Characterization at the Micro- and Nano-scale by SEM and SFM
- Fig. 1: Red-green stereo anaglyph of FESEM secondary electron micrographs of the PNIPAAm gel surface in the swollen state at RT at 2 kV acceleration Voltage. The stereo pair has been acquired by tilting the sample holder at ±6°C. Courtesy of Hawkes P.W., Spence J.C.H. (2007). The Science of Microscopy, vol 1, p. 235. With kind permission of Springer Science and Business Media. .
- Fig. 2: Scheme of the stimulated response of a “smart” hydrogel
- Fig. 3: The diagram shows the indentation versus the load at six different temperatures. The experimental data are represented by symbols. Each data set has been simulated using the Hertz theory for a sphere indenting a flat surface (solid lines). The Hertz theory has been used to determine the Young’s modulus at each temperature. The corresponding log-log presentation is given as insert (the graphs for 15°C, 20°C and 25°C are not shown for the sake of clarity). Reprinted with permission from J Phys Chem B 106:2861–2866, Copyright (2002) ACS.
- Fig. 4: Schemes of acting forces induced by the surface tension on the model structures sphere (a) and beam (b). Abbreviations: R – radius, γ – surface tension, F – force, - contact angle of the water meniscus, L – length of the beam, h – width of the beam, t – thickness of the beam, s – bending deflection.
- Fig.5: PVME hydrogels filled with BaTiO3 (a, b), Ni (c, d), PVDF (e, f) in the swollen (left) and deswollen state (right). The images are FESEM secondary electron micrographs recorded at 10 kV acceleration voltage. The bars correspond to 1 µm (a-d) and 0.2 µm (e, f), respectively. The figure is a courtesy of D. Theiss et al. Reprinted with permission from J Appl Polym Sci 98:2253–2265, Copyright (2005) Wiley Periodicals, Inc. .
- Table 1: Data used for calculation and estimated deformations for two simple cases. *A contact angle = 89.4°C was used for the estimate.
Hydrogels are water swollen, cross-linked polymeric structures, produced by the simple reaction of one or more monomers or by association of bonds such as hydrogen bonds and strong van der Waals interactions between chains. A large amount of today's research is focused on probably the most interesting among them, the so-called "smart" hydrogels. A representative of this class of hydrogels is a polymer system with a defined phase transition capable to abruptly swell to many times its original size or to collapse into a compact mass when stimulated externally . The following studies demonstrate some of the advantages and the strength of the complementary employment of Field Emission Scanning Electron Microscopy (FESEM) and Scanning Force Microscopy (SFM) in the characterization of hydrogels: Highly resolved structural information by FESEM are complemented by SFM using micrometer-sized probing spheres for quantitative measurements of the local micromechanical.
Unique Properties of Smart Hydrogels
Smart hydrogels react in response to an external stimulus in a manner similar to many living organisms rather than to not living organic matter. Some systems are reported to sense environmental changes such as an electric field, pH, temperature, salt content and solvent composition. In response to these external stimuli they undergo a reversible phase transition, leading to changes in their macroscopic size, optical appearance or elastic modulus (fig. 2). The unique properties of these soft materials have led to serious efforts in medical research and bioengineering, especially regarding the development of self-regulating drug delivery systems, on-off switches for enzymatic reactions, biosensors, purification matrices and biomedical actuators. For a detailed view on modern applications of smart hydrogels in medicine and biology see Peppas et al. .
The extremely large range of water uptake of these hydrogels (up to 103 times of their dry mass) on one hand and their distinct softness (Young´s modulus may be as small as some tens of kPa) on the other hand constitute real challenges for the characterization of their local structure, the caging of nano-scaled particles, and some micromechanical properties.
Micro- and Nano-structure
The smart hydrogel PNIPAAm [poly-(N-isopropylacrylamide)] was chosen as sample, because it has been extensively studied during the past years.
