Second harmonic imaging microscopy
Encyclopedia
Second-harmonic imaging microscopy (SHIM) is based on a nonlinear optical effect known as second-harmonic generation (SHG). SHIM has been established as a viable microscope
imaging contrast mechanism for visualization of cell
and tissue
structure and function. A second-harmonic microscope obtains contrasts from variations in a specimen’s ability to generate second-harmonic light from the incident light while a conventional optical microscope obtains its contrast by detecting variations in optical density, path length, or refractive index
of the specimen. SHG requires intense laser
light passing through a material with a noncentrosymmetric molecular structure. Second-harmonic light emerging from an SHG material is exactly half the wavelength (frequency doubled) of the light entering the material. While two-photon-excited fluorescence
(TPEF) is also a two photon process, TPEF loses some energy during the relaxation of the excited state, and SHG is energy conserving. Typically, an inorganic crystal is used to produce SHG light such as lithium niobate
(LiNbO3), potassium titanyl phosphate
(KTP = KTiOPO4), and lithium triborate
(LBO = LiB3O5). Though SHG requires a material to have specific molecular orientation in order for the incident light to be frequency doubled, some biological materials can be highly polarizable, and assemble into fairly ordered, large noncentrosymmetric structures. Biological materials such as collagen, microtubules, and muscle myosin
can produce SHG signals. The SHG pattern is mainly determined by the phase matching condition. A common setup for an SHG imaging system will have a laser scanning microscope with a titanium sapphire
mode-locked
laser as the excitation source. The SHG signal is propagated in the forward direction. However, some experiments have shown that objects on the order of about a tenth of the wavelength
of the SHG produced signal will produce nearly equal forward and backward signals.
. Also, since many biological structures produce strong SHG signals, the labeling of molecules with exogenous
probes is not required which can also alter the way a biological system functions. By using near infrared wavelengths for the incident light, SHIM has the ability to construct three dimensional
images of specimens by imaging deeper into thick tissues.
imaged various SHG crystals with a scanning optical microscope. The first biological imaging experiments were done by Freund in 1986 to study the orientation of collagen
fibers in rat
tail tendon
. In 1993, Lewis examined the second-harmonic response of styryl dye
s in electric field
s. He also showed work on imaging live cells.
In 2010 SHG was extended to whole-animal in vivo
imaging.
can be used to determine the orientation and degree of organization of proteins in tissues since SHG signals have well-defined polarizations. By using the anisotropy equation:
and acquiring the intensities of the polarizations in the parallel and perpendicular directions. A high
value indicates an anisotropic orientation where as a low 
value indicates an isotropic structure. In work done by Campagnola and Loew, it was found that collagen fibers formed well-aligned structures with an 
value.
It has also been used to prove that backpropagating action potentials invade dendritic spines without voltage attenuation, establishing a sound basis for future work on Long-term potentiation
. Its use here was that it provided a way to accurately measure the voltage in the tiny dendritic spines with an accuracy unattainable with standard two-photon microscopy.
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
imaging contrast mechanism for visualization of cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
and tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
structure and function. A second-harmonic microscope obtains contrasts from variations in a specimen’s ability to generate second-harmonic light from the incident light while a conventional optical microscope obtains its contrast by detecting variations in optical density, path length, or refractive index
Refractive index
In optics the refractive index or index of refraction of a substance or medium is a measure of the speed of light in that medium. It is expressed as a ratio of the speed of light in vacuum relative to that in the considered medium....
of the specimen. SHG requires intense laser
Laser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
light passing through a material with a noncentrosymmetric molecular structure. Second-harmonic light emerging from an SHG material is exactly half the wavelength (frequency doubled) of the light entering the material. While two-photon-excited fluorescence
Two-photon excitation microscopy
Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite...
(TPEF) is also a two photon process, TPEF loses some energy during the relaxation of the excited state, and SHG is energy conserving. Typically, an inorganic crystal is used to produce SHG light such as lithium niobate
Lithium niobate
Lithium niobate is a compound of niobium, lithium, and oxygen. Its single crystals are an important material for optical waveguides, mobile phones, optical modulators and various other linear and non-linear optical applications.-Properties:...
(LiNbO3), potassium titanyl phosphate
Potassium titanyl phosphate
Potassium titanyl phosphate or KTP is a nonlinear optical material which is commonly used for frequency doubling diode pumped solid-state lasers such as Nd:YAG and other neodymium-doped lasers. The material has a relatively high optical damage threshold , a great optical nonlinearity and excellent...
(KTP = KTiOPO4), and lithium triborate
Lithium triborate
Lithium triborate LBO is a non-linear optics crystal. It has a wide transparency range, moderately high nonlinear coupling, high damage threshold and desirable chemical and mechanical properties. This crystal is often used for second harmonic generation of Nd:YAG lasers...
