Furthermore, NLO processes based on multi-photon excitation overcome the use of staining, allowing one to obtain rich morphological/structural/molecular information from a sample, which shows nonlinear properties and/or distinctive chemical composition. Technological advances consist in the addition of automated scanners, vertical stages, high numerical aperture objectives (oil or water immersion), and corrective pinholes and filters to detect fluorescence over the volume of the specimen (500 μm) since the infrared excitation wavelengths inherently provide a reduced light scattering and absorption ( Konig, 2000 Sanderson et al., 2016). By exciting the fluorescent species with a lamp or a laser, it is possible to filter the longer wavelength (Stokes shifted) emitted signal, detecting it in either a widefield or a point scanning approach and reconstructing images ( Murphy, 2001 Lichtman and Conchello, 2005). In this technique, highly specific light-emitting probes (dye molecules, semiconductor nanoparticles, or fluorescent proteins) chemically bound to specific biological targets (i.e., DNA, phospholipids, and proteins) are administered to the live or fixed samples. Another common technique to enrich the information content achievable from a vital biological sample is fluorescence microscopy ( Combs and Shroff, 2017). To gain more details, biological specimens can be characterized by means of cyto-histopathological assays, which however, require fixative procedure to color specific cellular structures, thus affecting vitality. Nevertheless, these methods provide low amount of information from live samples, being limited to qualitative estimation on cell morphology and growth (e.g., % area occupied by the cells over time in the field of view) and being capable of simply distinguishing endoplasmic reticulum, nucleus and vesicles. However, monolayered cells appears transparent and contrast methods, like phase contrast microscopy, differential interference contrast microscopy or digital holography, are necessary to distinguish cells or parts of them ( Murphy, 2001). Among the existing microscopy techniques, brightfield microscopy is suitable to observe unlabelled vital and thin samples, and it is the easiest to use. The power of optical microscopy is to provide a non-invasive morphological and functional characterization of the observed living specimen over time ( Stephens, 2003). Since researchers are concerned to find high speed, effective, and non-invasive diagnostic tools to identify the state of biological samples, several micro-scale imaging techniques have been developed recently, aiming at applications usable by clinicians on human patients ( König et al., 2019). One of the most fascinating advancements in bioengineering is the possibility to observe and control the microscopic universe of cells in order to understand the biological mechanisms involved in physiological and pathological phenomena of life ( Vo-Dinh, 2003). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. 3Department of Physics, Politecnico di Milano, Milan, ItalyĪ recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode.2Istituto di Fotonica e Nanotecnologie (IFN) – CNR, Milan, Italy. ![]() Natta”, Politecnico di Milano, Milan, Italy
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