Aim 2: Improve existing nanowire-based injection systems by exploring the use of nanotubes incorporated into a fluidic system for cell injection
2 Experimental methods
Both wet and dry etching can etch in all directions at the same rate, isotropic etching, or at different rates in different directions, anisotropic etching. For wet etching, anisotropic etching typically follows crystal planes (some planes take longer to remove than others) and the resulting etch profile is determined by these facets . For RIE, etching parameters such as plasma composition and bias can be fine-tuned to generate specific etch slopes by balancing physical and chemical reaction mechanisms , .
On the other hand, it is not straight forward to use RIE to create e.g. overhang structures such as those described in Paper I.
15 reconstruction algorithm yields an image with very high contrast and can accommodate for a slightly out-of-focus sample. This inherent focus correction makes phase holographic microscopy superior to many standard microscopy systems: it can adjust for the slight focus drift that often occurs over long imaging sessions. Together, these traits enable extended time-lapse imaging (>96 h) without any adverse effects on the cells.
Figure 2.5 Phase holographic microscopy uses a laser beam which is split into a reference beam and a sample beam. After the sample beam has passed through the specimen, the two beams are recombined, giving rise to an interference pattern based on the phase changes the sample beam underwent on its path through the sample. This interference pattern can be used to construct a model of the sample and contains height information, enabling 3D reconstructions of the sample.
For our studies of cells cultured on GaP substrates with Gap nanowires, phase holographic microscopy is especially useful as it can be used to image cells on these surfaces (Figure 2.6). Normally, semiconductor materials are non-transparent to visible light, owing to a low band gap energy. For example, GaAs and InP have band gap energies around 1.5 eV, meaning they will absorb light with wavelengths below
~830 nm* while the ubiquitously used silicon has a bandgap of 1.12 eV , absorbing light with wavelengths below 1.1 μm. However, GaP has a relatively high band gap energy of 2.26 eV , allowing all light with a wavelength longer than 549 nm to pass unabsorbed. This transparency to long wavelengths means that it is possible to use transmitted light-based microscopy methods granted that the light used has a wavelength longer than 549 nm. Standard light based methods such as phase contrast microscopy, commonly uses full spectrum white light (including wavelengths above 549 nm) but, as seen in the phase contrast image of cells cultured on nanowires (Figure 2.6 a and c), this is not always enough. The poor image quality seen here is likely owing to scattering and absorption of shorter wavelengths of light in both the substrate and in the nanowires.
The commercial implementation of phase holographic microscopy provided by the company Phase Holographic Imaging, (PHI AB, Lund, Sweden), utilizes a red laser (633 nm ) to acquire image data, making it suitable to image cells on GaP. This long wavelength alone is not enough to image cells on our substrates because the rough backside of the samples will scatter the light, making image restoration impossible.
When we instead fabricated our nanowires on substrates that had been polished on both sides, it was possible to image the cells, as shown in Figure 2.6 (b and d). The high bandgap of GaP has enabled us to capture time-lapse images for up to 96 h of unlabelled cells on our nanowire surfaces, forming the backbone of Paper II and Paper IV. Note that, depending on the physical properties such as density and length, it was sometimes possible to use phase contrast microscopy (short and less dense nanowire arrays being preferable). We have also observed a decrease in image quality for phase holography related to nanowire geometry: increasing either nanowire density or nanowire length reduces image quality and it was not possible to image cells on nanowire arrays with a density of 10 nanowires μm-2 or more.
* Visible light has wavelengths in the range 400-700 nm.
Figure 2.6 Comparison between phase contrast microscopy (a, c) and phase holographic microscopy (b, d). Mouse fibroblasts (L929) cultured on GaP nanowires with a density of 0.1 μm-2 (diameter 80 nm, length 4 μm,) (a, b) and similar nanowires with a density of 1 μm-2 (diameter 80 nm, length 3.8 μm) (c, d). Phase holographic microscopy vastly improves the image quality; without it, imaging cells on many of the nanowire substrates would not be possible (depending on nanowire geometry). Scale bars are 50 μm.
Instead of relying on physical properties of cells to improve contrast in a biological sample as in phase holographic microscopy, it is common to add different fluorescent dyes that bind to specific cellular structures, enabling visualisation of these. The fluorescently labelled sample is imaged in a fluorescence microscope where the dyes are excited using light of a certain wavelength. If the incident photons have an energy corresponding to the difference between energy levels in the fluorophore, the photon energy can be absorbed by an electron, exciting the fluorophore (Figure 2.7 a). The excited electrons will lose a low amount of energy before relaxing to the ground state through the emission of a photon. Due to the energy loss, the emitted photon will have a longer wavelength than the photon used for excitation. This change in wavelength is referred to as Stoke’s shift (Figure 2.7 b) and is at the heart of fluorescence microscopy:
by using filters it is possible to illuminate the sample with excitation light while only collecting emitted light (Figure 2.7 c). When illumination and light capture occurs on the same side of the sample, the setup is referred to as an epifluorescence microscope (Figure 2.7 c), in contrast to a diafluorescence microscope where light source and collection are at opposite sides of the sample (not shown).
