Storable, thermally activated, near-infrared chemiluminescent dyes and dye-stained microparticles for optical imaging

Abstract

Imaging techniques are a vital part of clinical diagnostics, biomedical research and nanotechnology. Optical molecular imaging makes use of relatively harmless, low-energy light and technically straightforward instrumentation. Self-illuminating, chemiluminescent systems are particularly attractive because they have inherently high signal contrast due to the lack of background emission. Currently, chemiluminescence imaging involves short-lived molecular species that are not stored but are instead generated in situ, and they typically emit visible light, which does not penetrate far through heterogeneous biological media. Here, we describe a new paradigm for optical molecular imaging using squaraine rotaxane endoperoxides, interlocked fluorescent and chemiluminescent dye molecules that have a squaraine chromophore encapsulated inside a macrocycle endoperoxide. Squaraine rotaxane endoperoxides can be stored indefinitely at temperatures below -20 °C, but upon warming to body temperature they undergo a unimolecular chemical reaction and emit near-infrared light that can pass through a living mouse.

Figure 1. Thermal cycloreversion of 1EP to 1 and reverse photoreaction

a, Cycloreversion of 1EP releases singlet oxygen and emits near-infrared light. The encapsulated blue component in rotaxane 1 and 1EP is the squaraine 3. b, Partial ¹H NMR spectra (CDCl₃) showing photoconversion of 1 into 1EP: (top) 1, (middle) mixture of 1 and 1EP after irradiation with red light for 10 mins, (bottom) complete conversion to 1EP after irradiation for 30 mins.

Figure 2. False-colored pixel intensity maps at 38 °C with intensity scales in arbitrary units

a, Vial containing a solution of chemiluminescent 1EP in CDCl₃. b, Fluorescence micrograph of carboxylate functionalized polystyrene 1EP-microparticles (0.9 µm diameter), c, Chemiluminescence from polystyrene 1EP-microparticles that are aggregated in a vial of water (viewed from top), d, Chemiluminescence from carboxylate functionalized polystyrene 1EP-microparticles that are dispersed throughout a vial of water, e, f, g, Bright field, chemiluminescence, and fluorescence images, respectively, of a reverse-phase TLC plate with spots of 1EP. h, Chemiluminescence from a solution of 1EP viewed with different emission filters, i, Chemiluminescence (blue) and fluorescence (red, excitation = 650 nm) emission spectra of 1EP (1.5 mM, C₂D₂C1₄) at 65 °C.

Figure 3. Chemiluminescence and reflected fluorescence from 1EP-microparticles injected subcutaneously into the dorsal side of a nude mouse rear leg at 38 °C

a, b, c, Dorsal bright field, chemiluminescence, and fluorescence images (chemiluminescence and fluorescence TBR = 6.9 and 29, respectively). d, e, f, Ventral images which required light penetration through deeper tissue (chemiluminescence and fluorescence TBR = 6.8 and 4.4, respectively). N = 2

Figure 4. Chemiluminescence from 1EP at 38 °C penetrates through a living nude mouse

a, f, Experimental set-up for chemiluminescence and fluorescence imaging, respectively. b, g, Chemiluminescence and fluorescence pixel intensities from a small tube containing 1EP (250 nmol) in C₂D₂Cl₄. c, h, Photograph of mouse located above the tube. d, e, Pixel intensity map of chemiluminescence that is transmitted through the mouse (TBR = 11.6). i, Fluorescence intensity map of mouse located above the tube (TBR = 1.1). j, Fluorescence intensity map of mouse with no tube present.