Microscopy techniques for polymer nanoparticles

Microscopy techniques can be generally divided into three categories: optical, electron and scanning probe.97 Optical and electron microscopy use a beam of radiation (light or electrons) which is projected onto a sample to create images. As the wavelength of electrons can be 100,000 times shorter than light, the absolute resolution of electron microscopy is much higher than light optical microscopy (0.003 nm for 200 keV electrons, 150 nm for UV-light microscope λ = 200 nm).97 Scanning microscopes utilize a probe to scan each point of the object serially to form an image. The resolution of scanning microscopes always depends on the size of the probe used and the degree in which a change in the probe (position or voltage) can be detected. Figure 1.12 shows different types of images obtained from different microscopy techniques for a spherical particle.97

Figure 1.12 A schematic showing that the different types of images formed by different microscopy techniques for a spherical particle.97

1.5.1.1 Transmission electron microscopy

Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a thin specimen and interacts with the

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specimen as it passes through. An image is then formed as a result of the interaction of electrons. Nowadays TEM can be used to image samples in both the dry-state and solvated state.

The dry-state TEM requires drying samples onto substrates before imaging, which may cause changes in particle size, morphology, crystallization or even damage the sample.100 For example, solvated polymers such as the corona of micelles in solution will change their shape or length upon dehydration. However, given that dry-state TEM is relatively easy to perform and widely accessed, it is still commonly used in the research of polymer nanoparticles. In addition, the substrates on which the samples are dried are very important for gaining a better image contrast. In order to observe particles easily and clearly, samples should scatter more electrons than substrates. However, typical TEM grids use carbon based film which are about 40 nm in thickness and thus particles approaching similar sizes are always difficult to image.97 To improve the contrast, staining techniques have been developed. A number of stains (e.g., osmium tetroxide, ruthenium tetroxide, uranyl acetate, ammonium molybdate and phosphotungstic acid) are used, which selectively bind to the grid (negative staining) or particle (positive staining) to enhance the contrast. Staining appears to be a useful technique; however, stains sometimes can cause artefacts, limit resolution and obscure the internal structures of particles.100,101 An alternative method has been developed, where thinner but inexpensive supports, graphene102 and graphene oxide (GO)103 are used as substrates to image low contrast materials without staining. Previously O’Reilly’s group collaborated with Wilson’s group to show that GO-coated grids are very useful for adhering polymer particles from both aqueous and organic solutions and imaging these particles with good contrast (Figure 1.13).104 Moreover, Dyson and coworkers recently showed that a

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combination of exit wave reconstruction (EWR) and GO-TEM grids can be used to produce extraordinary images where polymer chains within nanostructures can be observed.105 In this thesis, all the dry-state TEM images were conducted using GO- coated TEM grids unless specified otherwise.

Figure 1.13 Vesicles prepared from PS250-b-PAA11 imaged by (a) uranyl acetate staining

and (b) unstained on a GO-TEM grid.97,104

As dry-state TEM cannot image solvated samples, cryo-TEM and in situ liquid TEM were developed to overcome this problem, by which samples could be kept in a solvated state while imaging. For the preparation of cryo-TEM grids, a small volume of sample solution is deposited on a lacey carbon grid, blotted to remove most of the solution and then rapidly plunged into a vitrification solvent (typically liquid ethane) to trap the particles in a solution-state. Once vitrified, the samples are kept, transferred, and imaged at liquid nitrogen temperature. Cryo-TEM is a powerful technique to show the structures of particles in their natural state, however, there are a few disadvantages of cryo-TEM. Cryo-TEM is only well-adapted to aqueous samples; samples in organic solvents are very difficult to image.106 In addition, cryo-TEM requires significant time and proficient skills to prepare and analyze the samples. In situ liquid TEM is an exciting method, where liquid samples are injected into a sealed TEM chamber and then imaged in solution. This method is

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ideal for observation of particle dynamics in real-time. However, currently this developing method is limited to metal-containing particles due to the poor contrast for polymeric particles.98,99

Electron tomography is a technique where a series of TEM images are taken at different tilt angles. These images are then reconstructed to provide a 3D representation of the studied particles. TEM images can be taken by both dry-state TEM104 and cryo-TEM,107 although for complex structures cryo-TEM is much better as there are minimized artefects. This method is invaluable in the study of particles with complex internal structures as it can provide internal details. For example, Figure 1.14, which is from Holder and Sommerdijk’s work, shows TEM images of bicontinuous micelles obtained by both dry-state TEM and cryo-TEM and their corresponding 3D reconstructions.80 However, ET is currently not widely used as it is rather challenging and hence requires expert skills in both computers and microscopy.

Figure 1.14 TEM analysis of bicontinuous micelles: a) Conventional TEM using negative staining; b) cryoTEM image of a vitrified film; c) gallery of z slices showing different cross

sections of a 3D SIRT (simultaneous iterative reconstruction technique) of a tomographic series recorded from the vitrified film in (b); d,e) visualization of the segmented volume

showing d) a cross section of the aggregate and e) a view from within the hydrated channels.80

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1.5.1.2 Atomic force microscopy

Atomic force microscopy (AFM) is a scanning probe technique which utilizes a cantilever with a sharp tip at its end. When the tip is in close proximity of a sample surface, a deflection of the cantilever is formed resulting from the forces between the tip and sample. The deflection is then measured typically using a laser spot which is reflected by the top surface of the cantilever into an array of photodiodes. AFM can operate in different imaging modes with different feedback mechanisms: contact mode and tapping mode, where the gentle tapping mode is more suitable for soft polymeric materials. It should be noted the x resolution (horizon, see Figure 1.15) in AFM is always limited by the size of tip owing to convolution effects (Figure 1.15), while z resolution (height, see Figure 1.15) is always high and accurate.97 Thus AFM is complementary to TEM as the height is hard to obtain using TEM unless changing the tilt angle. In addition, AFM is also capable of measuring extremely small particles and particles which have weak scattering in TEM and are therefore difficult to observe by TEM.97,108 Moreover, it is also possible to characterize hydrated samples by using liquid AFM.109 In this thesis, AFM is used to mainly measure the height of particles in a dry-state.

Figure 1.15 Schematic of an AFM tip measuring a spherical particle on a surface indicating how x resolution and z resolution.97

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1.5.1.3 Other microscopy techniques

Other microscopy techniques such as scanning TEM (STEM) and scanning electron microscopy (SEM) are also used to analyze polymer nanoparticles although these techniques are less widely used than TEM and AFM.97 In STEM, images are produced by a raster scan using a small beam of electrons. STEM mode is advantageous in chemical analysis by combining with energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS).97,110 Moreover, STEM is able to image in the high angle annular dark field (HAADF), which means that the image contrast is directly related to the electron density and the specimen thickness.104,111

SEM is also conducted using a small beam of electrons; however, the electrons have lower energy (1 – 30 keV for SEM and 100 – 300 keV for STEM) and the beam sizes are larger than those used in STEM. Therefore the resolution in SEM is poorer than STEM. However, SEM provides information on surface or near surface rather than internal structures and is therefore complementary to TEM/STEM techniques. In addition, SEM also allows for chemical analysis of the surface. Typically samples are coated with Au or Pt to prevent charging the sample surface, which causes image distortion.97