| Confocal laser scanning microscopy: CLSM is a technique for obtaining higher resolution images compared to classical video-microscopy with the ability to perform optical sectioning by adding a spatial pinhole in the confocal plane. It enables the 3D reconstruction of acquired objects from a vertical series of images. It was first patented in 1957 by Marvin Minsky and became continuously used in biological science from the 90th [70]. |
| Fluorescence correlation spectroscopy: in FCS, a laser beam is focused on the sample. The measured intensity fluctuations are analyzed by temporal autocorrelation. This analysis can be applied to really low concentration (nM range) of proteins and gives quantitative concentration and diffusion coefficient of fluorescent particle as well as the abnormality of the medium [71]. |
| Fluorescence recovery after photobleaching: FRAP consists in irreversibly inhibiting fluorescence in a restraint area of the sample by very fast exposure of this region to a high intensity laser creating two spatially distinct molecules population: fluorescent or bleached. If the molecules can move, there will be a redistribution of molecules until homogenization of population, and thus a fluorescence recovery in the bleached area. Analyzing this recovery give the diffusion coefficient and the mobile/immobile fraction of analyzed molecules [72]. |
| Genetically encoded biosensors: are a protein sequence flanked by fluorescent proteins, whose peptide sequence are sensitive to the presence of small molecules or to proteins activity that will alter its fluorescence properties. They can be introduced in cells, tissues or organisms and permits in vivo long term investigation of signaling pathways in space and time [73]. |
| Green fluorescent protein: GFP is an intrinsically fluorescent protein arising from a jellyfish (Aequoreavictoria). Its gene can be fused in-vitro to the gene of a protein of interest. This recombinant gene can then be introduced in a cell. A fluorescent fusion protein will then be synthesized and can be localized in-vivo using fluorescence microscopy. Variant from the GFP with different excitation and excitation properties allows following several proteins at the same time [74]. |
| Second harmonic generation: SHG is a second-order nonlinear phenomenon in which a fundamental wave is partially converted into a second-harmonic wave with half the initial wavelength. It appears under two photons excitation. It doesn't need any absorption process and no fluorescence labeling is necessary. It can be measured at exactly the half of excitation wavelength and the signal comes specifically from molecules which don’t show any centrosymmetry. It is thus of major interest for probing structures with high degree of orientation and organization either from extrinsic or intrinsic harmonophore such has collagen or tubulin [75]. |
| Two photons excitation microscopy: fluorescence imaging technique in which two photons carrying half the energy needed for traditional excitation can excite a fluorophore in one quantum event. From a practical point of view, it uses pulsed and red-shifted excitation sources, reducing scattering in the tissue and phototoxicity while increasing penetration depth compared to conventional fluorescence techniques [70,75]. It also benefits from optical sectioning capability while if the system is correctly set, the two photon effect only occurs at the focal plane. |
| Third harmonic generation: THG is a third-order nonlinear phenomenon in the fundamental wave is partially converted into a third harmonic wave with one third of the original wavelength. Contrarily to SHG, no geometrical rules are required and thus all material potentially can be used for THG. However, while THG signal is intrinsically weak, it is mainly used to image specific molecules such as lipids, myosins or collagen [75]. |
