Optical diagnostics of tissue pathology by multiphoton microscopy

Abstract

Advances in nonlinear optical microscopy have led to new imaging modalities that can be used for diagnosing pathological tissues. Intrinsic optical sectioning, reduced photodamage and greater specimen penetration depth allow three-dimensional, high-contrast, tissue imaging to be performed with multiphoton techniques. Although autofluorescence can be useful in imaging cellular architecture, it is the polarization-based technique of second harmonic generation (SHG) that is effective in visualizing non-centrosymmetric biological structures such as collagen and muscle fibers without extrinsic labeling. In addition, third harmonic generation (THG) offers another contrast mechanism for imaging the morphology of tissue constituents. These techniques offer the possibility of diagnosing normal and diseased states in selected epithelial tissues, often without the introduction of extrinsic labels. In this review, the physical principles of multiphoton fluorescence excitation, SHG and THG processes are discussed. In addition, the basic instrument set up of a multiphoton microscope along with recent advances in multiphoton microscopy instrumentation are presented. Examples from imaging tissue pathologies from organs such as skin, cornea, lung and the liver are illustrated. Finally, developments leading to miniaturized or endoscopic instrumentation that allows real-time diagnosis of tissue pathologies in accessible epithelial tissues are discussed. From this review, the reader will gain knowledge of the basic principles of multiphoton microscopy and the technical information of building a multiphoton instrument. In addition, the reader will appreciate the ability of this technique in the diagnostics of pathological tissues and gain insights into the future development of this technique. Intrinsic fluorescence and harmonic generation signals are capable of differentiating normal and pathological epithelial tissues. Further technical advances leading to improved imaging depths, multimodal imaging capabilities and endoscopic instrumentation may enable the effective diagnostics of selected epithelial tissues in the clinical setting.