Multimodal Nonlinear Optical (NLO) Imaging
Neil Anderson, Ph.D., Turan Erdogan, Ph.D.
Nonlinear optical (NLO) imaging is a powerful microscopy technique that has found increasing use in the field of biomedical optics. In NLO imaging, ultrafast laser excitation is used to exploit several nonlinear optical effects that can provide high-contrast imaging of biological samples. 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). 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. This approach is commonly known as multimodal NLO imaging. 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. 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. Fluorophores commonly...
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