**3. Nanofiber characterization methods**

Nanofibers can be produced by many different methods according to the area in which they are to be used. After choosing the appropriate production method for the application area and producing the nanofibers, some characterization studies are required to examine the quality, composition, morphology, and structure of the nanofibers. Characterization methods are still improving, and request for the establishment of effective techniques is continuously increasing. Therefore, commonly used methods for the characterization of nanofibers are described below.

#### **3.1 Morphological and structural characterization**

#### *3.1.1 Scanning electron microscopy (SEM)*

Generally, microscopic imaging techniques are routinely used to observe fiber diameters, alignment, porous structure, fiber morphology, and orientation. With

#### **Figure 8.**

*(a) Schematic image of scanning electron microscope, and (b) schematic image of transmission electron microscope. (Figures are reproduced from https://biorender.com/).*

scanning electron microscopy (SEM) imaging, high-resolution images of a scaffold surface can be obtained and surface properties (roughness, porosity, smoothness, etc.) can be determined.

In order to obtain a high-resolution image from scaffolds, samples have to be conductive, so sputtering with a thin layer of a conductive metal such as gold or titanium is a common modification for nonconductive samples. After sputtering, an electron gun is used to produce beams as a cathode source and focuses by electromagnetic lenses to an exact spot on the sample. Selected spot is shaped by deflection coils so that the surface of the sample can be scanned. This procedure depends on the interaction between the beam and the secondary electrons, which are produced from the sample. Interaction between the secondary electrons from the surface of the sample and the electron beam is monitored, amplified, and illustrated in the form of an image of the surface (**Figure 8a**) [45].

For the first evaluation of the nanofiber scaffolds, SEM is the most common characterization method due to its availability and the ease of use. It is possible to determine the porosity, the width, and length of pores on the surface, which can help understand the structure of the nanofibers [46]. To analyze the qualitative characteristics of the nanofibers, a convenient number of samples are needed to obtain statistical information about the materials.

Evaluation of nanofibers, cells, living organisms, or biological materials is also possible without any coating treatment required by using environmental SEM (ESEM). In this characterization method, the electron beam is wielded under water vapor environment. The ionization of water prevents the accumulation of the surface charges, which allows nonconductive materials to be evaluated without any modifications.

#### *3.1.2 Transmission electron microscopy (TEM)*

Transmission electron microscope (TEM) technology is considered one of the most important characterization techniques because of its ability to evaluate the interior structure of the samples. The pore structure of the scaffolds can be clearly seen by the images taken with TEM. The pore size and distribution of scaffolds are crucial parameters in tissue engineering field due to the fact that these parameters

#### *Nanofibers: Production, Characterization, and Tissue Engineering Applications DOI: http://dx.doi.org/10.5772/intechopen.102787*

directly affect the ability of the cells to penetrate through the pores of the material. Similar to SEM imaging, TEM also yields two-dimensional (2D) images of nanofibers and pores as well [45–47].

The common method includes transmitting electron beams through the ultrathin part of the samples, which causes a phase shift in portion of the electrons. When the incoming electron beam descends from the microscope column, it interacts with the sample fluorescent screen. And then the electron beam hits the sample, which leads a large amount of radiation to be emitted from the sample. This interaction causes the elastic and inelastic scattering of the emitted electrons. Images that take origin from the elastically scattered electrons allow the observation of the structure of the scaffolds or the defects at a high resolution (**Figure 8b**). Ultrathin samples are required for TEM evaluation (~20–200 μm) because electron beams are absorbed completely by the thick samples and no image can be formed. It is a very common characterization technique, but it is also a detrimental technique as well because of the possibility to damage the samples, especially the biological samples, by the electron beam going through them [45, 46].

#### *3.1.3 Atomic force microscopy (AFM)*

Atomic force microscopy (AFM) technique is mostly used for the evaluation of surface topography. The analytical capabilities of AFM are limited to the uppermost atomic layer of a sample because its operation is based on the interactions with the electron clouds of atoms at the surface. This technique also gives information about morphology, surface roughness, fiber orientation, and particle/grain distribution from the surface of the samples [45].

In this technique, a small tip is attached to a cantilever, and when the tip encounters with the sample surface, Van der Waals and electrostatic interactions between atoms at the tip and those on the surface create a force profile and cause attraction of the tip to the surface. A photodiode detector detects the changes and converts them into data, which are later to be converted into images (**Figure 9**) [45, 49].

