Bioinspired molecular dyes for biomedical fluorescent imaging

Fluorescence imaging can be conducted with long Stokes shift dyes that minimize crosstalk between the excitation source and fluorescent emission to improve the signal-to-background ratio. Regardless, researchers still form ...
The dyes showed near-infrared emissions with long Stokes shift and small molecular weight. The most promising dyes also showed rapid renal excretion and passing properties. The bioengineers modified the compounds for tumor imaging, fluorescence image-guided surgery, neurosurgery and pathological analysis. The findings contribute an essential small molecular dye category to the classical dyes.

Developing long Stokes shift dyes

Fluorescence imaging is widespread in preclinical biomedical research, as well as clinical pathology and fluorescence image-guided surgery. The low cost, easy platform offers minor light damage to the biological specimen for high detection sensitivity. The biomedical application of fluorescent imaging depends on the dyes with critical features, including absorption/emission profiles, absorption coefficient, quantum yield, Stokes shift, and photochemical stability.
Nevertheless, only a few dyes have shown optimal properties across all categories. The heavy crosstalk between the excitation and emission light can result in relatively low signal-to-background ratio. As a result, biochemists aim to develop long Stokes shift near infrared dyes for high signal-to-background ratio. In this work, Chen and the team described the first high-efficiency styrene oxazolone dyes (SODs) as long stokes shift dyes, to provide a new strategy for in vivo fluorescence imaging.

Experiments: Design, synthesis and characterization of styrene oxazolone dyes (SODs)

Long Stokes shift SOD for biomedical fluorescence imaging. (A) Chemical structure, molecular weight, max absorption, and emission wavelength, Stokes shift of typical commercial fluorescent dyes. (B) The absorption, fluorescence spectrum (left), chemical structure (middle), quantum yield, molar extinction coefficient (in water), computed isodensity surfaces of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), and cell imaging of the represent styrene oxazolone dye 9 (SOD9). (C) Triphenylphosphonium (TPP)–modified SOD9 for cell mitochondrial, in vivo head-neck cancer, and brain neuron imaging. a.u., arbitrary units; PI, post injection. Credit: Science Advances (2022). DOI: 10.1126/sciadv.abo3289
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Design and synthesis of SODs. (A) The chromophore chemical structures of GFP and RFP. (B) Synthesis of SODs (left) and the crystal structure of SOD10 (right). DIPEA, N,N-Diisopropylethylamine. (C) The chemical structures of SOD dyes. Ex., excitation; Em., emission; TICT, twisted intramolecular charge transfer; r.t., room temperature. Credit: Science Advances (2022). DOI: 10.1126/sciadv.abo3289
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Optical characteristics of SODs. The absorbance (A), fluorescence spectrum (B), and photostabilities (C) of SODs were measured in water with the concentration of 20, 12, and 10 μM, respectively (6G represents rhodamine 6G). (D) Density functional theory (DFT) optimized molecular orbital plots (HOMO and LUMO) of SOD9. (E) The optical properties summary of SOD dyes. Red is the maximum value, and blue is the minimum value of the same column. EtOH, ethanol. Credit: Science Advances (2022). DOI: 10.1126/sciadv.abo3289
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SOD9’s in vivo pharmacokinetics by fluorescence imaging. (A) SOD9 fluorescence imaging in NIH-3T3 cell (red) and merge with the nuclear stains Hoechst (blue). Scale bars, 10 μm. (B) Whole-body NIR imaging of nude mice (n = 3, prone and supine positions) after intravenous injection of SOD9 (2.5 mg/kg, 6.18 μmol/kg). The signal was collected in the 650- to 800-nm channel with an excitation at 500 nm. (C) The colored imaging (top) and fluorescence imaging of the nude mice with urine excretion 1.5 hours after intravenous injection of SOD9. (D) Comparison of bladder fluorescent intensities at different time points after intravenous injection of SOD9. Error bars, means ± SD (n = 3). (E) Ex vivo imaging of the major organs dissected after euthanizing animals at 2 hours after intravenous injection of SOD9 (10 mg/kg). Left: colored picture; right: fluorescence imaging. (F) Comparison of mean intensities for the major organs at 2 hours after intravenous injection of SOD9. Error bars, means ± SD (n = 3). Credit: Science Advances (2022). DOI: 10.1126/sciadv.abo3289
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SOD9-TPP for fluorescence image–guided surgery, brain neuroimaging, and on-site pathologic analysis. (A) Top: The colored picture of the orthotopic HNSCC mouse (SCC090; tumor marked with the red pentagram). Bottom left: The setup’s color photo of the confocal fluorescent endomicroscopy imaging–guided surgery. Bottom right: The setup’s colored photo of the confocal fluorescent endomicroscopy imaging of the resected tissue. (B) Confocal fluorescent endomicroscopy imaging of the dissected HNSCC tumor during fluorescence image–guided surgery of the mice 2 hours after intravenous injection of SOD9-TPP (5.0 mg/kg, 6.13 μmol/kg). Right, tumor; middle, tumor and normal tissue; left, normal tissue. (C) H&E staining of HNSCC tumor tissue sections. (D) The zoomed picture of (C). (E) The zoomed picture of (D). (F) Whole-body NIR imaging of nude mice (n = 3, prone and supine positions) after intravenous injection of SOD9-TPP (5.0 mg/kg, 6.13 μmol/kg); SOD9-TPP was found accumulated in the brain, BAT, and liver. (G) Different time points in vivo confocal fluorescent endomicroscopy imaging of brain neurons with the skull opened. Scale bars, 25 μm. (H) In vivo confocal fluorescent endomicroscopy imaging of major organs with abdomen and chest opened. Scale bars, 25 μm. Credit: Science Advances (2022). DOI: 10.1126/sciadv.abo3289
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