Fluorescence confocal microscopy is a common version of fluorescence microscopy used to collect 3D data from a sample. In this work, confocal microscopy was used in Papers I-III. In contrast to standard fluorescence microscopes, where all emitted light is collected, for confocal microscopy light is only collected from specific optical planes or slices, resulting in a much sharper image. By capturing several such optical slices by means of scanning in the vertical direction, it is possible to create a 3D reconstruction of the sample. The removal of the out-of-focus light is achieved by the insertion of a metal disc into the optical path. A small pinhole in the disc will ensure that only light from a specific focal plane can pass (Figure 2.7 d). The disc will also block light in the XY-plane, improving lateral resolution but imposing the need to scan across the sample, collecting emission light from one point at a time. This scanning is often implemented by scanning an excitation laser across the sample, collecting the emitted light one pixel at a time (confocal laser scanning microscopy (CLSM)). Another variant is the spinning disc confocal system where light from the sample is passed through an array of pinholes, collecting light from several points of a sample at once. By rotating the disc, light can be collected from the entire field of view using a CCD, greatly improving the acquisition rate at the cost of signal to noise ratio.
Fluorescence microscopy is based around the use of fluorophores, which come in a large variety and can be organic molecules adapted from plants and animals, semiconductor quantum dots, metal nanoparticles or fluorescent proteins among others . Some dyes bind to specific structures such as proteins, DNA or cell membrane. Those dyes that do not possess a high binding affinity are often attached to structures such as antibodies or toxins that do. It is not only this very specific labelling but also dyes that react to their surroundings that give rise to the great versatility of fluorescence based microscopy. Some dyes react to pH, transmembrane voltage or certain ions while other dyes are activated by unique enzymes, indicating their presence and function. Owing to all the research related to the development of genetic techniques and fluorescent dyes, fluorescence microscopy has truly become a workhorse in cell biology , –
. In this thesis, we have used dyes to stain cell structures such as DNA and actin filaments (Paper I-IV) and we have explored the use of functional dyes to assess cell respiration and generation of reactive oxygen species (ROS) (Paper II).
Figure 2.7 In fluorescence microscopy, the electrons in a fluorophore are excited by illumination with light (a). The excited electrons will lose a portion of their energy before relaxing to the ground state via the emission of a photon. This energy loss causes the emitted photon to have a longer wavelength than the incident light. This can be seen in the excitation and emission spectra (b) and is referred to as the Stoke’s shift. This shift is capitalized on in fluorescence microscopy (c). The use of filters makes it possible to illuminate a stained sample with short wavelength light (here blue) and only collect emitted, long wavelength light (here green). The excitation light is reflected toward the objective and sample by a dichroic mirror (a mirror which transmits light with wavelengths above or below a cut-off wavelength).
The excitation light will excite the fluorophores in the sample and the emitted light is collected by the objective and passes through the dichroic mirror. An emission filter ensures only emitted light can pass to the detector. In confocal microscopy a pinhole is placed in the light path to ensure a narrow focal depth (d) and scanning in the x- and y-directions ensures imaging of the entire sample. This is often combined with z-direction scanning to create 3D reconstructions of the specimen.
One major drawback of using dyes to investigate cell behaviour is phototoxicity , . Ideally, the excited electrons will lose their energy through radiative processes (i.e., the emission of a photon) but the high energy make these electrons very reactive.
Hence, excited dyes are prone to react with nearby molecules, in particular with oxygen.
This often leads to the generation of ROS, which then interacts with e.g. proteins and DNA, causing damage to the cells. These reactions limit the usefulness of dyes in living cells, even if the phototoxicity does not outright induce cell death, it can affect cell
behaviour such as mitosis . If studying living cells, the phototoxicity of fluorescent dyes need to be minimized by e.g. reducing dye concentration, exposure time, using oxygen scavengers or limiting staining and relying on white light, e.g. phase contrast microscopy, for the majority of the imaging –. In our work on time-lapse microscopy (Paper II and IV), we opted to remove fluorescence microscopy completely by using phase holographic microscopy, as outlined above.