The operation of AFM can be carried out by three modes depending on the application: contact, noncontact, and tapping modes. The contact mode measures the repulsion between the tip and the surface of the sample where the force of the tip against sample surface remains constant. At this mode, sensitive samples can be damaged because scanning requires constant contact of the tip to the surface. The noncontact mode on the other hand measures the attractive forces between the tip

*Atomic force microscopy setup. (Figure is reproduced with the permission from Deng et al. [48]).*

and the sample surface. Van der Waals forces between the tip and the sample surface are detected. Characterization of soft materials is often made with noncontact mode. At last, the tapping mode depends on the vertical oscillation of the tip. The tip contacts the surface of the sample and then lifts off at a certain frequency. Oscillation amplitude reduces as the tip contacts the surface due to the loss of energy. This mode overcomes problems with friction, adhesion, and electrostatic forces [49].

## *3.1.4 X-ray diffraction (XRD)*

X-ray diffraction (XRD) spectroscopy is a safe non-damaging characterization technique, which can be performed on wide range of materials such as minerals, metals, semiconductors, ceramics, polymers, etc. This technique is mostly applied to evaluate structural properties of the samples such as phase formation, crystallite size, lattice strain, contents of each phase, and crystal structure. The wavelength of X-Rays (0.5–50 A°) is similar to the distance between atoms in a solid, they are ideal for exploring atomic arrangement in crystal structure [46].

XRD, rather than measuring how the absorbance of X-rays affects the sample, examines how X-rays are diffracted from the atoms in a sample. Diffraction occurs when incident rays are scattered by atoms in a way that reinforces the waves (**Figure 10**). Working principle of XRD is basically a filament is heated to produce electrons in a cathode tube. By applying voltage, electrons are accelerated toward the sample and the sample is bombarded with electrons. Characteristic X-ray spectra are produced when electrons have enough energy to remove the inner shell electrons of the target sample. These X-rays are adjusted and located onto the sample, and the intensity of the reflected X-rays is recorded. Then these recorded signals are processed and converted to a count rate and directed to a printer or a computer monitor as an output [51].

**Figure 10.** *Working principle of X-ray diffraction. (Figure is adapted from Kaur et al. [50]).*

#### *3.1.5 Thermogravimetric analysis (TGA)*

Thermal methods can be examined under two categories: (a) differential thermal analysis and (b) thermogravimetric analysis (TGA). Differential analysis depends on the changes in heat content, which is measured as a function of increasing temperature. On the other hand, thermogravimetric analysis depends on the changes in weight, which is measured as a function of increasing temperature (**Figure 11**) [53].

TGA technique relies on the use of uniform heating to decompose all organic contents at high temperature, which eventually gives information about the

*Nanofibers: Production, Characterization, and Tissue Engineering Applications DOI: http://dx.doi.org/10.5772/intechopen.102787*

#### **Figure 11.**

*Thermogravmetric analysis diagram. (Figure is adapted from Loganathan et al. [52]).*

compositions of the sample. Mainly, by increasing the temperature at a constant rate, the decrease in the mass of the sample is recorded. The sample is located on a balance with a platinum melting pot, which is placed inside a furnace, and the procedure is generally carried on under air gas. As a result of this analysis, mass against temperature or time plot is obtained to measure the changes in the physical and chemical properties of the sample. The obtained data provide information about thermal stability of the remained sample, dehydration, pyrolysis, solid/gas interactions, etc. [54]

#### **3.2 Mechanical characterization**

Mechanical characterization of the scaffolds plays a critical role in tissue engineering applications. The designed scaffold's mechanical properties have to match the mechanical properties of the desired tissue. The mechanical strength of a scaffolds is crucial especially for in vivo applications, where the scaffold exposed mechanical loading repeatedly.

Most common characterization technique for nanofibrous scaffolds is tensile testing or nano-tensile testing. The theory is based on the attachment of the scaffold from both sides to the grips of the tensile testing machine and then pulling the scaffold with a constant rate until the rupture occurs (**Figure 12a**). The results give information about the stress-strain values, modulus, and strength of the scaffolds. But there are two limitations, which need to be overcome. Firstly, sample gripping is a problem because fibers tend to slip from the grips or break at the grips. These machines generally are not equipped to perform under micro sizes, so the small

#### **Figure 12.**

*(a) Tensile testing of nanofiber scaffolds, and (b) nanoscale bending test schematic. (a was author's unpublished thesis images, b was reproduced with the permission from Zhou et al. [55]).*

size of the specimen is a major limitation for this process. Second, alignment of the scaffolds is needed because randomly oriented fibers may lead to premature sample failure due to unwanted bending caused by misalignment [56].

