We present the results of combined fluorescence and optoacoustic monitoring of tumor treatment using novel photoactivatable multi-inhibitor liposomes with BPD and Irinotecan providing a synergetic effect of PDT and chemotherapeutic impact.
A novel approach to monitor photosensitizer accumulation and photobleaching in the course of photodynamic therapy (PDT) with the use of nanoconstructs based on the simultaneous fluorescence (FL) and optoacoustic (OA) imaging is implemented. A liposome nanoconstructs employed in this studies contain benzoporphyrin derivatives (BPD) which serve as a photosensitizer and secondly, as a fluorophore, and the fluorescent IRDye800 dye acting as an additional contrasting agent due to its high quantum yield. FL provides visualization of BPD and IRDye800 distribution, while OA principle allows for BPD-absorption based imaging of tumor and its vascular environment. We demonstrate the results of a preliminary in vivo study with combined FL and OA custom-made setups on a NUDE mouse with human glioblastoma U- 87. The results of this studies show a hemorrhage in the tumor area on the OA images obtained @532 nm after PDT that is not visually detected, but confirmed with the following histological verification. Fast nanoconstructs accumulation (< 10 min) was observed using FL imaging with the concentration in tumor only 10% higher than in surrounding tissues. We believe that the ratio of nanoconstructs accumulation in tumor can be significantly increased using target approach.
We propose a new approach to monitoring of photodynamic therapy (PDT) of glioblastoma with the use of targeted nanoconstructs containing a photosensitizer (PS) benzoporphyrin derivative (BPD) and IRDye800 dye, antibodies for efficient accumulation of the drug in a tumor, and a chemotherapeutic agent for combined effect on tumor cells. Monitoring of PDT is based on the simultaneous fluorescent and optoacoustic (OA) imaging. Fluorescent imaging provides visualization of fluorescence agents with high molecular sensitivity, and monitoring of the effectiveness of PDT by PS photobleaching. OA allows to examine the vascular pattern of the tumor environment, as well as assess the tumor depth. IRDye800 is a better contrast agent in comparison to BPD due to red shifted spectral characteristics and higher fluorescence quantum yield. The results of numerical simulations have been verified in phantom studies using fluorescence and optoacoustic experimental setups and an agar phantom with optical characteristics similar to those of murine brain.
The ability for noninvasive visualization of functional changes of a tumor’s oxygenation and circulatory system offers new advantages for prognosis and monitoring of the treatment efficacy. The results of breast cancer oxygen state study under chemotherapy action obtained by diffuse optical spectroscopy (DOS) in combination with Doppler ultrasonic imaging are presented. Complex use of optical and ultrasound methods gives complementary information about the size of the tumor node, peculiarities of its vascular bed, rate of its blood flow as well as oxygenation, and provide a picture of the tumor response to treatment. Comparison with tumor pathologic response allowed to identify differences in the changes of these parameters depending on the degree of pathological tumor response to chemotherapy. It was demonstrated that fourth and fifth degrees of therapeutic pathomorphism may be predicted by the increase of oxygen saturation level after the first cycle of chemotherapy. If the reduction or absence of the oxygen saturation dynamics is observed, first or second degree of pathological tumor response can be expected. Additional ultrasound investigation of the tumors may be useful for observation of the dynamics of tumor blood flow thereby for understanding the reasons of induced chemotherapy oxygenation changes. The proposed approach based on DOS and ultrasonography may be applied for monitoring of breast tumors under therapy and prediction of their sensitivity.
In this work we studied the efficiency of spatially modulated illumination in optical diffuse reflectometry (ODR) and analyzed various probing patterns. The optimal illumination pattern was determined from the series of Monte Carlo numerical experiments on structured illumination and comparison of the parameters of fluence distribution within tissue. We considered the following illumination profiles: sinusoidal patterns with different spatial frequencies k (1 - 2.5 mm-1); piecewise constant patterns with the fixed duty cycle w = 2 and various strip width (0.2 – 1 mm); piecewise constant patterns with the fixed strip width (0.1 mm) and various duty cycle (3 – 11). Assuming the same total incident intensity for all patterns, we observed the growth in intensity at depth with decreasing value of spatial frequency for sinusoidal pattern, the similar tendency of intensity distribution was for piecewise constant patterns with the fixed strip width (or duty cycle) and duty cycle increase (or increase of a strip width, respectively). The intensity distributions within the sample are almost identical for sinusoidal and piecewise constant patterns with matching peaks and lows. However, probing by piecewise constant profile of illumination provide more local information about sample’s properties. A prototype of an ODR system for probing with the structured illumination was designed. The system consists of PC-controlled optical projection system, polarization filters and a CCD camera. The approbation of the system was carried out on the optical phantoms with optical properties close to those of biological tissues and on skin of human volunteers.