PNIPAAm undergoes a reversible volume phase transition in response to external temperature changes. Inserted in water, the polymer matrix strongly shrinks as the temperature increases above the lower critical solution temperature (LCST), which is about 33°C. PNIPAAm hydrogel can be synthesized by free radical polymerisation (for the recipe see, e.g., Suzuki et al. .
SFM on the air-dried PNIPAAm at room temperature (RT) provides a rather smooth gel surface with nm-sized protrusions, similar to what is seen by FESEM. In addition to laterally resolved surface structural details, the SFM images provided quantitative topographic information: e.g., the height of the roundish protrusions varied from 5 nm to 15 nm. These protrusions may indicate regions with higher polymer density.
In case of the water-swollen PNIPAAm surface we only received "cloudy" irreproducible topographs, despite using less invasive imaging modes. It was found that the probing high aspect ratio SFM tips can easily penetrate the swollen gel surface even though small forces in the range of 1 nN were applied. To reduce the locally acting force during scanning, we replaced high aspect SFM tips by smooth glass spheres with diameters around 5 µm. Unfortunately, the avoidance of surface penetration using glass spheres as probes was accompanied by a significant reduction of lateral resolution during surface imaging.
In contrast, FESEM combined with state-of-the-art cryo-preparation techniques, is a very powerful tool to study the micro- and nanostructure of PNIPAAm hydrogels below and above the LCST. FESEM images reveal a characteristic sponge-like structure: Small cavities are confined by thin perforated membranes. Comparing the images of the deswollen state (35°C) with those of the swollen state (RT) reveals a noticeable difference. The mean cavity diameter remains not the same. A rough estimation leads to typical sizes of ~20 nm for the deswollen state and ~40 nm for the swollen state. Thus, the mean cavity diameter changes by a factor of ~2 and the mean cavity volume by ~8 when passing the phase transition temperature.
Although the SEM measurements satisfy in a precise determination of pore sizes, the high resolution SEM gains increasing importance in the determination of exact topography in a more general perspective. As SFM is mostly restricted to height variations less than ~ 10 µm and a scanning field smaller than ~ 100 x 100 µm², SEM enables the operator to visualize fields of bigger sizes and to locate particular details on the surface readily. Stereoscopic SEM images can be used to obtain information about topography and spatial structure as shown by a stereoscopic FESEM image of the PNIPAAm surface in figure 1.
SFM is perfectly suited to determine local mechanical properties such as Young's modul as it uses a force sensor to apply and accurately measure forces on the submicrometer scale. Local elastic properties of any sample surface can be obtained under ambient conditions in air and in water with high precision. In principle, these properties are revealed by force curves showing the indentation of the surface as the probe loads the sample. As mentioned before, the swollen hydrogel surface is as fragile to be easily penetrate the applying small loads. Therefore, the force sensing probe was modified by attaching an approximately 5 µm sized silica sphere to its extremity. The exact sphere diameter was measured individually from FESEM micrographs of the cantilever prior to its employment in the SFM experiment.
In figure 3 the measured indentation is plotted versus the loading force. Data sets are shown for six different temperatures. To do so, the forces were calculated by multiplying the cantilever deflection by the cantilever spring constant. Each data set was fitted to the Hertz model, according to the geometrical set-up of a smooth sphere and a flat surface. Thus, the local Young's modulus of a PNIPAAm gel surface is strongly affected by the phase transition occuring around 33°C. The local stiffness is more than 100 times higher in the deswollen state at 35°C than in the swollen state at 10°C .
Does ESEM Allow to Study Hydrogels?
The so-called Environmental Scanning Electron Microscope (ESEM) allows wet matter to be studied at low vacuum (up to ~ 50 torr) since about two decades. Water vapour is a common gaseous environment of wet specimens. The ionization of the gas reduces the electric charge build-up that occurs on insulating specimens being probed, thus, special sample preparation, especially conductive coating, is not needed. ESEM seems a suitable choice for studying the structure of water-swollen materials. However, since most of the wet hydrogels are mechanically sensitive structures, the polymer matrix can be deformed or may even collapse due to the effect of surface tension of water when decreasing the water level to make the hydrogel-structure accessible for ESEM-imaging. Surface tension induced effects of water during reducing the water level (represents gradual drying) was judged by calculating the acting forces for very small spheres and very thin beams, which represent simple structures and fine structural elements of more complex structures, respectively.
