Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.
An experimental setup for multicolor frequencydomain diffuse optical tomography (FD DOT) was created to visualize neoplasia of breast tissue and to estimate its size. The breast is gently pressed between two glass plates and 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 in a therapeutic transparency window 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. Moreover, it gives information about breast thickness, which is important for reconstruction. The sensitivity of the system enables one to detect the light propagated through tissue having thickness up to 8 cm. Studies on model media and preliminary in vivo experiments with normal breast and breast carcinoma were performed. An increase of scattering coefficient and total hemoglobin concentration is observed in the tumor area. This corroborates validity of the FD DOT method for breast cancer diagnosis.Examination couch equipped with FD DOT device in the Semashko Regional Hospital (Russia)
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.
Photodynamic therapy (PDT) has been successfully used in clinical practice for decades; however, clinical outcome data are not always consistent resulting in a great necessity for real-time monitoring to predict the therapy outcome.In a retrospective clinical study, 402 patients with non-melanoma skin malignancies were enrolled who underwent PDT treatment and fluorescence real-time imaging. The photosensitizer used was a chlorine e6 derivative (FotoditazinBaseline
Photodynamic therapy is one of the most promising methods for the treatment of oncological, inflammatory and degenerative diseases of the skin. This technique is based on light irradiation of a photosensitizer that has been injected into the patient's body prior to the procedure, with determination of the efficacy of treatment requiring proper assessment of the drug concentration in the tissue lesion and the extent of sensitizer photobleaching during irradiation. We have developed a compact and low cost device based on a fluorescence imaging for localizing the tumor in the patient's body, tracking the position of the tissue lesion during involuntary movements of the patient, estimating accumulation of the sensitizer in the tumor relative to the surrounding tissues and monitoring photobleaching of the sensitizer during laser irradiation. The system that has been created is compatible with any therapeutic laser and includes a single CCD camera and two LEDs, one in the excitation band and the other in the emission band of the 'Photoditazin' sensitizer. In this letter we also present the test results of the device in model experiments and in preliminary clinical trials. The results obtained clearly show the efficacy of the system for monitoring sensitizer photobleaching during photodynamic therapy.
A combination of approaches to the image analysis in cross-polarization optical coherence tomography (CP OCT) and high-resolution imaging by nonlinear microscopy and atomic force microscopy (AFM) at the different stages of atherosclerotic plaque development is studied. This combination allowed us to qualitatively and quantitatively assess the disorganization of collagen in the atherosclerotic arterial tissue (reduction and increase of CP backscatter), at the fiber (change of the geometric distribution of fibers in the second-harmonic generation microscopy images) and fibrillar (violation of packing and different nature of a basket-weave network of fibrils in the AFM images) organization levels. The calculated CP channel-related parameters are shown to have a statistically significant difference between stable and unstable (also called vulnerable) plaques, and hence, CP OCT could be a potentially powerful, minimally invasive method for vulnerable plaques detection.
Abstract. Diffuse optical spectroscopy (DOS) and its modification employing structured illumination are widely used in monitoring biotissue oxygenation. In such measurements it is important to know the probing volume for definite source-detector configuration; however, it cannot be measured directly. Monte Carlo simulations allow to trace the probing depth of each individual photon contributing to the signal, which provides a numerical solution for this problem. In this study we investigate distributions of photons over maximal depth reached in turbid media (probing depth) with optical parameters typical for cutaneous tissues at the wavelength of 600 nm. Different configurations of probing illumination are considered, such as collimated point source, one-dimension sinusoidal and rectangular patterns. For collimated point source and zero source-detector separation the number of collected photons monotonously decreases with the probing depth while a pronounced maximum in the distribution is manifested with the increase of source-detector separation. The position of this maximum shifts to higher depths with the decrease of µa. For one-direction sinusoidal and rectangular illumination patterns it is shown that when the photons are collected near the center of a bright stripe, the peak of the distribution remains close to the surface. When the photons are collected near the center of a dark stripe the peak shifts towards higher depths with the decrease in spatial duty cycle and spatial frequency of the illumination pattern. Employment of rectangular illumination pattern seems more efficient for DOS applications due to wider abilities for controlling probing depth.
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