Microscopy techniques

Microscopy is one of the most vital techniques for characterizing one dimensional nanostructures because it provides important conformational and morphological information. The techniques provides images of the synthesized nanostructures and in

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most cases complements other analytical methods of studying the properties of these materials. These direct imaging techniques such as fluorescence microscopy, transmission electron microscopy and atomic force microscopy provides a very convenient way of characterising a range of nanostructures in different environments.

2.6.1.1 Fluorescence microscopy

Fluorescence is an optical process that involves the relaxation of an excited electron to a lower energy orbital in an atom or molecule with the corresponding emission of a characteristic photon with wavelength larger than that of the excitation light. The emitted light has longer wavelength than absorbed light because light with shorter wavelength has higher energy and some energy is always dissipated as heat e.g. via vibrational modes. The energy change in the transition is referred to as the Stokes shift. The process showing this molecular transition can be explained using the Jablonski energy diagram presented in figure 2.1.

Figure 2.1 Jablonski diagram showing the changes in the electronic state of

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Figure 2.2 schematic representation of the basic light paths of a fluorescence

microscope125_{. (Where EM1 and EM2 are emission filters while DM1 and DM2 are the}

dichroic mirror).

This technique would be particularly useful because it makes use of the fluorescence of the templated material since transition metal chalcogenides are used as the templating material. The technique showed a successful templating process since bare DNA does not absorb 488 nm light, nor does it emit visible fluorescence.

2.6.1.2 Atomic force microscopy

Atomic force microscopy (AFM) is the most common scanning probe microscopy used for imaging conducting, semiconducting and non-conducting surfaces126_{. It was}

invented in 1985 by Binning, Quate and Gerber127_{. It is one of the most powerful and}

versatile nanoscale microscopy techniques and is used for studying many different kinds of samples. It can generate images with minimum sample preparation at submicron scale lateral resolution and sub-nanometre vertical resolution. It can also be used to obtain different kind of surface measurement (mechanical, electrical, magnetic and thermal properties). The AFM operates by measuring the deflection of a sharp tip attached to a flexible cantilever. The deflection is measured by reflecting a laser beam off the reflective back side of the cantilever and directing it to a split photodiode position detector.

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Figure 2.3 Schematic illustration showing laser light deflection off cantilever during

AFM measurement128_.

The AFM cantilevers and tips used as probes are normally made from Si or Si3N4 with

the tip radius in the range of a few nanometres. The images in AFM are acquired by measuring the lateral and vertical deflections of the cantilever between the tip and surface. If the stiffness of the cantilever is known, then the change in height of the tip is related to the force by Hooke’s law.

𝐹 = −𝑘𝑧 (2.1)

Where F is the force, z is the deflection of the cantilever and k is the stiffness of the cantilever. The deflection of the cantilever is monitored by the amount of laser beam light reflecting back to detector. The different signals of the deflection are collected and processed into a topographical image of the sample surface, usually in false colour.

Figure 2.4 The sample- tip separation showing the attraction and repulsion forces as

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The general scheme illustrating the different components and the working principle of an AFM is shown in figure 2.5. It consists of a piezoelectric scanner which controls image acquisition with precise control of the probe in relation to the sample surface in three dimensions. A cantilever with a tiny probe attached to it which scans across the surface when the cantilever is in motion. A split photodiode which measures the deflection of the cantilever, a laser with sets of mirrors and a z-axis feedback loop which controls the vertical movement to and from the sample, the control electronics and an attached computer system for data display and analysis. In this research, the AFM was used to acquire the morphological properties of the DNA templated nanowires and nanotubes such as diameter and height.

Figure 2.5 Schematic diagram of atomic force microscopy showing how topological

images of nanomaterials are obtained129_.

2.6.1.3 Transmission electron microscopy

The transmission electron microscope is a vital instrument for imaging ultrathin samples of nanostructures. In transmission electron microscopy (TEM), an electron gun with accelerating voltage typically within 100 - 400 kV with high penetrating power is channelled through a sample. The electrons undergo a range of scattering processes when in contact with the sample resulting in changes in their angular distribution and energy. The regions of the sample with higher electron density scatter

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the electrons more intensely and through a greater angle leading to the area appearing darker in the image7_{. The transmitted electron beam through the ultrathin sample is}

scattered into a non-uniform electron intensity after transmission. The non-uniform electron intensity is collected on the detector and is converted to a contrast image on the screen. The direct beam is used to form the bright field image while the diffracted beam forms the dark field images130_{. The process of the image formation using the}

direct beam in the bright field mode is presented in figure 2.6.

Figure 2.6 Schematic illustration showing the process of image formation in a typical

TEM in bright field image mode. The scattered electrons are indicated with the dash lines.

The objective aperture blocks the scattered electrons in order to improve the contrast. The primary beam interacts with the sample and produces other secondary signals too which can be used in determining the chemical composition and other relevant properties of the sample131_{. TEM experiments are carried out in an ultra-high vacuum}

environment to prevent the scattering of the electrons by atmospheric gas molecules and thereby reducing the electrons passing through the sample and further to the detector. TEM was used in this research to acquire the morphology of the templated material since DNA appears ‘transparent’ under the electron microscope.

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