Targeted fluorescent molecular imaging probes may provide an optimal means of detecting disease. Stable, organic fluorophores can be repeatedly excited in vivo by propagated light and consequentially can provide large signal-to-noise ratios (SNRs) for image detection of target tissues. In the literature, many small animal imaging studies are performed with a red excitable dye, Cy5.5, conjugated to the targeting component. We report the comparison of the in vivo fluorescent imaging performance of a near-IR (NIR) and a red-excitable dye. Epidermal growth factor (EGF) was conjugated with Cy5.5 [excitation/emission (ex/em), 660/710 nm] or IRDye® 800CW (ex/em: 785/830 nm) for imaging EGF receptor (EGFr) positive (MDA-MB-468) and/or negative (MDA-MB-435) human breast cancer cell lines in subcutaneous xenograft models. The conjugates were injected intravenously at 1-nmol-dye equivalent with and without anti-EGFr monoclonal antibody C225, preadministered 24 h prior as a competitive ligand to EGFr. Our images show that while both agents target EGFr, the EGF-IRDye® 800CW evidenced a significantly reduced background and enhanced the tumor-to-background ratio (TBR) compared to the EGF-Cy5.5. Immunohistochemistry shows that EGF causes activation of the EGFr signaling pathway, suggesting that prior to use as a targeting, diagnostic agent, potential deleterious effects should be considered.
Dynamic multi-wavelength fluorescence imaging was accomplished using a liquid crystal tunable filter (LCTF).
Since several different emission wavelengths can be selected by tuning the LCTF, two wavelength dynamic
fluorescence imaging was conducted in mice bearing human melanoma M21 and M21L after injection of a mixture
of (i) RGD peptide conjugated with a near-infrared (NIR) dye that targeted integrin &agr;v&bgr;3 and (ii) non-specific dye,
Cy5.5. Dynamic multi-wavelength imaging with LCTF can differentiate the uptake of the two different fluorescent
contrast agents between tumor and normal tissue ROIs in the M21 and M21L xenograft models. Although the LCTF
attenuated fluorescence signals by a factor of two when compared to holographic and bandpass filter sets used
previously, Tumor to background ratio (TBR) from NIR fluorescence images with a bandpass and holographic filter
were not statistically different from those acquired with the LCTF. Therefore, the benefit of spectral information as
well as dynamic multi-wavelength may outweigh the impact of the lower transmission efficiencies, and could enable
in vivo small animal imaging.
KEYWORDS: Tumors, Signal to noise ratio, Imaging systems, Near infrared, Optical imaging, Tissues, Luminescence, Signal detection, Nuclear imaging, Tissue optics
The cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) (c(KRGDf)), which is known to target v3 integrin, is dual-labeled with a radiotracer, 111indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, for continuous-wave (cw) imaging of v3 positive human M21 melanoma in xenografts. Twenty-four hours after administration of the dual-labeled peptide at a dose equivalent to 90 µCi of 111In and 5 nmol of near-infrared (NIR) dye, whole-body gamma scintigraphy and cw imaging was conducted. Image acquisition time was 15 min for the gamma scintigraphy images and 800 ms for the optical images acquired using an NIR sensitive intensified charge-coupled device. The results show that while the target-to-background ratio (TBR) of nuclear and optical imaging were similar for surface regions of interest and consistent with the origin of gamma and NIR radiation from a common targeted peptide, the signal-to-noise ratio (SNR) was significantly higher for optical than nuclear imaging. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets. While tomographic reconstructions are necessary to probe TBR, SNR, and contrast for interior tissues, this work demonstrates for the first time the direct comparison of molecular optical and planar nuclear imaging for surface and subsurface cancers.
Dynamic fluorescence images were obtained from a subcutaneous human Kaposi's sarcoma tumor (KS1767) model immediately following the intravenous injection of an integrin-targeting cyanine dye conjugate, Cy5.5-c(KRGDf). The fluorescence images, acquired via an intensified charge-coupled device detection system, were used in conjunction with a pharmacokinetic (PK) model to determine kinetic properties of target binding in the presence and absence of a competitive ligand, free c(KRGDf). The results indicate that the conjugate dye behaves similarly in normal tissue to the free Cy5.5 dye while it possesses increased uptake in tumor tissue. The change in pharmacokinetic parameters obtained from dynamic imaging of Cy5.5-c(KRGDf) after administration of c(KRGDf) as a competitive ligand to the integrin receptor suggests that (i) the increased uptake of Cy5.5-c(KRGDf) is molecularly specific and that (ii) receptor turnover occurs within 24 h. In addition, PK analysis enables quantification of an in vivo c(KRGDf) binding constant attributable to integrin binding. In vivo pharmacokinetic analysis based on rapid and dynamic optical imaging may be potentially useful for evaluating the presence and turnover rate of disease markers that are potential targets of molecular medicine.
