In the last decade, there has been a fruitful marriage of traditional chemical nonlinear optical (NLO) spectroscopic methods with biological microscopy. For example, chemists have long used modalities such as Second Harmonic Generation (SHG), Third Harmonic Generation (THG), Coherent anti-Stokes Raman (CARS), and multiphoton excitation (MPE) and are now applying these to optical imaging of cells and tissues. A strong emphasis has been on developing these methods as quantitative tools not just for basic science research, but also as future diagnostic tools for clinical applications because of the submicron resolution afforded by optical wavelengths. In contrast, clinical tomographic modalities such as computed tomography (CT), MRI, and positron emission tomography (PET) are limited to ~1 mm. This capability is particularly relevant given the size scales of cells/tissues, which display architectures ranging from ~50 nm for organelles to tens of microns for whole cells, and alterations on these scales in the 3D tissue environment accompany many diseases.
Historically, clinical imaging at the cellular level has been performed by pathologists on ex vivo biopsies removed by the surgeon. While histology remains the “gold standard”, its accuracy depends on the experience and interpretation skill of the pathologist. Furthermore, histological samples are fixed and sliced into thin sections (~5–10 μm), making spatial correlations difficult.
An important potential role exists for quantitative optical microscopy approaches that eliminate the pitfalls of both noninvasive imaging (lack of resolution/specificity) and traditional histology (subjectivity). High resolution nonlinear optical microscopy can meet many of these challenges in imaging and quantifying tissue structural changes during disease progression. For example, collagen in the extracellular matrix (ECM), upon which epithelial cells are supported, is highly altered in cancer, connective tissue diseases, autoimmune disorders, and cardiovascular diseases. In this Feature, we will focus on SHG imaging microscopy for quantitative analysis of diseased states.