Another characterization technique that is widely used is bending test for nanofibers (nanoscale three-point bending test). The capability of an AFM system to apply forces in the nano/pico-Newton range and measure the deformation in the range of Angstroms has made this characterization method very useful. The nanofiber sample is produced or deposited on a substrate with holes in it. Then the nanofiber is positioned such that a part of it is suspended over a hole. The adhesion between the sample and the substrate is enough for the test to be performed without a failure (**Figure 12b**). With three-point bending test, Young modulus and fracture strength can be obtained. The downside of this method is that it is only limited to samples that can be produced using AFM anodization [45, 56].

#### **3.3 Chemical characterization**

#### *3.3.1 X-ray photoelectron spectroscopy (XPS)*

X-ray photoelectron spectroscopy (XPS) technique is one of the most powerful characterization techniques because of the ability of giving chemical information about the surface of the material, both elemental and molecular composition (**Figure 13a**). It can also differentiate chemical states of the same element to determine their depth distribution at a thickness between 5 and 10 nm. Useful electron signal is obtained only from a depth around 10–100 A° on the surface. Basically, the surface is irradiated by hitting the core electrons of the atoms. X-ray absorption causes the removal of an electron from one of the innermost atomic orbitals, and the kinetic energy of the emitted electron is recorded. The recorded kinetic energy is then converted into a spectrum by a computer. Binding energies of the elements from the sample will be determined according to the peaks from the spectrum. In literature, the kinetic energy and binding energy values assigned to each element can be found. This method often requires an argon ion bombardment step to eliminate surface impurities [45, 59].

#### *3.3.2 Fourier's transform infrared spectroscopy (FTIR)*

Fourier's transform infrared spectroscopy (FTIR) is a technique used to collect an infrared spectrum of an emission or an absorption of a solid, liquid, or gas. It is used to identify organic, inorganic, and polymeric materials utilizing IR light to

#### **Figure 13.**

*(a) Working principle of X-ray photoelectron spectroscopy, and (b) schematic of Fourier's transform infrared spectroscopy. (a was adapted from Seyama et al. [57], b was adapted from Lee et al. [58]).*

#### *Nanofibers: Production, Characterization, and Tissue Engineering Applications DOI: http://dx.doi.org/10.5772/intechopen.102787*

scan samples. Standard FTIR setup is composed of a source, sample cell, detector, amplifier, A/D converter, and a computer (**Figure 13b**).

IR radiation is passed through the sample, and the emitted radiation could be absorbed and/or transmitted from the sample. Changes in the patterns of the absorption bands pinpoint a change in the composition of the material. So, the obtained signals are amplified, changes got detected by the detector, converted by the A/D converter, and as a result, a spectrum will be obtained. The obtained spectrum provides information about chemical composition of the material because the wavelength of absorbed light indicates characteristics of the chemical bonds. Just like fingerprints, two individual molecular structures cannot generate same IR spectrum. Every molecule has a specific fingerprint, which makes this technique a valuable tool for chemical identification. Also, this feature makes FTIR very preferable for many analyses such as determining the components in a mixture, identifying unknown materials, detecting contaminants in a material, finding additives, or determining the quality of a sample [60].

#### *3.3.3 Raman spectroscopy (RS)*

Raman spectroscopy (RS) method is based on irradiating a sample with a powerful laser source consisting of a monochromatic beam and measuring the scattered beam from a specific angle. During light scattering, the energy of most of the scattered light becomes equal to the energy of light interacting with the specimen. This type of elastic scattering is called Rayleigh scattering. In addition to elastic scattering, if a small part of the scattered light includes inelastic scattering, it is called Raman scattering. In Raman scattering, the excess or decrease in the energy of the scattered light relative to the energy of the light interacting with the molecule is as much as the energy difference between the energy levels of the molecule interacting with the light. This excess or scarcity at the energy levels is called the Raman shift. These shifts are measured in Raman spectroscopy (**Figure 14**) [62].

This method is used to evaluate vibrational and rotational frequency modes in physical and chemical systems. The intensity of Raman scattering depends on the change in polarizability. RS is suitable for the qualitative and quantitative analysis of organic, inorganic, and biological systems. With the obtained spectrum, unknown material identification, material differences, crystallinity, and material amount can be determined [60].

**Figure 14.** *Working principle of Raman scattering. (Figure was adapted from Kim [61]).*