In our work, several dyes have been used to assess aspects of cell behaviour, as summarised in Table 2.1. To visualize cells and investigate their morphology, the DNA stain bisbenzimide (Hoechst 33342) has been used together with phalloidin* conjugated to the dye fluorescein isothiocyanate (FITC) in Papers I-IV to label cell nuclei and the actin cytoskeleton, respectively. In Paper II, functional dyes were used to assess ROS generation, double-strand DNA (dsDNA) breaks and cell respiration.
ROS was detected using the fluorescein derived compound carboxy-H2DCFDA.
Upon entering cells, intracellular esterases will remove two carboxyl groups, rendering the compound membrane impermeable . In the presence of ROS, the altered carboxy-H2DCFDA will be converted to native fluorescein, a membrane impermeable fluorophore, thereby labelling cells with high ROS content. Resazurin, commonly referred to by its trade name AlamarBlue™, is used to study cell respiration. When added to a cell culture, resazurin will enter the cells and be reduced to resorufin via interactions with the electron transport chain in the mitochondria , converting it from a weak, blue fluorophore to a strong, red fluorescent compound. In this case, both compounds are membrane permeable, i.e. resorufin is able to leave the cells, giving the medium a visible pink hue. By measuring the red fluorescence from a cell culture, the level of respiration can be assessed. In Paper II, we investigated dsDNA breaks using antibody labelling. When a dsDNA break is detected by the cell, the histone subunit H2AX will be phosphorylated, forming γ-H2AX, triggering the assembly of a DNA repair complex . The γ-H2AX can be selectively labelled using antibodies, enabling an assessment of the ongoing level of DNA repair and, by association, DNA damage.
Propidum iodide (PI) and ethidium homodimer-1 (EthD-1) are two membrane impermeable dyes that are often used to stain the DNA of cells with disrupted membranes . These dyes are often combined with calcein-acetomethoxyl (calcein AM) to assess the viability of cells in a culture. Similar to carboxy-H2DCFDA, calcein AM is a modified version of the fluorophore calcein, where several ester groups have been attached, turning the dye non-fluorescent and membrane permeable . Upon entering a cell with enzymatic activity, the ester groups are removed by enzymes, returning calcein to its natural, fluorescent, membrane impermeable state. Adding both
* Phalloidin is a toxin extracted from death cap mushrooms which binds strongly to cytoskeletal actin filaments.
21 PI or EthD-1 and calcein AM to a cell culture has the combined effect of turning living cells green (calcein) while dead or dying cells turn red or orange (PI or EthD-1, respectively).
Table 2.1 Fluorophores used in this thesis and cited literature to visualize cells and evaluate their function.
Dye Target Description Used in
FITC/phalloidin Actin filaments Membrane impermeable. Used after fixation and permeabilization.
Bisbenzimide (Hoechst 33342)
DNA Membrane permeable. Used to label live or fixed cells.
ROS Activated to a fluorescent compound by ROS generated in live cells.
AlamarBlue™ Electron transport chain
Cell respiration converts resazurin to resorufin, causing a shift in emission wavelength and intensity
Antibodies against γ-H2AX
γ-H2AX Phosphorylated version of histone H2AX, indicative of DNA repair
DNA Membrane impermeable , –
EthD-1 DNA Membrane impermeable –
Calcein AM Cytosol Becomes membrane impermeable and fluorescent upon modification by intracellular enzymes
, , , , , , 
Scanning electron microscopy
Though incredibly versatile and extensively used, optical microscopy suffers from one major drawback: the resolution limit. As light from the sample travels through the microscope towards the detector, it will be refracted and the minimum focus spot is limited to approximately half the wavelength of the light* used , in practice around 200-300 nm. This limit means it is not possible to resolve structures that are closer to each other than this distance without using emerging super resolution techniques like STORM  or STED  (though limited by diffraction, special procedures enable position determination with greater accuracy, circumventing the resolution limit).
To circumvent the problem of the resolution limit, electron microscopes have been developed. These microscopes do suffer from the same limitations but the wavelength of electrons can be much shorter than that of (visible) light, and a higher resolution is
* Diffraction limits resolution to approximately λ/2∙n∙sin(θ), where λ is the wavelength of light, n is the refractive index of the medium between objective and specimen and θ is the collection angle of the objective.
readily obtainable. In a standard SEM operating with an accelerating voltage of 20 kV, the wavelength of the electrons is 0.001 nm. In practice, the resolution in SEM is limited by the poor quality of the electron lenses but can reach values down to single nanometres . The improvement in resolution compared to optical microscopy makes electron microscopy an indispensable tool in nanofabrication and is used on a daily basis. In this thesis, SEM has been used to evaluate the results of nanowire growth, nanofabrication (Papers I-IV) and to image the interactions between cells and nanowires (Papers I, II and IV).
