Vladimir I. Plehanov

Fluorescence diffuse tomography for detection of red fluorescent protein expressed tumors in small animals.

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.

Multicolor frequency-domain diffuse optical tomography for detection of breast cancer

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.

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

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.

Frequency-Domain Optical Diffusion Tomography of Fluorescent Proteins

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.

Fluorescence lifetime imaging of deep-seated fluorophore in turbid medium

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.

Frequency domain fluorescence diffuse tomography of small animals

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.

Frequency-domain photon density wave setup with multicolor illumination at 684, 794, and 1060 nm

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.

Frequency domain fluorescent diffuse tomography of small animals with DsRed2-expressed tumors

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.