Second window for in vivo imaging

Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.

The intrinsic optical parameters absorption coefficient mu(a), scattering coefficient micros, anisotropy factor g, and effective scattering coefficient micros were determined for human red blood cell (RBC) suspensions of hematocrit 33.2% dependent on the oxygen saturation (SAT O(2)) in the wavelength range 250 to 2,000 nm, including the range above 1,100 nm, about which there are no data available in the literature. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte Carlo simulation were carried out for SAT O(2) levels of 100 and 0%. In the wavelength range up to 1,200 nm, the absorption behavior is determined by the hemoglobin absorption. The spectral range above the cells' absorption shows no dependence on SAT O(2) and approximates the absorption of water with values 20 to 30% below the respective values for water. Parameters micros and g are significantly influenced by the SAT O(2)-induced absorption changes. Above 600 nm, micros decreases continuously from values of 85 mm(-1) to values of 30 mm(-1) at 2,000 nm. The anisotropy factor shows a slight decrease with wavelengths above 600 nm. In the spectral regions of 1,450 and 1,900 nm where water has local absorption maxima, g shows a significant decrease down to 0.85, whereas micros increases.