Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping

Abstract

The use of near-infrared or infrared photons is a promising approach for biomedical imaging in living tissue. This technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores. Here we show that the fluorescence emission of type II quantum dots can be tuned into the near infrared while preserving absorption cross-section, and that a polydentate phosphine coating renders them soluble, disperse and stable in serum. We then demonstrate that these quantum dots allow a major cancer surgery, sentinel lymph node mapping, to be performed in large animals under complete image guidance. Injection of only 400 pmol of near-infrared quantum dots permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using excitation fluence rates of only 5 mW/cm(2). Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential of near-infrared quantum dots for biomedical imaging.

Figure 1

Physical and optical properties of aqueous-soluble, NIR type II QDs. (a) TEM image of water-dispersed NIR QDs. (b) Molar extinction coefficient (solid curve; left axis) and photoluminescence intensity (dashed curve; right axis) of NIR QDs in PBS, pH 7.4. (c) Gel-filtration chromatography of protein standards (left) and NIR QDs (right) in PBS, pH 7.4. Standards included thyroglobulin (669 kDa; solid curve), alcohol dehydrogenase (150 kDa; thick solid curve) and ovalbumin (44 kDa; dotted curve). NIR QDs had an effective molecular weight of 440 kDa. (d) Fluorescence stability of 1 μM organic NIR fluorophore IRDye78-CA (left) and 1 μM NIR QDs (right), in 100% FCS, as a function of excitation fluence rate and time. Note that the abscissa for IRDye78-CA is in seconds and that for NIR QDs is in minutes. (e) Fluorescence stability of 1 μM NIR QDs, in 100% FCS, at 37 °C over time.

Figure 2

NIR QD sentinel lymph node mapping in the mouse and pig. (a) Images of mouse injected intradermally with 10 pmol of NIR QDs in the left paw. Left, pre-injection NIR autofluorescence image; middle, 5 min post-injection white light color video image; right, 5 min post-injection NIR fluorescence image. An arrow indicates the putative axillary sentinel lymph node. Fluorescence images have identical exposure times and normalization. (b) Images of the mouse shown in a 5 min after reinjection with 1% isosulfan blue and exposure of the actual sentinel lymph node. Left, color video; right, NIR fluorescence images. Isosulfan blue and NIR QDs were localized in the same lymph node (arrows). (c) Images of the surgical field in a pig injected intradermally with 400 pmol of NIR QDs in the right groin. Four time points are shown from top to bottom: before injection (autofluorescence), 30 s after injection, 4 min after injection and during image-guided resection. For each time point, color video (left), NIR fluorescence (middle) and color-NIR merge (right) images are shown. Fluorescence images have identical exposure times and normalization. To create the merged image, the NIR fluorescence image was pseudocolored lime green and superimposed on the color video image. The position of a nipple (N) is indicated.

Figure 3

Post-resection inspection of the surgical field and evaluation of NIR QD lymph node retention. (a) Post-resection evaluation of the surgical field. Shown are color video (left), NIR fluorescence (middle) and color-NIR merge (right) images. Arrows indicate the resected sentinel lymph node. (b) NIR QD retention by the resected SLN (S) and the next lymph node (N) in the chain is shown in this bisected specimen. (c) Histologic analysis of frozen sections of the SLN in b. Shown are two representative hematoxylin and eosin (H + E)-stained sections and consecutive unstained sections photographed on a NIR fluorescence microscope.