Effective breast cancer treatment requires the assessment of metabolic changes of tumor tissue during chemo- and hormonotherapy for prediction tumor response. Evaluation of the dynamics of tumor oxygen state (by diffuse optical spectroscopy imaging) and tumor vasculature (by ultrasound investigation in power Doppler mode) was performed before treatment beginning and before the second cycle of chemotherapy in 16 patients who received preoperative chemotherapy. Changes of these indicators were compared then with tumor pathologic response. Breast tumors demonstrated different dynamics of tumor oxygenation depending on the changes of tumor tissue. The increase of the tumor oxygenation after the first cycle of chemotherapy was observed in five of six patients with grade 4 and 5 of pathologic tumor response. Decrease of the oxygenation level was revealed in one patient with the 4th degree of tumor response. Variable changes of the oxygenation level were mentioned in the patients with moderate (the 3d degree) tumor response. Tumor oxygenation decreased or was unchanged in case of 1 or 2 degree of tumor response in five of six cases. The study of the tumor blood vessels didn't reveal any correlation between vasculature changes and tumor response under the performed treatment. The trend of tumor oxygenation in early time after treatment beginning might be a predictive criterion of tumor sensitivity to chemotherapy.
For correct identification of fluorophores in fluorescence lifetime imaging invivo it is important to account for widening of fluorescent kinetics curve due to light scattering and absorption in turbid medium. This widening leads to the difference between real and measured lifetimes of a fluorescent agent. We studied this effect for media with different optical properties and lifetimes corresponding to those of real fluorophores applying Monte-Carlo simulation. We found that for the fluorophore depths up to 15 mm for reduced scattering coefficient varying from 0.15 to 4.8 mm-1 and absorption coefficient varying from 0.0025 to 0.08 mm-1 this difference is insignificant for long-lived fluorophores (typical fluorescent proteins), however, it should be taken into account for fluorophores with lifetimes of several hundred picoseconds. Results of numerical simulation are confirmed by the results of the model experiment.
Correct identification of different fluorophores in the fluorescence lifetime imaging in vivo requires accounting for distortion of the measured fluorescent kinetics curve due to light scattering and absorption in medium. This distortion induces the difference between real and measured lifetimes of a fluorophore. We obtained analytical expression based on diffuse approximation of radiation transfer equation that allows to refine estimating the lifetime of a fluorophore. It was shown that our approach can be applied both for analytic kinetics curves obtained by diffuse approximation, Monte Carlo simulated curves and results of model experiment. Analytical and Monte Carlo simulated curves were obtained for media with different optical properties and lifetimes corresponding to those of real fluorophores. Results of numerical simulation are confirmed by the results of the model experiment.
Diffuse Optical Tomography (DOT) is based on acquiring information from multiply scattered light which penetrates
into the tissue up to depths of several centimeters. This technique allows for imaging of absorbing and scattering
inclusions inside tissue and distinguishing between them after computer processing of an image. An experimental setup
for multicolor frequency-domain diffuse optical tomography (FD DOT) to visualize neoplasia of breast tissue and to
estimate its size has been created. A breast is scanned in the transilluminative configuration by a single source and
detector pair. Illumination at three wavelengths (684 nm, 794 nm, and 850 nm) which correspond to different parts of the
absorption spectrum provides information about concentration of the main absorbers (oxygenated hemoglobin,
deoxygenated hemoglobin, and fat/water). Source amplitude modulation at 140 MHz increases spatial resolution and
provides separate reconstruction of scattering and absorption coefficients. In vivo study of breast carcinoma has been
performed. Maps of 2D distributions of reconstructed absorption and scattering coefficients and concentration of
hemoglobin have been obtained. An increase of absorption and scattering coefficient, total hemoglobin concentration and
decrease of blood oxygen saturation is observed in the tumor area in comparison with the surrounding tissue. We can
conclude that FD DOT technique confirms a possibility of detecting neoplastic changes.
A fluorescence diffuse tomography (FDT) setup for monitoring tumor growth in small animals has been created. In this setup an animal is scanned in the transilluminative configuration by a single source and detector pair. To remove stray light in the detection system, we used a combination of interferometric and absorption filters. To reduce the scanning time, an experimental animal was scanned using the following algorithm: (1) large-step scanning to obtain a general view of the animal (source and detector move synchronously); (2) selection of the fluorescing region; and (3) small-step scanning of the selected region and different relative shifts between the source and detector to obtain sufficient information for 3D reconstruction. We created a reconstruction algorithm based on the Holder norm to estimate the fluorophore distribution. This algorithm converges to the solution with a minimum number of fluorescing zones. The use of tumor cell lines transfected with fluorescent proteins allowed us to conduct intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Ibr, Mel-Kor, and human embryonic kidney HEK293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. The emission of red fluorescent proteins (RFPs) in the long-wave optical range permits detection of deep-seated tumors. In vivo experiments were conducted immediately after subcutaneous injection of fluorescing cells into small animals.
KEYWORDS: Tumors, Luminescence, Sensors, 3D modeling, Point spread functions, Fluorescent proteins, Tomography, In vivo imaging, Reconstruction algorithms, Fluorescence tomography
Capabilities of tumor detection by different optical methods can be significantly improved by labeling of tumors with
fluorescent markers. Creation of tumor cell lines transfected with fluorescent proteins provides the possibility not only to
detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and
human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. Emission of
RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body
imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable
noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in
small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated
light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. An optimizing algorithm
for scanning of an experimantal animal is suggested. In vivo experiments were conducted immediately after the
subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the
presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug
responce.
