Gold nanoshells with NIR plasmon resonance can be modified to simultaneously enhance conjugated NIR
fluorescence dyes and T2 contrast of embedded iron-oxide nanoparticles, and molecularly targeted to breast and other
cancers. We calibrated the theranostic performance of magneto-fluorescent nanoshells, and contrasted the performance
of molecularly targeted and untargeted nanoshells for breast cancer therapy, employing MCF-7L and their HER2 overexpressing
derivative MCF-7/HER2-18 breast cancer cells as in vitro model systems. Silica core gold nanoshells with
plasmon resonance on ~810 nm were doped with NIR dye ICG and ~10 nm iron-oxide nanoparticles in a ~20 nm epilayer
of silica. A subset of nanoshells was conjugated to antibodies targeting HER2. Cell viability with varying laser
power levels in presence and absence of bare and HER2-targeted nanoshells was assessed by calcein and propidium
iodide staining. For MCF-7L cells, increasing power resulted in increased cell death (F=5.63, p=0.0018), and bare
nanoshells caused more cell death than HER2-targeted nanoshells or laser treatment alone (F=30.13, p<0.001). For
MCF-7/HER2-18 cells, death was greater with HER2-targeted nanoshells and was independent of laser power. This
study demonstrates the capability of magneto-fluorescent nanocomplexes for imaging and therapy of breast cancer cells,
and the advantages of targeting receptors unique to cancer cells.
In this paper, we report an intraoperative approach by combining optical coherence tomography (OCT) and position
detection to detect and characterize ovarian cancers. A total of 18 ovaries were studied ex vivo. Based on histopathology
result, they were classified into normal and malignant groups, respectively. On average positron count rate of 8.0-fold
higher was found between malignant and normal ovaries. OCT imaging of ovaries revealed many detailed morphologic
features that could be potentially valuable for detecting early malignant changes in ovarian tissue. Optical scattering
coefficients of these ovaries were estimated from OCT A-lines. Normal ovarian tissue showed higher scattering
coefficient than that of malignant ovarian tissue. Using a threshold of 2.00 mm-1 for all ovaries, a sensitivity of 100% and a
specificity of 100% were achieved. This initial data shows our intraoperative probe based on OCT and positron detection
has a great potential for ovarian cancer detection and characterization.
Optical scattering coefficient from ex-vivo unfixed normal and malignant ovarian tissue was quantitatively extracted by
fitting optical coherence tomography (OCT) A-line signals to a single scattering model. 1097 average A-line
measurements at a wavelength of 1310nm were performed at 108 sites obtained from 18 ovaries. The average scattering
coefficient obtained from normal group consisted of 833 measurements from 88 sites was 2.41 mm-1 (±0.59), while the
average coefficient obtained from malignant group consisted of 264 measurements from 20 sites was 1.55 mm-1 (±0.46).
Using a threshold of 2 mm-1 for each ovary, a sensitivity of 100% and a specificity of 100% were achieved. The amount of
collagen within OCT imaging depth was analyzed from the tissue histological section stained with Sirius Red. The average
collagen area fraction (CAF) obtained from normal group was 48.4% (±12.3%), while the average CAF obtained from
malignant group was 11.4% (±4.7%). Statistical significance of the collagen content was found between the two groups
(p < 0.001). The preliminary data demonstrated that quantitative extraction of optical scattering coefficient from OCT
images could be a potential powerful method for ovarian cancer detection and diagnosis.
Optical scattering coefficient from ex vivo unfixed normal and malignant ovarian tissue was quantitatively extracted by fitting optical coherence tomography (OCT) A-line signals to a single scattering model. 1097 average A-line measurements at a wavelength of 1310 nm were performed at 108 sites obtained from 18 ovaries. The average scattering coefficient obtained from the normal tissue group consisted of 833 measurements from 88 sites was 2.41 mm−1 (±0.59), while the average coefficient obtained from the malignant tissue group consisted of 264 measurements from 20 sites was 1.55 mm−1 (±0.46). The malignant ovarian tissue showed significant lower scattering than the normal group (p < 0.001). The amount of collagen within OCT imaging depth was analyzed from the tissue histological section stained with Sirius Red. The average collagen area fraction (CAF) obtained from the normal tissue group was 48.4% (±12.3%), while the average CAF obtained from the malignant tissue group was 11.4% (±4.7%). A statistical significance of the collagen content was found between the two groups (p < 0.001). These results demonstrated that quantitative measurements of optical scattering coefficient from OCT images could be a potential powerful method for ovarian cancer detection.
