Abstract
Here, we present a new class of third harmonic generation (THG) imaging probes that can be activated with precise spatiotemporal control using non-linear excitation. These probes consist of lipid-coated perfluorocarbon nanodroplets with embedded visible chromophores. The droplets undergo phase transition from liquid to gas upon heating mediated by two-photon absorption of NIR light by the embedded dyes. Resulting microbubbles provide a sharp, local refractive index mismatch, which makes an excellent source of THG signal. Potential applications of these probes include activatable THG agents for biological imaging and "on-demand" delivery of various compounds under THG monitoring.
Highlights
Advances in imaging technologies and respective contrast agents have greatly expanded our understanding of tissue structure, cell functions and interactions as well as subcellular events from basic cell biology to oncology [1–5]
third harmonic generation (THG) occurs at interphases, typically with a refractive index mismatch, that results in conversion of three photons of an excitation light into a single emitted photon with triple the energy (1/3 of the excitation wavelength) [12]
Typical preparations resulted in fairly uniform size distributions of nanodroplets with average sizes at the major distribution peak of 379 ± 96 nm and 306 ± 80 nm for PFC nanodroplets with embedded Cyanine 3 (Cy3) chromophores (ND-Cy3) and “blank” PFC nanodroplets without dye molecules (ND-B) specimen, respectively (Fig. 1(c))
Summary
Advances in imaging technologies and respective contrast agents have greatly expanded our understanding of tissue structure, cell functions and interactions as well as subcellular events from basic cell biology to oncology [1–5]. Most contrast agents in life sciences today rely on the principle of single- or two-photon excitation. While these agents are extremely useful in generating imaging contrast, there are constraints in the number of unique targets that can be imaged due to spectral overlap in the visible light range. The development of multiphoton microscopy with pulsed near-red and infrared laser excitation sources enabled in vivo imaging of live cells and tissue structures at high resolution, low phototoxicity, and reasonable depth of penetration (300-1000 μm) [7–11]. SHG occurs when biological structures containing asymmetric repetitive units (e.g. striated muscle and collagen fibers) convert two photons of an excitation light source into a single emitted photon at double the energy (1/2 of the excitation wavelength). THG does not suffer from photobleaching or generation of reactive oxygen species [12] and allows detection of intrinsic complex tissue structures with subcellular resolution, including interstitial tissue, cell surfaces, and microvesicles in live animals [13]
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