Second-harmonic imaging microscopy
SHIM has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function.
[1] 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, either inherent or induced externally, for example by an electric field.
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.
While some biological materials such as collagen, microtubules, and muscle myosin[3] can produce SHG signals, even water can become ordered and produce second-harmonic signal under certain conditions, which allows SH microscopy to image surface potentials without any labeling molecules.
Thus, 2PEF is a non coherent process, spatially (emitted isotropically) and temporally (broad, sample-dependent spectrum).
The first biological imaging experiments were done by Freund and Deutsch in 1986 to study the orientation of collagen fibers in rat tail tendon.
In 2006, Goro Mizutani group developed a non-scanning SHG microscope that significantly shortens the time required for observation of large samples, even if the two-photons wide-field microscope was published in 1996 [10] and could have been used to detect SHG.
The non-scanning SHG microscope was used for observation of plant starch,[11][12] megamolecule,[13] spider silk[14][15] and so on.
[16][17] In 2019, SHG applications widened when it was applied to the use of selectively imaging agrochemicals directly on leaf surfaces to provide a way to evaluate the effectiveness of pesticides.
In work done by Campagnola and Loew,[19] it was found that collagen fibers formed well-aligned structures with an
SHG being a coherent process (spatially and temporally), it keeps information on the direction of the excitation and is not emitted isotropically.
Therefore, thicker structures will appear preferentially in forward, and thinner ones in backward: since the SHG conversion depends at first approximation on the square of the number of nonlinear converters, the signal will be higher if emitted by thick structures, thus the signal in forward direction will be higher than in backward.
However, the tissue can scatter the generated light, and a part of the SHG in forward can be retro-reflected in the backward direction.
[20] Then, the forward-over-backward ratio F/B can be calculated,[20] and is a metric of the global size and arrangement of the SHG converters (usually collagen fibrils).
The orientation ϕ of the filaments in the plane XY of the image can also be extracted from p-SHG by FFT analysis, and put in a map.
[25][26] Collagen (particular case, but widely studied in SHG microscopy), can exist in various forms : 28 different types, of which 5 are fibrillar.
One of the challenge is to determine and quantify the amount of fibrillar collagen in a tissue, to be able to see its evolution and relationship with other non-collagenous materials.
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.
[29] SHG microscopy and its expansions can be used to study various tissues: some example images are reported in the figure below: collagen inside the extracellular matrix remains the main application.
[37] SHG is usually coupled to other nonlinear techniques such as Coherent anti-Stokes Raman Scattering or Two-photon excitation microscopy, as part of a routine called multiphoton microscopy (or tomography) that provides a non-invasive and rapid in vivo histology of biopsies that may be cancerous.
[38] The comparison of forward and backward SHG images gives insight about the microstructure of collagen, itself related to the grade and stage of a tumor, and its progression in breast.
[40] Even if SHG microscopy has contributed a lot to breast cancer research, it is not yet established as a reliable technique in hospitals, or for diagnostic of this pathology in general.
[39] The r ratio (see #Orientational anisotropy) is also used [41] to show that the alignment of fibrils is slightly higher for cancerous than for normal tissues.
[43] Changes in collagen ultrastructure in pancreatic cancer can be investigated by multiphoton fluorescence and polarization-resolved SHIM.
[44] SHG microscopy was reported for the study of lung, colonic, esophageal stroma and cervical cancers.
[45][46] In particular, the anisotropic alignment of collagen fibers allowed the discrimination of healthy dermis from pathological scars in skin.
[50] The ability of SHG to image specific molecules can reveal the structure of a certain tissue one material at a time, and at various scales (from macro to micro) using microscopy.
For instance, the collagen (type I) is specifically imaged from the extracellular matrix (ECM) of cells, or when it serves as a scaffold or conjonctive material in tissues.
[51] Also, SHG coupled to other multiphoton techniques can serve to monitor the development of engineered tissues, when the sample is relatively thin however.
