Microscopic methods

Microscopes are used in polymer characterisation to probe the microstructural and nanostruc-tural morphologies of species that are not visible to the human eye. This is made possible through the magnification of the test sample using a combination of illumination sources and

CHAPTER4. EXPERIMENTAL METHODS AND SAMPLE CHARACTERISATION

various types of lenses. The instrument generates images having resolutions that depend on the type of illumination source and the method of image generation (Campbell et al., 2000).

4.2.5.1 Optical Microscopy (OM)

Optical microscopes are used in CNF characterisation to study the evolution of fibril morphology with increased processing (Chinga-Carrasco, 2013). They are also used to detect the presence of fibrils within the microscopic range (Desmaisons et al., 2017). Aqueous CNF suspensions are diluted and often stained with dyes to improve the contrast. The major components of a compound optical microscope, as shown in the schematic in Figure 4.9, include an illumination source, an objective lens, an eyepiece, a photomicrographic system and a specimen stage (Campbell et al., 2000; Stuart, 2008; Yang et al., 2013). In the Köhler illumination system used in most microscopes, the visible rays transmitted from a tungsten-halogen lamp are first collected by the collector lens and condensed by the condenser lens before being illuminated on the sample (Yang et al., 2013).

Figure 4.9: Schematic representation of a light optical microscope

The objective lens then magnifies the object image and projects it to the eyepiece, which further magnifies the object. When coupled with a camera, an image that can be viewed and sent to a computer screen is created. Convex lenses that are corrected for chromatic and spherical aberrations are conventionally used in optical microscopes. The total magnification of the

CHAPTER4. EXPERIMENTAL METHODS AND SAMPLE CHARACTERISATION

object is a product of the objective lens and eyepiece magnification. Up to 1000x magnification can be achieved with most modern microscopes (Wegerhoff et al., 2006). Images can be viewed in the bright field or dark field, depending on how evenly the visible rays are illuminated (Yang et al., 2013).

A Leitz DMRX transmitted light microscope from Leica Microsystems was used to study the effect of the number of passes through the high shear processor on the morphology of the cellulose fibres. A photographic image and the specification of the microscope are provided in Table 4.9. The 10x objective lens was used in this study, in addition to the 10x standard eyepiece, bringing the magnification to a 100x capacity.

Table 4.9: Photograph and technical details of the Leitz DMRX optical microscope

Optical microscope

Manufacturer: Leica Microsystems (Wetzlar GmbH)

Model: Leitz DMRX

Visible radiation source: Tungsten halogen lamp, mercury arc lamp

Eyepiece magnification: 10x

Objective lens magnification: 20x and 50x

4.2.5.2 Field Emission – Scanning Electron Microscopy (FE-SEM)

To view the nanofibrils created from cellulose fibres, an instrument that is capable of producing higher resolution images is required. This can be achieved using SEM, which collects sample images by scanning a beam of electron across the sample. The schematic representation of the image generation from an SEM is shown in Figure 4.10.

CHAPTER4. EXPERIMENTAL METHODS AND SAMPLE CHARACTERISATION

Figure 4.10: Schematic representation of an SEM

In standard SEM, the electron beam is generated by heating a tungsten and lanthanum hexaboride filament using an electric current. However, for FE-SEM, cold cathode field emitters from tungsten wires are used (Campbell, Pethrick and White, 2000). This ensures improved brightness and highly resolved images. When the beam of electrons interacts with a sample, backscattered electrons, secondary electrons and X-rays are produced. These are detected to form an image of the area scanned (Campbell et al., 2000; Stuart, 2008; Goldstein et al., 2004).

The SEM column containing the electron beam, condenser lens and electromagnetic lens is kept under a low vacuum. The condenser and electromagnetic lens serve respectively to condense and demagnify the beam diameter so that highly resolved images can be produced.

The SEM sample holder is called a stub. To prepare samples for SEM analysis, the SEM stub is first covered with a carbon black disc, onto which a mica disc is attached. The mica is cleaved with a tape to reveal a fresh flat layer. The sample to be tested is highly diluted (if in the liquid state) and deposited on mica disc surface. The sample is left to dry overnight in the sample box before image collection.

CHAPTER4. EXPERIMENTAL METHODS AND SAMPLE CHARACTERISATION

A major problem that is associated with the scanning of a non-conducting polymeric mater-ial is surface charging, which affects the quality of image produced. To circumvent this problem, CNF and other polymeric materials are thinly coated with conducting metals, such as gold, gold-palladium or with carbon (Campbell et al., 2000)

In this research an S-4800 FE-SEM from Hitachi was used to study the dimensions and morphology of CNF materials. A Zeiss Supra 40 VP from Carl Zeiss at the University of Genoa was used in a study of the porosity that was created within the CNF/alginate composite hydro-gels after freeze-drying. The images collected at the University of Genoa was done under the guidance of Laura Negretti. A photographic image and the specification of the Hitachi S-4800 unit that serves as a representative for the two instruments used are provided in Table 4.10.

Table 4.10: Photograph and technical details of S-4800 FE-SEM

FE-SEM

Much higher resolution images are acquired from TEM. Hence, its use in CNF characterisation to study the morphology and dimensions of nanofibrils (Foster et al., 2018). There is a similarity in the instrumentation used in TEM transmittance and those used optical light microscopy. Major differences lie in the radiation source used and in the type of lens used. In TEM, an electron beam is accelerated from hairpin halogen lamps and electromagnetic lenses are used (Stuart, 2008). TEM also uses a condenser lens, a specimen stage, an objective lens and a projector system. However, a vacuum environment is required to ensure that only scattered electrons

CHAPTER4. EXPERIMENTAL METHODS AND SAMPLE CHARACTERISATION

from the sample under test are detected (Campbell et al., 2000). TEM can resolve images down to the atomic level of 0.2 nm because of the higher energy of the electron beam, making it a valuable tool in polymer characterisation (Campbell et al., 2000). A very thin layer of sample, deposited on a carbon film that is coated on a metal grid, is required for TEM analysis. As with optical microscopy, the samples for TEM can be negatively stained or positively stained to improve the contrast. Photographic images can be acquired from the camera placed under the projector system or sent to a computer screen.

Negative staining is usually carried out with uranyl acetate. However, the radioactive prop-erties of this staining agent limit its use for sample staining. Alternatively, phosphotungstic acid can be used. In this study, TEM image analysis was carried out using Zeiss Leo EM900 unit from Carl Zeiss. A photographic image and specification of the instrument are shown in Table 4.11.

The analysis was conducted at the Consiglio Nazionale Delle Ricerche, Genoa, Italy under the guidance of Dr. Lucia Conzatti.

Table 4.11: Photograph and technical details of Zeiss Leo EM900 TEM

TEM

Manufacturer: Carl Zeiss

Model: Zeiss Leo EM900

Electron beam source: Tungsten halogen lamp, mercury arc lamp

Accelerating voltage: 20 – 80 kV