Linear dichroism (LD) or diattenuation is the difference between

absorption Absorption may refer to: Chemistry and biology *Absorption (biology), digestion **Absorption (small intestine) *Absorption (chemistry), diffusion of particles of gas or liquid into liquid or solid materials *Absorption (skin), a route by which su ...

of light polarized parallel and polarized perpendicular to an orientation axis. It is the property of a material whose

transmittance Electromagnetic radiation can be affected in several ways by the medium in which it propagates. It can be Scattering, scattered, Absorption (electromagnetic radiation), absorbed, and Fresnel equations, reflected and refracted at discontinui ...

depends on the orientation of

linearly polarized In electrodynamics, linear polarization or plane polarization of electromagnetic radiation is a confinement of the electric field vector or magnetic field vector to a given plane along the direction of propagation. The term ''linear polarizati ...

light incident upon it. As a technique, it is primarily used to study the functionality and structure of

molecule A molecule is a group of two or more atoms that are held together by Force, attractive forces known as chemical bonds; depending on context, the term may or may not include ions that satisfy this criterion. In quantum physics, organic chemi ...

s. LD measurements are based on the interaction between matter and light and thus are a form of electromagnetic

spectroscopy Spectroscopy is the field of study that measures and interprets electromagnetic spectra. In narrower contexts, spectroscopy is the precise study of color as generalized from visible light to all bands of the electromagnetic spectrum. Spectro ...

. This effect has been applied across the EM spectrum, where different

wavelength In physics and mathematics, wavelength or spatial period of a wave or periodic function is the distance over which the wave's shape repeats. In other words, it is the distance between consecutive corresponding points of the same ''phase (waves ...

s of light can probe a host of chemical systems. The predominant use of LD currently is in the study of bio-

macromolecules A macromolecule is a "molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass." Polymers are physi ...

(e.g.

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

) as well as synthetic

polymers A polymer () is a substance or material that consists of very large molecules, or macromolecules, that are constituted by many repeating subunits derived from one or more species of monomers. Due to their broad spectrum of properties, b ...

Basic information

Linear polarization

LD uses

light, which is light that has been polarized in one direction only. This produces a wave, the

electric field vector An electric field (sometimes called E-field) is a physical field that surrounds electrically charged particles such as electrons. In classical electromagnetism, the electric field of a single charge (or group of charges) describes their capacit ...

, which oscillates in only one plane, giving rise to a classic

sinusoidal wave A sine wave, sinusoidal wave, or sinusoid (symbol: ∿) is a periodic wave whose waveform (shape) is the trigonometric sine function. In mechanics, as a linear motion over time, this is '' simple harmonic motion''; as rotation, it corresponds ...

shape as the light travels through space. By using light parallel and perpendicular to the orientation direction it is possible to measure how much more energy is absorbed in one dimension of the molecule relative to the other, providing information to the experimentalist. As light interacts with the molecule being investigated, should the molecule start absorbing the light then electron density inside the molecule will be shifted as the electron becomes photoexcited. This movement of charge is known as an

electronic transition In atomic physics and chemistry, an atomic electron transition (also called an atomic transition, quantum jump, or quantum leap) is an electron changing from one energy level to another within an atom or artificial atom. The time scale of a qua ...

, the direction of which is called the electric transition polarisation. It is this property for which LD is a measurement. The LD of an oriented molecule can be calculated using the following equation:- :LD = A_║- A_┴ Where A_║ is the

absorbance Absorbance is defined as "the logarithm of the ratio of incident to transmitted radiant power through a sample (excluding the effects on cell walls)". Alternatively, for samples which scatter light, absorbance may be defined as "the negative log ...

parallel to the orientation axis and A_┴ is the absorbance perpendicular to the orientation axis. Note that light of any wavelength can be used to generate an LD signal. The LD signal generated therefore has two limits upon the signal that can be generated. For a chemical system whose electric transition is parallel to the orientation axis, the following equation can be written: :LD = A_║- A_┴ = A_║ > 0 For most chemical systems this represents an electric transition polarised across the length of the molecule (i.e. parallel to the orientation axis). Alternatively, the electric transition polarisation can be found to be perfectly perpendicular to the orientation of the molecule, giving rise to the following equation: :LD = A_║- A_┴ = - A_┴ < 0 This equation represents the LD signal recorded if the electric transition is polarised across the width of the molecule (i.e. perpendicular to the orientation axis), which in the case of LD is the smaller of the two investigable axes. LD can therefore be used in two ways. If the orientation of the molecules in flow is known, then the experimentalist can look at the direction of polarisation in the molecule (which gives an insight into the chemical structure of a molecule), or if the polarisation direction is unknown it can be used as a means of working out how oriented in flow a molecule is.

UV linear dichroism

Ultraviolet (UV) LD is typically employed in the analysis of biological molecules, especially large, flexible, long molecules that prove difficult to structurally determine by such methods as

NMR Nuclear magnetic resonance (NMR) is a physical phenomenon in which atomic nucleus, nuclei in a strong constant magnetic field are disturbed by a weak oscillating magnetic field (in the near and far field, near field) and respond by producing ...

and

X-ray diffraction X-ray diffraction is a generic term for phenomena associated with changes in the direction of X-ray beams due to interactions with the electrons around atoms. It occurs due to elastic scattering, when there is no change in the energy of the waves. ...

DNA

is almost ideally suited for UV LD detection. The molecule is very long and very thin, making it very easy to orient in flow. This gives rise to a strong LD signal. DNA systems that have been studied using UV LD include DNA-

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

complexes and DNA-

ligand In coordination chemistry, a ligand is an ion or molecule with a functional group that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's el ...

complexes, the formation of the latter being easily observable through kinetic experiments.