In SEM, free electrons are generated from an electron source (such as a heated tungsten filament) and accelerated over a voltage (typically 1-20 kV) . The electrons pass through electromagnetic lenses that shape the beam into a circular cross section and focus it onto the sample. As the electrons impinge on the specimen, secondary electrons are generated and collected by an electron detector. Alternately, electrons from the incident beam that have scattered back toward the electron source can be collected using a back-scatter (BS) detector. To generate an image of the specimen, a set of electromagnetic coils are used to scan the beam across the sample while recording the signal in each position. The strength of the recorded signal is proportional to the number of electrons, either secondary or back-scattered, that reach the detector. This number depends on material composition, topography and location of the detector.
For BS electrons, the atomic weight of the sample plays a major role, enabling image contrast based on atomic species; heavy atoms such as the gold used for nanowire growth will appear very bright and can even be seen through lighter materials, such as those composing biological material (Figure 2.9 c, d)
SEM sample preparation
A sample imaged using SEM needs to be conductive in order to prevent charge up: the accumulation of electrons which will deflect the incident beam, giving rise to artefacts and poor image quality. Further, the SEM needs to operate in vacuum to prevent the electrons from scattering and reacting with air molecules. For semiconductor samples, these restrictions pose no problem but cell cultures need special sample preparation.
First the sample is dehydrated and then it is coated with a conductive layer. The dehydration typically proceeds by replacing the liquid covering the fixed cells with ethanol at increasing concentrations up to ≥99.5%. The final ethanol solution can either be allowed to evaporate under ambient conditions or replaced with liquid CO2
using a critical point dryer (CPD). By heating the liquid CO2 under pressure, the system will reach a critical point in the phase diagram of CO2 from which the liquid CO2 can evaporate without an increase in volume. For many samples, CPD is a means of avoiding sample deformations that can be caused by the expansion of an evaporating liquid . For monolayer cell cultures, such sample distortions are minor, but when studying cells on nanowires, CPD can help prevent nanowires from adhering to one
23 another. If allowed to dry in air, the receding ethanol often causes nanowires to bend and cling together and sometimes even break, depending on the nanowires’ physical properties such as density, diameter and length (Figure 2.8). After dehydration, the sample is placed in a sputter coater (see Section 2.1) and a thin, conducting layer of metal is deposited on top to improve imaging by preventing charge up.
Figure 2.8 If nanowire samples are dehydrated by allowing ethanol to evaporate under ambient conditions, the nanowires may cling together (a), though for certain geometries, such as sparse (b) or short nanowires, this will not happen. To prevent nanowire clinging, the ethanol can be replaced with CO2 (l) which is then heated until it evaporates in a CPD (c). Scale bars are 2μm, tilt is 30 ° (a, b) or 60 ° (c).
One of the strengths of optical microscopy is the use of dyes to improve contrast. Since contrast in SEM is based on geometry and atomic weight, fluorescent dyes do not work here. Instead, dedicated contrast enhancing fixatives and dyes based on heavy atomic species have been developed for electron microscopy, such as osmium tetroxide and uranyl acetate which were used in Paper II. Osmium tetroxide is primarily incorporated into fatty tissue like the cell membrane while uranyl acetate reacts with phosphates and amine groups, thereby labelling nucleic acids and many proteins . To improve specificity, antibodies labelled with metal nanoparticles are manufactured, making it possible to locate specific cellular structures in electron microscopy .
Focused ion beam tomography
In SEM, only the surface of a sample is imaged and, though this gives useful information, it is occasionally of interest to investigate the interior of a sample. For biological samples, sectioning tools such as microtomes are used to cut tissue into thin sections, which can then be imaged. For cells cultured on brittle semiconductor substrates, such as those studied in this thesis, this is not an option since the sample shatters, preventing the creation of thin sections. In Paper II, we circumvented this issue by using focused ion beam (FIB) tomography to section cells on nanowire substrates inside an SEM during operation, experiments carried out by our collaborators Carsten Købler and Kristian Mølhave at Denmark’s Technical University. In some commercial SEM systems, an ion source has been installed, enabling the use of a FIB to manipulate a sample using e.g. FIB-assisted material
deposition . Alternately, the FIB can be used to sputter material from a precisely defined region of a sample. That is, the sample can be imaged in SEM and a small area of the specimen can be selected for removal in situ , . In Paper II, Købler and Mølhave combined FIB tomography with heavy metal stains to generate cross sectional images of cells cultured on nanowires with nanometre resolution, allowing us to study the intracellular cell-nanowire interactions with nanoscale resolution.