Jessica P. Houston

Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents

The deep tissue propagation of near-infrared (NIR) light between 700-900 nm offers new opportunities for diagnostic imaging when employing sensitive detection techniques and NIR excitable fluorescent agents that target and report disease and metabolism. Herein, we highlight approaches for illuminating tissues and monitoring the re-emitted fluorescence for tomographic reconstruction, strategies for developing fluorescent dye constructs, and clinical opportunities for fluorescence-enhanced NIR optical imaging.

Quality analysis of in vivo near-infrared fluorescence and conventional gamma images acquired using a dual-labeled tumor-targeting probe

The cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) (c(KRGDf)), which is known to target alpha(v)beta3 integrin, is dual-labeled with a radiotracer, (111)indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, for continuous-wave (cw) imaging of alpha(v)beta3 positive human M21 melanoma in xenografts. Twenty-four hours after administration of the dual-labeled peptide at a dose equivalent to 90 microCi of (111)In 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.

Improved Excitation Light Rejection Enhances Small-Animal Fluorescent Optical Imaging

Small-animal fluorescence-enhanced imaging involves the detection of weak fluorescent signals emanating from nanomolar to picomolar concentrations of exogenous or endogenously produced fluorophore concurrent with the rejection of an overwhelmingly large component of backscattered excitation light. The elimination of the back-reflected excitation light of the collected signal remains a major and often unrecognized challenge for further reducing the noise floor and increasing sensitivity of small-animal fluorescence imaging. Herein, we show that the combination of three-cavity interference and holographic super notch filters with appropriate imaging lenses to collimate light improves rejection of excitation light, enabling more accurate imaging. To assess excitation leakage, the “out-of-band (S(Λ x ))” to “in-band (S(Λ m )–S(Λ x ))” signal ratio from phantom studies and the target-to-background ratio (TBR) from in vivo animal imaging was acquired with and without collimating optics. The addition of collimating optics resulted in a 51% to 75% reduction in the ratio of (S(Λ x ))/(S(Λ m )–S(Λ x )) for the phantom studies and an improvement of TBR from 11% to 31% and of signal-to-noise ratio from 11% to 142% for an integrin-targeting conjugate in human glioma xenografts.

The influence of improved interference filter performance for molecular imaging using frequency domain photon migration measurements

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.

In vivo pharmacokinetic analysis for fluorescently labeled RGD peptide targeted to the α vβ 3 integrin in Kaposi's sarcoma

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.

In vivo pharmacokinetic analysis for fluorescently labeled RGD peptide targeted to the α v β 3 integrin in Kaposi"s sarcoma

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.

Dual-modality imaging in vivo with an NIR and gamma emitter using an intensified CCD camera and a conventional gamma camera

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.

Depth penetration and molar sensitivity for near-infrared fluorescence-enhanced optical imaging

Non-invasive, in vivo imaging modalities are valuable diagnostic indicators of tissue abnormalities, disease, metabolic changes, and other cellular anomalies that occur beneath the skins surface. Frequency domain photon migration imaging (FDPM) is a maturing optical tool that is based upon the propagation of near infrared (NIR) radiation through tissue and scattering media. Fluorescence-enhanced FDPM exploits a NIR light source for the excitation of a fluorescent contrast agent for detection. The research presented by this contribution seeks to further develop fluorescence-enhanced FDPM for cancer screening via sentinel lymph node mapping. Sentinel lymph node mapping involves the localization and resection for biopsy of the sentinel node to determine cancer spread. Investigating the propagation of NIR fluorescence deep within tissue-like scattering media will provide evidence to support the sensitivity of NIR imaging for sentinel lymph node localization, particularly when the sentinel lymph node is located up to 4 cm below the skin surface. The work presented herein provides a systematic examination of FDPM detection of signals originating from deeply embedded fluorescent inclusions within a tissue-mimicking phantom. Two dimensional multipixel images of embedded fluorescent targets are examined to determine if detection of fluorescence is possible over a reflectance geometry provided using an intensified charge coupled device camera system (ICCD). Data collected show fluorescence from targets containing 1 mM, 0.01 mM and 1 nM can be located when originating from 1 to 7-cm below the imaged surface, and as few as 0.01 femtomoles of ICG can be detected by the ICCD system, with proper choice of rejection filters.