In reference to the schemes in figure 4 the maximum induced force Fmax is applied by the surface tension if the contact angle of the water meniscus with the surface of the structure = 0. Assuming the surface tension of water at 21°C is γ = 0.726 mN/cm, the maximum induced force amounts about 2.3 nN in case of a sphere with a diameter of 10 nm and it amounts about 15 nN in case of a thin beam of 100 nm. The surface tension induced forces for structures others than spheres and beams with comparable size are in the same order of magnitude.
Some few numerical estimates are summarized in table 1, based on data obtained from FESEM micrographs (e.g. fig. 1; nano-scale hydrogel structures) and from ESEM micrographs (Zhao et al. ; micro-scale hydrogel structures).
The estimates proved ESEM being critical in case of hydrogel imaging in the water-swollen state, since features sticking out of the water can be artificially deformed by the surface tension. For a detailed discussion see .
FESEM of Filled Hydrogels
The capability to swiftly swell and shrink is a crucial parameter for application areas such as drug delivery, sensors, valves and pumps. It is common sense that the creation of a hydrogel with fast response depends on the reduction of its characteristic dimension. With other words: the smaller the faster. K.-F. Arndt succeeded in synthesizing micro- and nano-scale hydrogels with fast stimulation-reaction pathways. These hydrogels were successfully used as components for analytical devices, e.g. "a lab-on-a-chip". In this case, the thermosensitive hydrogel actuator was directly placed in a micro-sized fluid channel. Increasing the temperature of the water above the LCST rapidly causes a deswelling of the gel. Thus, the valve opens and the fluid can flow .
However, the velocity of stimulation-reaction pathways for bulk thermo-sensitive hydrogels can also be increased by the temperature of the surrounding liquid environment. One interesting strategy, e.g., is to prepare smart hydrogels in the presence of ferroelectric micro- and nanoparticles .
In particular, barium titanate (BaTiO3) and poly (vinylidene fluoride) (PVDF) as ferroelectric particles and Ni powder as ferromagnetic particles can be incorporated in the polymer matrix as "fillers". Then an alternating electric or magnetic field leads to a heating of the surrounding phase, thus influencing the swelling behaviour directly. Filling of hydrogels leads to materials with strongly modified characteristics . In the above presented study, the fillers are incorporated in a PVME matrix and fixed, either because of the size (the mesh size of the gel was observed to be smaller than the particles) or covalent bonding (in case of PVDF). FESEM proves also in this case to be perfectly suited to study the location of Ni, BaTiO3, and PVDF submicrometer particles in PVME hydrogel in the swollen and the deswollen state at high resolution (cf. fig. 5).
Information about the fine surface structures of wet hydrogels obtained by SFM or ESEM seems unreliable since the imaging force and the surface tension may create artificially deformed structures, respectively. The strength of SFM regarding swollen soft materials focuses rather on the characterization of micromechanical properties than on high resolution imaging of the surface topography. In contrast, FESEM preferentially at low acceleration voltages in combination with state-of-the-art cryo-preparation and thin conductive coating with an almost homogeneous extremely thin film, e.g. Pt/C, are presently the only combination of techniques, which allow the structural characterization of hydrogels in different states down to a few nanometers.
References are available from the authors.
Dr. Thomas Matzelle
Chimie-Physique des Matériaux
Université Libre de Bruxelles
GIT Verlag GmbH & Co. KG
Univ.-Prof Dr. Rudolf Reichelt
Institut für Medizinische Phaysik und Biophysik
Universitätsklinikum, Westfälische Wilhelms-Universität