Several phantom and in vivo small animal imaging studies have been performed to detect the re-emitted fluorescence signal arising from micro to pico molar concentrations of fluorophore by employing band-pass and band-rejection filters. However, elimination of the back-reflected excitation light still remains a major challenge for further reducing the noise floor in fluorescence imaging. Furthermore, despite the well-known deterioration of interference filter performance as the angle of incidence deviates from zero degrees, most studies do not employ collimated light optical design required for efficient excitation light rejection using interference filters. In this study, we measured quantities in frequency domain data for the combination of three-cavity interference and holographic super notch filters. To assess excitation leakage, the “out-of-band (S (λx ) )” to “in-band (S (λm ) - S (λx ) )” signal ratio, AC amplitude (IAC ), and phase delay (δ-δ*) measured from a gain modulated, intensified CCD imaging system with and without collimating optics was evaluated. The addition of collimating optics resulted in a reduction of 82% to 91% of the out-of-band to in-band ratio for the phantom studies and an increase of 1.4 to 3.7 times of the target-to-background ratio (T:B) for small animal studies.
KEYWORDS: Tumors, Luminescence, Acquisition tracking and pointing, In vivo imaging, Brain, Photoacoustic tomography, Neuroimaging, Near infrared, Head, Signal detection
We present a dual modality imaging technique by combining photoacoustic tomography (PAT) and near-infrared (NIR) fluorescence imaging for the study of animal model tumors. PAT provides high-resolution structural images of tumor angiogenesis, and fluorescence imaging offers high sensitivity to molecular probes for tumor detection. Coregistration of the PAT and fluorescence images was performed on nude mice with M21 human melanoma cell lines with αvβ3 integrin expression. An integrin αvβ3-targeted peptide-ICG conjugated NIR fluorescent contrast agent was used as the molecular probe for tumor detection. PAT was employed to noninvasively image the brain structures and the angiogenesis associated with tumors in nude mice. Coregistration of the PAT and fluorescence images was used in this study to visualize tumor location, angiogenesis, and brain structure simultaneously.
The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the αvβ3 integrin was assessed from xenografts bearing a subcutaneous human Kaposi’s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi’s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.
KEYWORDS: Tumors, Signal to noise ratio, Cameras, Optical imaging, In vivo imaging, Near infrared, Image quality, Imaging systems, Luminescence, Melanoma
Fluorescence-enhanced optical imaging measurements and conventional gamma camera images on human M21 melanoma xenografts were acquired for a "dual-modality" molecular imaging study. The avb3 integrin cell surface receptors were imaged using a cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) [c(KRGDf)] probe which is known to target the membrane receptor. The probe, dual-labeled with a radiotracer, 111Indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, was injected into six nude mice at a dose equivalent to 90mCi of 111In and 5 nanomoles of near-infrared (NIR) dye. A 15 min gamma scan and 800 millisecond NIR-sensitive ICCD optical photograph were collected 24 hours after injection of the dual-labeled probe. The image quality between the nuclear and optical data was investigated with the results showing similar target-to-background ratios (TBR) based on the origin of fluorescence and gamma emissions at the targeted tumor site. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets measured by surface regions of interest.
KEYWORDS: Tumors, Luminescence, In vivo imaging, Signal detection, Imaging systems, Cancer, Data acquisition, Charge-coupled devices, Animal model studies
The specificity of a novel EGF-Cy5.5 fluorescent optical probe was assessed using CW fluorescence imaging accomplished via an ICCD camera. Imaging was performed on mice with MDA-MB-468 cancer, known to overexpress EGFr, and contrasted against an analogous cell line, MDA-MB-435, that does not express EGFr. Fluorescence images on mice bearing s.c. inoculated tumors were obtained every 6 seconds for a period of 20 minutes following i.v. injection of ICG, Cy5.5, or EGF-Cy5.5 and every 24 hrs thereafter for up to 192 hrs. In addition, mice with MDA-MB-468 tumors were injected i.v. with anti-EGFr antibody C225 24 hrs prior to injection of EGF-Cy5.5. Monitoring the time-sensitive fluorescence intensity confirms that ICG and Cy5.5 show no favorable binding to tumor, regardless of EGFr expression level. In contrast, EGF-Cy5.5 exhibits selective accumulation only in the MDA-MB-468 tumor. Moreover, tumor uptake of EGF-Cy5.5 was blocked by pre-injection of C225 antibody, demonstrating specificity of the targeted contrast agent. Data further demonstrate that ICG and Cy5.5 fluorescence is completely absent from the tumor site, regardless of EGFr expression level, 24-hrs post injection. Similarly, little EGF-Cy5.5 fluorescence was detected in the EGFr-negative tumor after 24 hrs, however, for the MDA-MB-468 tumor, EGF-Cy5.5 fluorescence did not reach undetectable levels until 192 hrs.
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