Elsevier

Biomaterials

Volume 31, Issue 26, September 2010, Pages 6823-6832
Biomaterials

Dual-emissive quantum dots for multispectral intraoperative fluorescence imaging

https://doi.org/10.1016/j.biomaterials.2010.05.030Get rights and content

Abstract

Fluorescence molecular imaging is rapidly increasing its popularity in image guided surgery applications. To help develop its full surgical potential it remains a challenge to generate dual-emissive imaging agents that allow for combined visible assessment and sensitive camera based imaging. To this end, we now describe multispectral InP/ZnS quantum dots (QDs) that exhibit a bright visible green/yellow exciton emission combined with a long-lived far red defect emission. The intensity of the latter emission was enhanced by X-ray irradiation and allows for: 1) inverted QD density dependent defect emission intensity, showing improved efficacies at lower QD densities, and 2) detection without direct illumination and interference from autofluorescence.

Introduction

Visual assessment of disease is often one of the first diagnostic lines in medicine. Especially surgical interventions demand a “see-and-treat” approach. Hence it is common that surgical interventions based on non-invasive imaging technologies e.g. radioguided sentinel lymph node (SLN) dissections are performed in combination with superficial visual guidance using a dye such as patent blue [1]. Recent developments in surgical image guidance have resulted in the inclusion of fluorescent, or rather luminescent, dyes [2], [3].

The requirement of only superficial optical detection in the SLN procedure warrants the use of visible dyes with limited tissue penetrating properties. Indeed, several fluorescent imaging agents that emit in the visible range (450–650 nm) have been described for their potential in imaging applications e.g. fluorescein (in clinical use) [4], 5-aminovulinic acid (in clinical use) [5], dendrimers [6], microspheres [7] and quantum dots (QDs) [8], [9], [10]. The latter (QDs) are not only brightly fluorescent, with a size tunable emission color, but also have proven to be a versatile platform for further functionalization with e.g. tumor targeting moieties [10].

Approaches using luminescent probes in general require an external optical excitation, resulting in background signals induced by reflectance, scattering or autofluorescence of tissue [11]. Moreover, the tissue absorbance of light leads to strong signal attenuation of both the excitation and emission signals [11]. For this reason the mainstream focus is on the use of near infra red (NIR; ≥800 nm) in fluorescence-based image guidance [12]. As an alternative to NIR imaging agents, the luminescent signal to background ratio (SBR) at increased tissue depths can also be improved with probes that do not require real-time excitation and emit in the tissue transparent range above 650 nm [13]. Examples of such probes are “self-illuminating” [13] and “defect-emission”[14] based imaging agents.

Light emissions ≥650 nm become rapidly invisible to the human eye [15] and require specialized camera based imaging. On the other hand, to enable visual detection during (complex) surgical procedures an imaging agent should absorb or emit light between 400 and 650 nm.

Combining superficial visual and “deep” far red/NIR image guidance requires either “cocktails” of dyes, or dual-emissive luminescent imaging agents that have an emission between 400 and 650 nm and one above 650 nm.

Multispectral imaging is a technical extension in fluorescence imaging that allows for the detection of multiple emissions in a single imaging session. Spectral splitting of signals is a.o. used to: visualize multiple dyes at once, increase the detection specificity, and enable more quantitative measurements [16]. Because of these properties it is also an upcoming imaging technology in surgical image guidance [17]. In multispectral imaging the emission detection is typically performed after excitation at a single excitation wavelength [18]. Hence, dedicated QD based imaging agents for multispectral imaging have been reported in the form of “large” (1.2 μm) polymer particle assemblies containing multiple QDs; [19] most commonly used QDs have a similar absorbance trend with increased absorption values <400 nm. However, size matters during the (bio) distribution and clearance of an imaging agent; Choi et al. have demonstrated that smaller particles have better clearance properties [20]. Grabolle et al. have stated that the multispectral imaging technology would also benefit significantly from the incorporation of lifetime dependant emissions [21]. The use of lifetime imaging may help filter out the short-lived autofluorescence. Hence, a combination of lifetime dependence and multispectral imaging would allow for signal discrimination using both the emission wavelength and the emission lifetime, improving the accuracy of fluorescence imaging even further. The ideal multispectral imaging agent for surgical guidance to the SLN should thus be a “small” particle with at least two emissions that have respective short and long emission lifetimes.

Despite the obvious advantages of imaging guidance during surgical interventions there are still challenges to be met in the development of luminescent imaging agents that allow for combined visual (superficial) and deep tissue imaging. In this manuscript we present (lifetime dependant) multispectral InP/ZnS QDs that meet both these requirements and that are applied in SLN procedures.

Section snippets

Reagents

Indium chloride, tris(trimethylsilyl)phosphine (TMS)3P, hexadecylamine (HDA), stearic acid, sulfur, dodecanethiol, and 1-octadecene (ODE) were obtained from Sigma Aldrich. Zinc undecanoate was synthesized following an adapted metal carboxylic acid method published by Pradhan et al. [22] using triethylamine as the base. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (18:0 PEG2 PE) was obtained from Avanti Polar Lipids. d-luciferine fire fly potassium salt

Generation of InP/ZnS QDs

InP/ZnS QDs were prepared and characterized as described in the experimental section, adapted from the procedure of Xu et al. [23]. The InP/ZnS QDs collected from the reaction mixture exhibit a fluorescence quantum yield (ηF) of ≈70% in chloroform. To allow for optimal visual detection sensitivity (520–550 nm range), we tuned the exciton emission of the InP/ZnS QDs towards that of fluorescein (λmax = 520 nm), a dye clinically used for visual image guidance during surgical procedures (see above).

Discussion

In general the fluorescent probe development focuses on NIR-dyes that cannot be detected by eye as these give an increased tissue penetration because of their longer emission wavelength and reduced autofluorescence in vivo. For reference, the NIR penetration does not exceed the (still superficial) cm range and thus does not compare to 3D imaging modalities such as e.g. positron emission tomography. Based on our own experience with the NIR-dye ICG (both in preclinical SLN studies and recently

Conclusion

We have demonstrated the manufacture of InP/ZnS QDs with both a short-lived visible (520 nm) and an induced long-lived far-red defect emission (650 nm). InP/ZnS QDs provide the possibility to exploit a number of exciting (optical) properties. First, X-ray irradiation can be used to enhance the defect emission signal intensity. Second, these particles have lifetime dependent emissions wherein the exciton emission has a “short” lifetime and the defect emission a “long” lifetime. Third, the QD

Acknowledgments

This research is supported, in part, by the Technology Foundation, applied science division of NWO and the technology program of the Ministry of Economic Affairs (Grant No. STW BGT 7528 Veni; FvL), a KWF-translational research award (Grant No. PGF 2009-4344; FvL), the European Community’s Seventh Framework Program (FP7/2007-2013; TB), and the Marie-Curie Research Training Network NANOMATCH (Grant No. MRTN-CT-2006-035884; AAdM). We gratefully acknowledge the Nichia cooperation for providing us

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