All three conditions can be satisfied using established techniques in linear optical microscopy.
Ideally optical microscopy should provide high lateral spatial resolution, three-dimensional volume sectioning, and high image contrast. Optical microscopy is a ubiquitous imaging technique that has found widespread use across the life sciences and is emerging as a highly valuable tool in biomedical applications. II. Nonlinear Optical Imaging (NLO) Techniques Fluorophores commonly used in combination with NLO fluorescence imaging will also be discussed. In this article, we discuss the emergence of NLO imaging and how it has been facilitated by advances in three key technology areas: ultrafast lasers high-performance, hard-coated optical filters and high-sensitivity detectors. One of the key advantages of NLO imaging is the ability to employ both label and label-free imaging strategies when probing the form and function of complex biological samples, such as individual cells and tissue. This approach is commonly known as multimodal NLO imaging. Although each imaging mode can be used separately, advances in ultrafast lasers and laser systems, coupled with innovative developments in hard-coated optical filter design, now allow the near-seamless combination of several NLO modalities into a single, unified microscope platform. Common NLO imaging modalities are two- and three-photon fluorescence (2P & 3P), second- and third-harmonic generation (SHG & THG), and coherent Raman scattering (CRS) in the form of either Coherent Anti-Stokes Raman Scattering (CARS) or Stimulated Raman Scattering (SRS). In NLO imaging, ultrafast laser excitation is used to exploit several nonlinear optical effects that can provide high-contrast imaging of biological samples. Nonlinear optical (NLO) imaging is a powerful microscopy technique that has found increasing use in the field of biomedical optics. Multimodal Nonlinear Optical (NLO) Imaging
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