, B., 2000. Inhibition of Ostwald ripening in local anesthetic emulsions by using hydrophobic excipients in the disperse phase. Int. J.
Pharm. 200, 249260.
Whittinghill, J.M., Norton, J., Proctor, A., 1999. A fourier transform infrared spectroscopy study on the effect of temperature on soy lecithin-stabilized emulsions.
J. Am. Oil Chem. Soc. 76, 13931398.
Yakovlev, S., Misra, M., Shi, S., Firlar, E., Libera, M., 2010. Quantitative nanoscale water
mapping in frozen-hydrated skin by low-loss electron energy-loss spectroscopy.
Ultramicroscopy 110, 866876.
Yan, Y.L., Zhang, N.S., Qu, C.T., Liu, L., 2005. Microstructure of colloidal liquid aphrons
(CLAs) by freeze fracture transmission electron microscopy (FF-TEM). Colloids
Surf. A 264, 139146.
Yilmaz, E., Borchert, H.H., 2005. Design of a phytosphingosine-containing, positivelycharged nanoemulsion as a colloidal carrier system for dermal application of
ceramides. Eur. J. Pharm. Biopharm. 60, 9198.
Zeevi, A., Klang, S., Alard, V., Brossard, F., Benita, S., 1994. The design and characterization of a positively charged submicron emulsion containing a sunscreen
agent. Int. J. Pharm. 108, 5768.
Zhou, H., Yue, Y., Liu, G., Li, Y., Zhang, J., Gong, Q., Yan, Z., Duan, M., 2010. Preparation
and characterization of a lecithin nanoemulsion as a topical delivery system.
Nanoscale Res. Lett. 5, 224230.
Zhu, Y., Egerton, R.F., Malac, M., 2001. Concentration limits for the measurement of
boron by electron energy-loss spectroscopy and electron-spectroscopic imaging. Ultramicroscopy 87, 135145.