| Abstract [eng] |
Nonlinear microscopy utilizes light phenomena occurring in electromagnetic fields of extremely high intensity to provide new imaging capabilities. Multi-photon excitation fluorescence (MPEF) and second harmonic generation (SHG) are among them. The former occurs when a pigment molecule absorbs two or more photons and emits one of higher frequency, but the latter only appears in specific materials, which are lacking the center of symmetry. This property is not common in nature, especially in live organisms. However fibrillar proteins myosin and collagen are capable of SHG and require no chemical processing to be discernible. Since these proteins have vital roles, ability to noninvasively inspect them leads to potential use cases in medicine. Since inception of nonlinear microscopy, exclusively only scanning microscopes were used. Biological matter is translucent to near infrared light (commonly used in nonlinear microscopy) so deeper penetration into the sample is possible. Effectiveness of second-order nonlinear phenomena is proportional to the square of excitation light intensity, thus optical sectioning can be achieved, which enables 3D imaging without a pinhole. However, a scanning microscope is inherently slow, because it can only image one point at a time. Wide field microscopy, on the other hand, captures the whole image at once and is much faster. This technology can find it's use in real-time imaging of kinetic processes, e.g. muscle contraction, or high throughput of histological samples in medical environment. Polarimetric SHG methods are more sensitive to subtle molecular structure alterations, usually not visible by other means. The less a disease is developed, the easier it is to treat it. |