Fluorescent compounds for selective cancer cell marking are used for development of novel medical diagnostic methods,
investigation of the influence of external factors on tumor growth, regress and metastasis. Only special tools for turbid
media imaging, such as optical diffusion tomography permit noninvasive monitoring of fluorescent-labeled tumor
alterations deep in animal tissue. In this work, the results of preliminary experiments utilizing frequency-domain
fluorescent diffusion tomography (FD FDT) experimental setup in small animal are presented. Low-frequency modulated
light (1 kHz) from Nd:YAG laser with second harmonic generation at the wavelength of 532 nm was used in the setup.
The transilluminative planar configuration was used in the setup. A series of model experiments has been conducted and
show good agreement between theoretical and experimental fluorescence intensity. Models of deep tumors were created
by two methods: (1) glass capsules containing fluorophore solution were inserted into esophagus of small animals to
simulate marked tumors; (2) a suspension of transfected HE&Kgr;293-Turbo-RFP cells was subcutaneously injected to small
animal. The conducted experiments have shown that FD FDT allows one to detect the presence of labeled tumor cells in
small animals, to determine the volume of an experimental tumor, to perform 3D tumor reconstruction, as well as to
conduct monitoring investigations. The obtained results demonstrate the potential capability of the FD FDT method for
noninvasive whole-body imaging in cancer studies, diagnostics and therapy.
An experimental setup for multicolor frequency-domain optical diffuse tomography was designed to visualize soft
biological tissue inhomogeneities at the distance of up to 6 cm. Scanning is performed by independent electronically
controlled shift of source and detector placed in transmission mode. Employing illumination at three wavelengths (684,
794, and 1060 nm) which correspond to predominating absorption of oxygenated hemoglobin, deoxygenated
hemoglobin and water provides determination of component composition of an inhomogeneity. Source power
modulation at 140 MHz increases spatial resolution (compared to CW imaging) and improves quality of reconstruction
procedure. Studies on model media and preliminary in vivo experiments were performed.
KEYWORDS: Tumors, Luminescence, 3D modeling, Point spread functions, Sensors, Fluorescent proteins, Tomography, In vivo imaging, Tissue optics, Scattering
Conventional optical imaging is restricted with tumor size due to high tissue scattering. Labeling of tumors by
fluorescent markers improves sensitivity of tumor detection thus increasing the value of optical imaging dramatically.
Creation of tumor cell lines transfected with fluorescent proteins gives the possibility not only to detect tumor, but also to
conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney
HEK-293 Phoenix were transfected with DsRed-Express and TurboRFP genes. Emission of RFP in the long-wave
optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special
tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the
internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An
animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser
with second harmonic generation at the 532 nm wavelength. In vivo experiments were conducted immediately after the
subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the
presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug
responce.
The main applications of fluorescent proteins (FPs) are monitoring tumor growth, angiogenesis, metastases formation and effects of new classes of drugs. Different types of tomography allow fluorescence imaging of tumors located deep in human or animal tissue. These techniques were used for investigation of the distribution of near-infrared fluorescent probes, but only a few works are devoted to fluorescence tomography in visible light. In this work, preliminary results of the frequency domain fluorescent diffuse tomography (FD FDT) method in application to DsRed2 protein as a fluorescent agent are presented. For the first step of our experiments we utilized second harmonic generation of Nd:YAG laser (532 nm) modulated by low frequency (1 kHz) in the experimental setup. The transilluminative planar configuration was used in the setup. A series of model experiments has been conducted and show good agreement between theoretical and experimental fluorescence intensity. Post mortem experiments with capsules containing DsRed2 and scattering solution introduced into esophagus of rats to simulate tumor formation have been conducted. The results of these experiments show that sensitivity of the setup is sufficient to detect DsRed2 in concentrations similar to those in FP-expressed tumor, but the contrast is not enough high to separate fluorescence of DsRed2 and surrounding tissues. The setup can be significantly improved by utilizing high-frequency modulation (110 MHz using acousto-optical modulator) of the excitation light and precise phase measurements due to difference in fluorescence life-time of FPs and surrounding tissues. An algorithm of processing a fluorescent image based on calculating zero of maximum curvature was employed for detection of fluorescent inclusions boundaries in the image.
We present preliminary results of the frequency domain fluorescent diffuse tomography (FD FDT) method in application to fluorescent proteins. For first step in the experimental setup we utilized light-emitting diode (530 nm wavelength) modulated with low frequency (18 kHz). A model experiments with capsules containing DsRed suspension in scattering medium has been conducted. The results of post mortem experiments with capsules containing DsRed, introduced into abdominal cavity of mice to simulate tumors inside animal body, are presented. An algorithm of processing fluorescent image based on calculating zero of maximum curvature has been applied to detect fluorescent inclusions boundaries on the image.
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