TEM is a highly useful technique for studying particles and structures of nanoscopic dimensions as it examines their internal structure and provides detailed microstructural information of the material. By switching between different imaging modes422 TEM can study e.g. the chemical composition, crystal orientation or electronic structure of a sample, in addition to the conventional absorption-based imaging. TEM can distinguish and image objects in the approximate size range 1 nm-5 m. Using high resolution transmission electron microscopy (HRTEM), also called phase-contrast, images with even greater resolution have been achieved, enabling study of the periodic structure of solids, complex structures and crystal defects down to the atomic level. For a sample to be suitable for TEM analysis it needs to be extremely thin, typically <100 nm, and in extreme cases such as HRTEM even <50 nm or <10 nm. Since high-vacuum conditions are required for the electrons to travel unhindered the sample should furthermore be stable in vacuum and able to withstand radiation damage by the electron beam.
The components of a TEM are assembled into a vertical microscope column with an electron gun positioned at the top of the instrument.421 Condenser lenses situated below the electron gun demagnify the electron beam, limiting its diameter, hence, controlling the intensity of the illumination and the size of the illuminated sample area. The sample, usually mounted on a thin electron-transparent plastic or carbon film supported by a copper mesh grid, is illuminated by accelerating an electron beam at a potential difference range 40-400 kV onto the sample. The applied voltage depends on the nature of the specimen and the desired information. The electrons interact with the specimen as they pass through it and form an image on the underlying fluorescent screen or photographic plate. One of the main requirements of the sample is that it needs to be ultra-thin in order for the transmitted electrons to have sufficient intensity for creating an interpretable image within a certain time limit, which generally is a function of the electron energy and the average atomic number of the sample.
Contrast formation in TEM is largely dependent on which imaging technique is used. The most common operation mode is bright-field imaging, in which only the transmitted electrons contribute to image-formation.423 The result is a two-dimensional image where thicker specimen regions or regions with atoms of higher atomic number appear darker and thinner specimen
regions or regions with a lower atomic number appear brighter on the screen.
Dark-field imaging exploits the scattering of electrons from the sample into locations in the back focal plane and can be useful e.g. in identifying lattice defects in crystals. By selecting certain reflections and excluding the unscattered beam and by projecting the back focal plane instead of the imaging plane onto the image device, a diffraction pattern, which appears dark at the locations lacking sample scattering can be created. For thin single-crystal samples, diffraction produces an image dotted pattern, which provides information about crystal orientation and space group symmetries in the crystal. In polycrystalline or amorphous materials diffraction causes a ring-pattern.143
8 Flow cytometry
424–426Fluorescence-based flow cytometry (FCM) is an effective technique for analyzing the physical and chemical characteristics of biological material, such as cells or other biological components in the same size range, based on their fluorescent characteristics. Thousands of cells can be quantitatively analyzed, counted and sorted each second,427 as the cells are illuminated by a laser beam and pass through an electronic detection device in a fluid stream, called the sheath fluid. The liquid flow is regulated so that the cells are well separated and only one cell at a time passes the fluorescence measuring station, making it possible to record the fluorescent signal of each individual cell.426 When the stream flows through the laser beam fluorescent molecules in or on the surface of the cell can be exited and emit light. The emitted light is detected by the forward scattering (FSC) and sideway scattering (SSC) detectors as well as by one or several fluorescence detectors. While FSC provides information about cell size or volume, SSC describes the granularity or inner structure of the cell.
Each cell produces a separate signal that is converted by an analog-to-digital conversion detector system to electrical signals that can be processed and analyzed by a computer. The acquired data can then be viewed on a linear or logarithmic scale as a two-dimensional dot plot or as a single-dimension histogram. Based on fluorescence intensity one or several regions of interest, i.e.
cell populations with similar properties, can be chosen or “gated” for analysis.424 When using fluorescence as the means to obtain information about a sample it is always important taking into consideration factors such as fluorochrome quenching, bleaching and photon saturation that might potentially interfere with the measurement.428
9 Confocal laser scanning microscopy
429,430Confocal laser scanning microscopy (CLSM) is a valuable instrument for non-destructive examination of thin (0.5-1.5 m) optical sections within or on the surface of a wide range of both living and fixed biological specimens. By scanning multiple layers, so-called stacks of the sample, also 3D-images of the object can be produced. Modern confocal fluorescence microscopes have 3-5 lasers with adjustable excitation intensity and several detectors, each able to collect emission in a certain wavelength range. One or several of the lasers and detectors may be operated simultaneously and the microscopes are able to detect fluorescence emission in the wavelength range 400-750 nm.431 Due to the wide array of existing fluorochromes suitable for labeling of biological specimens, a variety of molecules as well as cellular and sub-cellular components, can be fluorescently labeled and simultaneously identified and imaged with high precision using CLSM.
Figure 30. Schematic diagram of the optical pathway and main components in a CLSM.431
Within the instrument both the fluorescence and scattered light from the examined specimen is collected by the objective lens and passed forward to a beam splitter, which separates the light and carries only a part of it into the detector. The detector filtrates the light, letting through only the selected fluorescent wavelengths, while blocking the primary excitation wavelength.
The signal then passes through a pinhole in the conjugate plane, allowing only
light from the focal plane to enter the detector, thus, contributing to creating a sharper image. The final signal passes through a photodetection device, which converts the light into an electric signal that can be registered by a computer.430,431 The beam path and principal component of a CLSM is illustrated in Figure 30.
CLSM can additionally be used for studying varying cellular structures and dynamics by techniques, such as Stimulated emission depletion (STED) microscopy, Fluorescence-lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET) and Fluorescence recovery after photobleaching (FRAP). STED microscopy enables enhancement of the image resolution by minimizing the area of illumination at the focal point by selectively deactivating the peripheral fluorescence.432 This allows for highly detailed analysis of various structures in biological systems. In FLIM, the exponential decay, i.e. the lifetime of the fluorescent signal, instead of its intensity, is recorded and used for producing an image.433 The technique is useful e.g. when investigating the intracellular degradation of fluorescently labeled samples and has the advantage of reducing the effects of photon scattering in thicker samples. FRET describes a mechanism, where energy is transferred from a donor chromophore to an acceptor chromophore through radiationless dipole-dipole coupling.434 Since the distance required for interaction is extremely short (≤10 nm), FRET is highly useful for studying the colocalization of different structures. FRAP is performed by selectively illuminating a small area of a specimen at a low light intensity, while the emitted fluorescence is measured. 435 The illumination is then briefly increased to a very high level to rapidly bleach the fluorescent molecules in that region.
The recovery of fluorescence intensity, by diffusion of new unbleached fluorescent molecules into the bleached region, is then monitored to obtain information about the molecular transfer dynamics into the specified region.436