Fibrous protein

Fibrous protein In molecular biology, fibrous proteins or scleroproteins are one of the three main classifications of protein structure (alongside globular and membrane proteins). Fibrous proteins are made up of elongated or fibrous polypeptide chains which for ...

s, such as proteins involved in Alzheimer's disease and

prion A prion () is a Proteinopathy, misfolded protein that induces misfolding in normal variants of the same protein, leading to cellular death. Prions are responsible for prion diseases, known as transmissible spongiform encephalopathy (TSEs), w ...

proteins fulfil the requirements for UV LD in that they are a class of long, thin molecules. In addition,

cytoskeletal The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all Cell (biology), cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane ...

proteins can also be measured using LD.

Membrane proteins

The insertion of

membrane protein Membrane proteins are common proteins that are part of, or interact with, biological membranes. Membrane proteins fall into several broad categories depending on their location. Integral membrane proteins are a permanent part of a cell membrane ...

s into a

lipid membrane The lipid bilayer (or phospholipid bilayer) is a thin polar membrane made of two layers of lipid molecules. These membranes form a continuous barrier around all cells. The cell membranes of almost all organisms and many viruses are made of a l ...

has been monitored using LD, supplying the experimentalist with information about the orientation of the protein relative to the lipid membrane at different time points. In addition, other types of molecule have been analysed by UV LD, including

carbon nanotube A carbon nanotube (CNT) is a tube made of carbon with a diameter in the nanometre range ( nanoscale). They are one of the allotropes of carbon. Two broad classes of carbon nanotubes are recognized: * ''Single-walled carbon nanotubes'' (''S ...

s and their associated ligand complexes.

Alignment methods

Couette flow

The

Couette flow In fluid dynamics, Couette flow is the flow of a viscosity, viscous fluid in the space between two surfaces, one of which is moving tangentially relative to the other. The relative motion of the surfaces imposes a shear stress on the fluid and ind ...

orientation system is the most widely used method of sample orientation for UV LD. It has a number of characteristics which make it highly suitable as a method of sample alignment. Couette flow is currently the only established means of orientating molecules in the solution phase. This method also requires only very small amounts of analysis sample ( 20 - 40 μL) in order to generate an LD spectrum. The constant recirculation of sample is another useful property of the system, allowing many repeat measurements to be taken of each sample, decreasing the effect of noise on the final recorded spectrum. Its mode of operation is very simple, with the sample sandwiched between a spinning tube and a stationary rod. As the sample is spun inside the cell, the light beam is shone through the sample, the parallel absorbance calculated from horizontally polarised light, the perpendicular absorbance from the vertically polarised light. Couette flow UV LD is currently the only commercially available means of LD orientation.

Stretched film

Stretched film linear dichroism is a method of orientation based on incorporating the sample molecules into a polyethylene film. The polyethylene film is then stretched, causing the randomly oriented molecules on the film to ‘follow’ the movement of the film. The stretching of the film results in the sample molecules being oriented in the direction of the stretch.

Associated techniques

Circular Dichroism

LD is very similar to

Circular Dichroism Circular dichroism (CD) is dichroism involving circular polarization, circularly polarized light, i.e., the differential Absorption (electromagnetic radiation), absorption of left- and right-handed light. Left-hand circular (LHC) and right-hand ci ...

(CD), but with two important differences. (i) CD spectroscopy uses circularly polarized light whereas LD uses linearly polarized light. (ii) In CD experiments molecules are usually free in solution so they are randomly oriented. The observed spectrum is then a function only of the

chiral Chirality () is a property of asymmetry important in several branches of science. The word ''chirality'' is derived from the Greek language, Greek (''kheir''), "hand", a familiar chiral object. An object or a system is ''chiral'' if it is dist ...

or asymmetric nature of the molecules in the solution. With biomacromolecules CD is particularly useful for determining the secondary structure. By way of contrast, in LD experiments the molecules need to have a preferential orientation otherwise the LD=0. With biomacromolecules flow orientation is often used, other methods include stretched films, magnetic fields, and squeezed gels. Thus LD gives information such as alignment on a surface or the binding of a small molecule to a flow-oriented macromolecule, endowing it with different functionality from other spectroscopic techniques. The differences between LD and CD are complementary and can be a potent means for elucidating the structure of biological molecules when used in conjunction with one another, the combination of techniques revealing far more information than a single technique in isolation. For example, CD tells us when a membrane peptide or protein folds whereas LD tells when it inserts into a membrane.

Fluorescence detected Linear Dichroism

Fluorescence Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with colore ...

-detected linear dichroism (FDLD) is a very useful technique to the experimentalist as it combines the advantages of UV LD whilst also offering the

confocal In geometry, confocal means having the same foci: confocal conic sections. * For an optical cavity consisting of two mirrors, confocal means that they share their foci. If they are identical mirrors, their radius of curvature, ''R''mirror, equals ' ...

detection of the fluorescence emission.Gabor Steinbach, Istvan Pomozi, Otto Zsiros, Aniko Pay, Gabor V. Horvat, Gyozo Garab ‘Imaging Fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope’ 2008, Cytometry Part A, 73A : 202-208. FDLD has applications in microscopy, where can be used as a means of two-dimensional surface mapping through differential polarisation spectroscopy (DPS) where the

anisotropy Anisotropy () is the structural property of non-uniformity in different directions, as opposed to isotropy. An anisotropic object or pattern has properties that differ according to direction of measurement. For example, many materials exhibit ve ...

of the scanned object allows an image to be recorded. FDLD can also be used in conjunction with intercalating fluorescent dyes (which can also be monitored using UV LD). The intensity difference recorded between the two types of polarised light for the fluorescence reading is proportional to the UV LD signal, allowing the use of DPS to image surfaces

References

Spectroscopy