Abstract
Second harmonic generation (SHG) and third harmonic generation (THG) microscopies have emerged as powerful imaging modalities to examine structural properties of a wide range of biological tissues. Although SHG and THG arise from very different contrast mechanisms, the two are complimentary and can often be collected simultaneously using a modified multiphoton microscope. In this review, we discuss the needed instrumentation for these modalities as well as the underlying theoretical principles of SHG and THG in tissue and describe how these can be leveraged to extract unique structural information. We provide an overview of recent advances showing how SHG microscopy has been used to evaluate collagen alterations in the extracellular matrix and how this has been used to advance our knowledge of cancers, fibroses, and the cornea, as well as in tissue engineering applications. Specific examples using polarization-resolved approaches and machine learning algorithms are highlighted. Similarly, we review how THG has enabled developmental biology and skin cancer studies due to its sensitivity to changes in refractive index, which are ubiquitous in all cell and tissue assemblies. Lastly, we offer perspectives and outlooks on future directions of SHG and THG microscopies and present unresolved questions, especially in terms of overall miniaturization and the development of microendoscopy instrumentation.
Highlights
Multiphoton microscopy (MPM) has revolutionized biological imaging since its modern inception in the early 1990s, where the first breakthroughs utilized two-photon excited fluorescence (TPEF) to probe live cells and tissues [1]
We provide an overview of recent advances showing how second harmonic generation (SHG) microscopy has been used to evaluate collagen alterations in the extracellular matrix and how this has been used to advance our knowledge of cancers, fibroses, and the cornea, as well as in tissue engineering applications
While it is more common to pair SHG and TPEF microscopies given the vast array of engineered fluorescent proteins and autofluorescent extracellular matrix (ECM) proteins, combining SHG and third harmonic generation (THG) microscopies has its advantages
Summary
Multiphoton microscopy (MPM) has revolutionized biological imaging since its modern inception in the early 1990s, where the first breakthroughs utilized two-photon excited fluorescence (TPEF) to probe live cells and tissues [1] This technology largely solved long-standing problems of confocal microscopy including photobleaching and penetration depth limitations. While it is more common to pair SHG and TPEF microscopies given the vast array of engineered fluorescent proteins and autofluorescent extracellular matrix (ECM) proteins (e.g., elastin), combining SHG and THG microscopies has its advantages. These include increased imaging depths due to longer excitation wavelengths and applicability to a wide range of tissues, even those that do not autofluoresce. In this review, we will focus on the fundamental principles and emerging applications of SHG and THG microscopies
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