We have developed a novel nitroimidazole indocyanine dye conjugate for tumor-targeted hypoxia fluorescence tomography. The hypoxia probe has been evaluated in vitro using tumor cell lines and in vivo with tumor targeting in mice. The in vitro cell studies were performed to assess fluorescence labeling differences between hypoxia and normoxia conditions. When treated with the hypoxia probe, a fluorescence emission ratio of 2.5-fold was found between the cells incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed by comparing the cells treated with indocyanine dye alone. In vivo tumor targeting in mice showed that the fluorescence signals measured at the tumor site were twice those at the normal site after 150 min post-injection of the hypoxia probe. On the other hand, the fluorescence signals measured after injection of indocyanine dye were the same at tumor and normal sites. In vivo fluorescence tomography images of mice injected with the hypoxia probe showed that the probe remained for more than 5 to 7 h in the tumors, however, the images of mice injected with indocyanine only dye confirmed that the unbound dye washed out in less than 3 h. These findings are supported with fluorescence images of histological sections of tumor samples using a Li-COR scanner and immunohistochemistry technique for tumor hypoxia.
In diffuse optical tomography (DOT), a typical perturbation approach requires two sets of measurements obtained at the lesion breast (lesion or target site) and a contra-lateral location of the normal breast (reference site) for image reconstruction. For patients who have a small amount of breast tissue, the chest-wall underneath the breast tissue at both sites affects the imaging results. In this group of patients, the perturbation, which is the difference between measurements obtained at the lesion and reference sites, may include the information of background mismatch which can generate artifacts or affect the reconstructed quantitative absorption coefficient of the lesion. Also, for patients who have a single breast due to prior surgery, the contra-lateral reference is not available. To improve the DOT performance or overcome its limitation, we introduced a new method based on an exogenous contrast agent and demonstrate its performance using animal models. Co-registered ultrasound was used to guide the lesion localization. The results have shown that artifacts caused by background mismatch can be reduced significantly by using this new method.
Tumor hypoxia is an important indicator of tumor metabolism and tumor response to various forms of therapy. Currently,
no imaging modality exists that can directly map tumor hypoxia non-invasively. We present an ultrasound guided diffuse
optical imaging technique for precisely measuring the tumor oxygenation. The approach employs ultrasound structural
information as a-prior knowledge for diffuse optical imaging. Hypoxia mapping is achieved using endogenous
chromophores such as oxy- and deoxy- hemoglobin in the tissue. Because oxy- and deoxy- hemoglobin respond
differently at different wavelengths, four different laser diodes of wavelengths 740 nm, 780 nm, 808 nm and 830 nm
were used for mapping tumor hypoxia by diffuse optical imaging. Hypoxia model experiments were performed using
phantoms at different oxygenation conditions (Hemoglobin oxygen saturation: 14%-92%) representing the hemoglobin
oxygenation range in tissue. Targets of different sizes mimicking different development stages of breast tumors, 1.0 cm
to 2.5 cm diameter in 0.5 cm steps, were tested to validate the oxygen saturation measurement accuracy with target size.
The absolute deviations between the estimated hemoglobin oxygen saturations from absorption maps and oxygen
measurements obtained using a pO2 electrode were less than 8% over the measured range of oxygen saturations (14% -
92%). An inhomogeneous cocentric blood phantom of deoxygenated center core and oxygenated outer shell was imaged
and deoxy- and oxy- hemoglobin maps revealed corresponding distributions which correlate with inhomogeneous
deoxy- and oxy- distributions frequently seen in advanced breast cancers located in the depth range of 1-3 cm.
