Polychromatic in vivo imaging of multiple targets using visible and near infrared light

Abstract

Conventional diagnostic imaging methods such as X-ray CT, MRI, and nuclear medicine are inherently monochromatic meaning that they can depict only one molecular target at a time. Optical imaging has the unique ability to be polychromatic and therefore multi-color imaging employing targeted agents conjugated to fluorophores of varying wavelength enables multiple simultaneous readouts thus providing greater multiplexed information. Numerous successful multicolor imaging techniques have recently been reported using optical imaging in in vivo animal disease models, thus adding to a growing body of research supporting the clinical viability and applicability of these technologies. Herein, we review multicolor optical imaging from the basic chemistry and physics perspective and then extend this to biological and medical applications.

Keywords: Cancer; Endoscope; Fluorescence; Fluorescence-guidance; Molecular imaging; Multi-color; Surgery.

Figure 1

Schema of variable groups of fluorescent and luminescent materials separated by size.

Figure 2

A. Basis of spectrally resolved multi-color imaging. B. An practical comparison of single excitation versus multiple excitation spectrally resolved multi-color NIR imaging technologies using Cy5-, Alexa Flore 700-, and Cy7-conjugated monoclonal antibodies. Multiple excitation spectrally resolved multi-color NIR images (lower images) showed higher signal-to-noise ratios in all three spectral images than single excitation spectrally resolved multi-color NIR images.

Figure 3

In vivo four color imaging of a single peritoneally disseminated ovarian tumor (Halo-SHIN3). By using four different fluorophore-conjugated Halo-tag ligands (Alexa Flore 488, TAMRA, Dynamic 633, and IRDye800), tumors produced from a single cell line can display distinct colors.

Figure 4

Multi-color image of a peritoneal ovarian cancer micro-nodule. Intraperitoneally injected GSA-rhodamine Green probe shows mostly the surface tumor nodules. In contrast, intravenously injected trastuzumab-Alexa Fluor 680 only shows HER2+ part of viable cancer cells, which are expressing RFP.

Figure 5

Visible five color lymphatic drainage imaging by using five different visible Quantum dots (540, 560, 580, 600, 650). The image was taken by a conventional digital camera through a long pass filter, therefore, similar image can be seen by our own eyes.

Figure 6

A “cocktail” of three antibody-NIR fluorophore conjugates can simultaneously diagnose tumors expressing distinct receptors. A431 (EGFR+), 3T3/HER2 (HER2+), SP2/Tac (CD25+) and LS174T (triple negative control) were subcutaneously implanted. The ‘cocktail’ of daclizumab-Alexa700 (anti-CD25), trastuzumab-Cy7 (anti-HER2) and cetuximab-Cy5.5 (anti-HER1) was injected 24 hours prior to imaging. The respective tumors with their distinct receptors could be simultaneously distinguished by spectral unmixing: A431 (HER1) in cyan; 3T3/HER2+ (HER2) in magenta; SP2/Tac (CD25) in yellow; LS174T is seen as colorless.

Figure 7

Multi-color fluorescence-guided simulation surgery of HER2+ and HER2− peritoneally disseminated ovarian cancers. SHIN3-RFP (HER2−/RFP+) and SKOV3 (HER2+/RFP−) tumors were grown in the abdomen of a mouse. GSA-rhodamine Green probe detects both tumors shown in green. Trastuzumab-Alexa Fluor 680 probe shows only HER2+/RFP− tumors shown in blue.