Optimizing quantitative in vivo fluorescence imaging with near-infrared quantum dots

Abstract

Quantum dots (QDs) are fluorescent nanoparticles with broad excitation and narrow, wavelength-tunable emission spectra. They are used extensively for in vitro fluorescence imaging studies and more recently for in vivo small animal and pre-clinical studies. To date there has been little concern about the selection of QD size (and thus emission wavelength peak) and excitation wavelengths, as they have little relevance to the results of in vitro studies. In vivo imaging, however, poses additional constraints, such as the scattering and absorption by tissue, which may influence the signal intensity at the body surface. Here, we demonstrate that longer-wavelength excitation and emission yield less quantization error in measured relative fluorescence intensity, using three near-infrared QDs (QD655, QD705 and QD800) applied to in vivo lymphatic imaging, and a range of excitation wavelengths from the blue to the red. Statistically significant differences in quantization error were observed between nearly all pairs of excitation wavelengths (445-490, 503-555, 575-605, 615-665 and 671-705 nm). Similarly, quantization error decreased with longer emission wavelengths (655, 705 and 800 nm). Light absorbance and scattering were demonstrated to be more potent factors than absorbance efficiency of QDs in producing quantization error in the measured fluorescence intensity. As a result, while wavelengths can be adjusted for qualitative experiments, the longest possible wavelengths should be used if quantification is desired during QD imaging experiments.

Figure 1.

Absorbance spectra (dashed lines) and emission spectra (solid lines) for QD655 (blue), QD705 (green) and QD800 (red).

Figure 2.

Relative fluorescence intensities of the lymph nodes in each image [ex vivo (a, d; white light and fluorescence images), in vivo with deep red (b, e), red, yellow, green and blue excitation, ex vivo with skin covering with deep red (c, f), red, yellow, green and blue excitation] were calculated relative to the standard node, which was set as the brightest node for each mouse in the ex vivo image. The ex vivo relative intensities (S_n) were used as the gold standards and the quantization error (En) in fluorescence intensity (I_n) was calculated relative to those intensities (E_n = |(S_n − I_n)/S_n| for each n, or lymph node).

Figure 3.

Relative fluorescence intensity quantization error for in vivo lymph nodes relative to ex vivo standards, at different excitation wavelengths for QD800 (red), QD705 (green) and QD655 (blue).

Figure 4.

Relative fluorescence intensity quantization error for ex vivo lymph nodes with skin covering relative to ex vivo standards, at different excitation wavelengths for QD800 (red), QD705 (green) and QD655 (blue).