KEYWORDS: Tumors, Carbon nanotubes, Photoacoustic imaging, Photoacoustic spectroscopy, Absorption, Hypoxia, Near infrared, Single walled carbon nanotubes, In vivo imaging, Digital signal processing
Development of new and efficient contrast agents is of fundamental importance to improve detection sensitivity of
smaller lesions. Within the family of nanomaterials, carbon nanotubes (CNT) not only have emerged as a new
alternative and efficient transporter and translocater of therapeutic molecules but also as a photoacoustic molecular
imaging agent owing to its strong optical absorption in the near-infrared region. Drugs, Antibodies and nucleic acids
could functionalize the CNT and prepare an appropriate system for delivering the cargos to cells and organs. In this
work, we present a novel photoacoustic contrast agent which is based on a unique hypoxic marker in the near infrared
region, 2-nitroimidazole -bis carboxylic acid derivative of Indocyanine Green conjugated to single walled carbon
nanotube (SWCNT-2nitroimidazole-ICG). The 2-nitroimidazole-ICG has an absorption peak at 755 nm and an extinction
coefficient of 20,5222 M-1cm-1. The conjugation of this marker with SWCNT shows more than 25 times enhancement of
optical absorption of carbon nanotubes in the near infrared region. This new conjugate has been optically evaluated
and shows promising results for high contrast photoacoustic imaging of deeply located tumors. The conjugate
specifically targets tumor hypoxia, an important indicator of tumor metabolism and tumor therapeutic response. The
detection sensitivity of the new contrast agent has been evaluated in-vitro cell lines and with in-vivo tumors in mice.
We present tumor hypoxia mapping by diffuse optical fluorescence tomography. A novel 2-nitroimidazole bis-carboxylic
acid indocyanine dye conjugate has been developed for tumor-targeted hypoxia fluorescence imaging. The hypoxia
probe has been evaluated in-vitro using 4T1 tumor cell lines and in-vivo tumor targeting in mice. In-vivo tumor targeting
in six mice demonstrated that a measured half-life of 2-nitroimidazole-indocyanine dye wash out in the tumor was
significantly longer (112±32.37 minutes) than that of bis-carboxylic acid indocyanine dye (69.75±14.01 minutes). The
bis-carboxylic acid indocyanine dye was completely washed out from the tumor site within 3-5 hours post-injection, but
2-nitroimidazole-ICG remained for up to 21 hours in the tumor site. Near infrared fluorescence images of mice tumors
showed a 2.6-fold contrast of dye uptake with hypoxic conjugate injection (7.46±1.68 μM) compared to that with
indocyanine dye injection (2.9±0.60 μM). The in-vitro cell studies were performed to assess fluorescence labeling
comparing hypoxia to normoxia conditions. A fluorescence emission ratio of 2.5-fold was found between the cells
treated with the 2-nitroimidazole-indocyanine dye and incubated under hypoxia compared to the cells in normoxia
condition. Hypoxia specificity was also confirmed when compared to cells treated with unconjugated indocyanine dye
alone. Fluorescence images acquired using a Li-COR scanner from harvested tumor samples support the in vivo
monitoring and imaging results.
Ovarian cancer has the lowest survival rate of the gynecologic cancers with a 5-year survival of about 50% in the United
States. With current screening and diagnostic abilities for ovarian cancers, most of the diagnosed patients are already with
advanced stages and the majority of them will die of this deadly disease. In this paper, we report a multimodal imaging
approach which combines optical coherence tomography (OCT) and positron detection for early ovarian cancer detection.
The dual modality system has the capability of providing both functional and morphological images simultaneously. While
the positron detection provides the metabolism activity of the ovary due to the uptake of radiotracer, the OCT provides the
high resolution (25μm X 25μm X 12μm - longitudinal X lateral X axial in air) structural imaging at 20k A-lines per second.
