Linear dichroism
Encyclopedia
Linear dichroism is a spectroscopic technique that is primarily used to study the functionality and structure of molecules. LD can be defined as the difference in absorption of light polarized parallel and perpendicular to an orientation axis . LD measurements are based on the interaction between matter and light and thus are a form of electromagnetic spectroscopy
.
This effect has been applied across the EM spectrum, where different wavelengths of light can probe a host of chemical systems. The predominant use of LD currently is in the study of bio-macromolecules (e.g. DNA) as well as synthetic polymers.
LD uses linearly polarized light, which is light that has been polarized in one direction only. This produces a wave, the electric field vector, which oscillates in only one plane, giving rise to a classic sinusoidal wave 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 photo-excited. This movement of charge is known as an electronic transition, 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 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 known it can be used as a means of working out how oriented in flow a molecule is.
DNA
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 complexes and DNA-ligand complexes, the formation of the latter being easily observable through kinetic experiments.
Fibrous protein
Fibrous proteins, such as proteins involved in Alzheimer’s disease and prion proteins fulfil the requirements for UV LD in that they are a class of long, thin molecules. In addition, cyto-skeletal proteins can also be measured using LD.
Membrane proteins
The insertion of membrane proteins into a lipid membrane 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 nano-tubes and their associated ligand complexes.
The Couette flow
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 background 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.
LD is very similar to Circular Dichroism
(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
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 can 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
-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
detection of the fluorescence emission. 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
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
IR Linear Dichroism
X-Ray Linear Dichroism Microscopy
Spectroscopy
Spectroscopy is the study of the interaction between matter and radiated energy. Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, e.g., by a prism. Later the concept was expanded greatly to comprise any interaction with radiative...
.
This effect has been applied across the EM spectrum, where different wavelengths of light can probe a host of chemical systems. The predominant use of LD currently is in the study of bio-macromolecules (e.g. DNA) as well as synthetic polymers.
Basic information
Linear polarizationLD uses linearly polarized light, which is light that has been polarized in one direction only. This produces a wave, the electric field vector, which oscillates in only one plane, giving rise to a classic sinusoidal wave 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 photo-excited. This movement of charge is known as an electronic transition, 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 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 known it can be used as a means of working out how oriented in flow a molecule is.
UV linear dichroism
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 and X-ray diffraction.DNA
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 complexes and DNA-ligand complexes, the formation of the latter being easily observable through kinetic experiments.
Fibrous protein
Fibrous proteins, such as proteins involved in Alzheimer’s disease and prion proteins fulfil the requirements for UV LD in that they are a class of long, thin molecules. In addition, cyto-skeletal proteins can also be measured using LD.
Membrane proteins
The insertion of membrane proteins into a lipid membrane 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 nano-tubes and their associated ligand complexes.
Alignment methods
Couette flowThe Couette flow
Couette flow
In fluid dynamics, Couette flow refers to the laminar flow of a viscous fluid in the space between two parallel plates, one of which is moving relative to the other. The flow is driven by virtue of viscous drag force acting on the fluid and the applied pressure gradient parallel to the plates...
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 background 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 DichroismLD is very similar to Circular Dichroism
Circular dichroism
Circular dichroism refers to the differential absorption of left and right circularly polarized light. This phenomenon was discovered by Jean-Baptiste Biot, Augustin Fresnel, and Aimé Cotton in the first half of the 19th century. It is exhibited in the absorption bands of optically active chiral...
(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 (chemistry)
A chiral molecule is a type of molecule that lacks an internal plane of symmetry and thus has a non-superimposable mirror image. The feature that is most often the cause of chirality in molecules is the presence of an asymmetric carbon atom....
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 can 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
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
-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
Confocal
In geometry, confocal means having the same foci.* For an optical cavity consisting of two mirrors, confocal means that they share their foci...
detection of the fluorescence emission. 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
Anisotropy is the property of being directionally dependent, as opposed to isotropy, which implies identical properties in all directions. It can be defined as a difference, when measured along different axes, in a material's physical or mechanical properties An example of anisotropy is the light...
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
IR Linear Dichroism
X-Ray Linear Dichroism Microscopy