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# 5: Experimental Characterization - Spectroscopy & Microscopy

• 5.1: Model Membranes vs. Biological Membranes
Model-based studies have proven to be very useful in understanding basic biophysical properties of biological membranes. They provide a foundation upon which new hypotheses can be generated and tested in living cells. A solid foundational understanding of simple synthetic membranes should allow scientists to develop experimental and computational tools to test more complex properties of biological membranes.
• 5.2: Supported and Tethered Membranes
Model lipid bilayers or synthetic lipid bilayers are membranes that are created in vitro. In vivo studies of lipid bilayers and the proteins embedded within them can be difficult due to the complexity of the cellular dynamics of membranes. Model bilayers use biological membranes, along with artificial constraints, to investigate the structure and function of lipid bilayers. Supported and tethered membranes are two types of model bilayers frequently used in model studies.
• 5.3: Styrene Maleic Acid Lipid Particles (SMALP) Technology
Styrene maleic acid lipid particles (SMALPs) are disc-shaped lipid assemblies on the nano-scale made from the interaction of membrane bilayers and styrene maleic acid (SMA) copolymer. SMALP technology holds great potential as a biochemical tool as it allows solubilization of membrane bilayers and its embedded constituent into discs without any use of detergents. SMALPs are thought to be superior to other disc-forming techniques.
• 5.4: Lipid Probes
Lipid labeling offers an alternative to destructive methods and allow for measurement made in native environments without immobilization. There are three major types of lipid probes: fluorescent probes can be used for identifying target membranes or lipids, spin-probes are useful for measuring membrane dynamics, and isotopic probes are often used for membrane composition analysis.
• 5.5: Fluorescence on Membranes
The study of membranes poses a unique challenge in science due to their complex and fluid nature. Until recently, the majority of experiments was conducted in either fixed samples or on population levels that did not give insight into individual events. However, recent advances in fluorescence microscopy techniques have allowed scientists to visualize microscopic perturbations of individual vesicles and have allowed the study of membrane dynamics at higher spatiotemporal resolution than before.
• 5.6: Near-field scanning optical microscopy (NSOM)
Near-field scanning optical microscopy(NSOM), also called scanning near-field optical microscopy(SNOM), is a scanning probe technique that overcomes the diffraction barrier in traditional far-field optical microscopy. Conventional optical microscopy techniques are limited by the diffraction of the light and the resolution is limited to roughly 250 nm, which make it very difficult to resolve the domains or clusters in the cellular membranes.
• 5.7: Single Molecule Tracking
Single Molecule Tracking (SMT), also known as Single Particle Tracking (SPT) or Single Dye Tracking (SDT), refers to a class of non-invasive techniques that involve direct spatial observation of individual molecular or particle entities as a function of time. This Wiki page offers a broad overview of the some of the working principles, technical aspects, and biophysical applications of SMT.
• 5.8: FTIR on Membranes
Fourier Transform Infrared (FTIR) Spectroscopy is a widespread, relatively cheap technique for studying the structure of compounds through chemical bond vibrations. Since there are already pages dedicated to FTIR and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) this page will not go into the details of the theory associated with these techniques, but will instead describe these techniques in relation to lipid membranes.
• 5.9: Raman Spectroscopy on Membranes
Biochemical compounds of interest like lipids have been studied and their corresponding Raman shifts have been characterized. Lipids are characterized as oily molecules that are soluble in non-polar solvents and are not miscible in water. Some common lipids that can be studied are Cholesterol, saturated and fatty acids, Triglycerides and Phospholipids and many more.
• 5.10: Nuclear Magnetic Resonance (NMR) Theory & Solution NMR
Nuclear magnetic resonance (NMR) occurs when nuclei in an unmoving magnetic field are disturbed by an oscillating magnetic field; the nuclei generate an electromagnetic signal, whose frequency depends on the magnetic field applied. This happens near resonance, where the frequency of oscillation aligns with the frequency of the nuclei. The magnetic field strength, chemical environment, and isotope affect resonance.
• 5.11: Solid-state NMR
The ssNMR studies both isotropic and anisotropic interactions.  One can interpret the spectrum to understand interactions between nuclei and further construct the structure of condensed matter. It can be used as a powerful tool to solve membrane protein structures.
• 5.12: Electron Paramagnetic Resonance (EPR) of Membranes
Electron paramagnetic resonance (EPR) is a technique with applications in multiple branches of science, including physics, biology, and chemistry. The basic concepts of EPR are known to be analogous with Solid-state NMR, except it is electron spins that are excited as opposed to spins of atomic nuclei.
• 5.13: Membrane X-ray Scattering
Membrane scattering encompasses a huge variety of methods for characterizing lipid membranes. For example, a few popular techniques include X-ray or neutron reflectivity and diffraction, Brewster angle microscopy, ellipsometry, X-ray interferometry, infrared reflection-adsorption spectroscopy, and vibrational sum frequency generation spectroscopy. These methods can be used with a variety of different model membranes types.

Thumbnail: S. cerevisiae septins revealed with fluorescent microscopy utilizing fluorescent labeling. Image used with permission (Public Domain, Spitfire ch,).