(LBO = LiB3O5). Though SHG requires a material to have specific molecular orientation in order for the incident light to be frequency doubled, some biological materials can be highly polarizable, and assemble into fairly ordered, large noncentrosymmetric structures. Biological materials such as collagen, microtubules, and muscle myosin
Myosin
Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. They are responsible for actin-based motility. The term was originally used to describe a group of similar...
can produce SHG signals. The SHG pattern is mainly determined by the phase matching condition. A common setup for an SHG imaging system will have a laser scanning microscope with a titanium sapphire
Ti-sapphire laser
Ti:sapphire lasers are tunable lasers which emit red and near-infrared light in the range from 650 to 1100 nanometers. These lasers are mainly used in scientific research because of their tunability and their ability to generate ultrashort pulses...
mode-locked
Modelocking
Mode-locking is a technique in optics by which a laser can be made to produce pulses of light of extremely short duration, on the order of picoseconds or femtoseconds ....
laser as the excitation source. The SHG signal is propagated in the forward direction. However, some experiments have shown that objects on the order of about a tenth of the wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...
of the SHG produced signal will produce nearly equal forward and backward signals.
Advantages
SHIM offers several advantages for live cell and tissue imaging. SHG does not involve the excitation of molecules like other techniques such as fluorescence microscopy therefore, the molecules shouldn’t suffer the effects of phototoxicity or photobleachingPhotobleaching
Photobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing...
. Also, since many biological structures produce strong SHG signals, the labeling of molecules with exogenous
Exogenous
Exogenous refers to an action or object coming from outside a system. It is the opposite of endogenous, something generated from within the system....
probes is not required which can also alter the way a biological system functions. By using near infrared wavelengths for the incident light, SHIM has the ability to construct three dimensional
3D computer graphics
3D computer graphics are graphics that use a three-dimensional representation of geometric data that is stored in the computer for the purposes of performing calculations and rendering 2D images...
images of specimens by imaging deeper into thick tissues.
History
Before SHG was used for imaging, the first demonstration of SHG was performed in 1961 by P. A. Franken, G. Weinriech, C. W. Peters, and A. E. Hill at the University of Michigan, Ann Arbor using a quartz sample. In 1968, SHG from interfaces was discovered by Bloembergen and has since been used as a tool for characterizing surfaces and probing interface dynamics. In 1974, Hellwarth and Christensen first reported the integration of SHG and microscopy by imaging SHG signals from polycrystalline ZnSe. In 1977, Colin SheppardColin Sheppard
Colin Sheppard is Professor in the Division of Bioengineering and Faculty of Engineering for National University of Singapore . He has held joint appointments with the Departments of Biological Sciences and Diagnostic Radiology...
imaged various SHG crystals with a scanning optical microscope. The first biological imaging experiments were done by Freund in 1986 to study the orientation of collagen
Collagen
Collagen is a group of naturally occurring proteins found in animals, especially in the flesh and connective tissues of mammals. It is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content...
fibers in rat
Rat
Rats are various medium-sized, long-tailed rodents of the superfamily Muroidea. "True rats" are members of the genus Rattus, the most important of which to humans are the black rat, Rattus rattus, and the brown rat, Rattus norvegicus...
tail tendon
Tendon
A tendon is a tough band of fibrous connective tissue that usually connects muscle to bone and is capable of withstanding tension. Tendons are similar to ligaments and fasciae as they are all made of collagen except that ligaments join one bone to another bone, and fasciae connect muscles to other...
. In 1993, Lewis examined the second-harmonic response of styryl dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....
s in electric field
Electric field
In physics, an electric field surrounds electrically charged particles and time-varying magnetic fields. The electric field depicts the force exerted on other electrically charged objects by the electrically charged particle the field is surrounding...
s. He also showed work on imaging live cells.
In 2010 SHG was extended to whole-animal in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
imaging.
Applications
SHG polarization anisotropyAnisotropy
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...
can be used to determine the orientation and degree of organization of proteins in tissues since SHG signals have well-defined polarizations. By using the anisotropy equation:
and acquiring the intensities of the polarizations in the parallel and perpendicular directions. A high
value indicates an anisotropic orientation where as a low value indicates an isotropic structure. In work done by Campagnola and Loew, it was found that collagen fibers formed well-aligned structures with an value.It has also been used to prove that backpropagating action potentials invade dendritic spines without voltage attenuation, establishing a sound basis for future work on Long-term potentiation
Long-term potentiation
In neuroscience, long-term potentiation is a long-lasting enhancement in signal transmission between two neurons that results from stimulating them synchronously. It is one of several phenomena underlying synaptic plasticity, the ability of chemical synapses to change their strength...
. Its use here was that it provided a way to accurately measure the voltage in the tiny dendritic spines with an accuracy unattainable with standard two-photon microscopy.
Sources
- P. J. Campagnola, H.A. Clark, W.A. Mohler, A. Lewis and L.M. Loew, “Second-harmonic Imaging Microscopy of Living Cells,“ J. Biomed. Opt. 6, 277–286 (2001)
- P. J. Campagnola, H.A. Clark, W.A. Mohler, A. Lewis and L.M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,“ Nature Biotech. 21, 1356–1360 (2003)
- P. Stoller, K.M. Reiser, P.M. Celliers, & A.M. Rubenchik, “Polarization-modulated second harmonic generation in collagen.” Biophys. J. 82, 3330–3342 (2002)
- M. Han, G. Giese, and J. F. Bille, “Second harmonic generation imaging of collagen fibrils in cornea and sclera,” Opt. Express 13, 5791–5797 (2005)