Total 18 ovaries obtained from 10 patients classified as normal, abnormal and malignant ovarian tissues were characterized
ex vivo. Positron counts of 1.2-fold higher was found between abnormal and normal ovaries and 3~30-fold higher was
found between malignant and normal ovaries. OCT imaging of malignant and abnormal ovaries revealed many detailed
morphologic features that could be potentially valuable for detecting early malignant changes in the ovary.
We demonstrate the feasibility of fluorescence imaging of deeply seated tumors using mice injected with an angiogenesis tracer, a vascular endothelial growth factor conjugated with the infrared dye cyanine 7 (VEGF/Cy7). Our optical-only imaging reconstruction method separately estimates the target depth, and then applies this information to reconstruct functional information such as fluorophore concentration. Fluorescence targets with concentrations as low as sub-25 nM are well reconstructed at depths up to 2 cm in both homogeneous and heterogeneous media with this technique.
We present a robust technique for diffuse optical fluorescence imaging of tumors in mice and tissue
simulating fluorescence phantoms. The detection optics, which is a crucial part of a frequency domain
fluorescence imaging system, with appropriate optical filters for efficient rejection of the excitation light, is
demonstrated. The image reconstruction is divided into two parts; i.e. reconstructing the target locations
such as size and position, and reconstructing the functional information such as fluorophore concentration
and image reconstruction. The structural parameters i.e. tumor size and locations of the targets are
recovered by a chi-square fitting technique by fitting the experimental data into analytically generated data.
Having the structural information beforehand, the images are reconstructed by using our dual-mesh
technique. The fluorescence images of targets of few tens of nanomolar fluorophore concentrations in both
homogeneous and heterogeneous media are reconstructed in this study.
The statistical and characteristic features of the polarized fluorescence spectra from cancer, normal and benign
human breast tissues are studied through wavelet transform and singular value decomposition. The discrete
wavelets enabled one to isolate high and low frequency spectral fluctuations, which revealed substantial randomization
in the cancerous tissues, not present in the normal cases. In particular, the fluctuations fitted well
with a Gaussian distribution for the cancerous tissues in the perpendicular component. One finds non-Gaussian
behavior for normal and benign tissues' spectral variations. The study of the difference of intensities in parallel
and perpendicular channels, which is free from the diffusive component, revealed weak fluorescence activity in
the 630nm domain, for the cancerous tissues. This may be ascribable to porphyrin emission. The role of both
scatterers and fluorophores in the observed minor intensity peak for the cancer case is experimentally confirmed
through tissue-phantom experiments. Continuous Morlet wavelet also highlighted this domain for the cancerous
tissue fluorescence spectra. Correlation in the spectral fluctuation is further studied in different tissue types
through singular value decomposition. Apart from identifying different domains of spectral activity for diseased
and non-diseased tissues, we found random matrix support for the spectral fluctuations. The small eigenvalues of
the perpendicular polarized fluorescence spectra of cancerous tissues fitted remarkably well with random matrix
prediction for Gaussian random variables, confirming our observations about spectral fluctuations in the wavelet
domain.
Fluorescence intensity fluctuations in the visible wavelength regime in normal, benign, and cancerous human breast tissue samples are studied through wavelet transform. The analyses have been carried out in unpolarized, parallel and perpendicularly polarized channels, for optimal tissue characterization. It has been observed that polarized fluorescence data, particularly the perpendicular components, differentiate various tissue types quite well. Wavelet transform, because of its ability for multiresolution analysis, provides the ideal tool to separate and characterize fluctuations in the fluorescence spectra at different scales. We quantify these differences and find that the fluctuations in the perpendicular channel of the cancerous tissues are more randomized as compared to their normal counterparts. Furthermore, for cancerous tissues, the same is very well described by the normal distribution, which is not the case for normal and benign samples. It has also been observed that, up to a certain point, fluctuations at larger scales are more sensitive to tissue types. The differences in the average, low-pass wavelet coefficients of normal, cancerous, pericanalicular, and intracanalicular benign tissues are also pointed out.
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