Mikhail Kleshnin

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.

Optimal wavelengths for optoacoustic measurements of blood oxygen saturation in biological tissues.

The non-invasive measurement of blood oxygen saturation in blood vessels is a promising clinical application of optoacoustic imaging. Nevertheless, precise optoacoustic measurements of blood oxygen saturation are limited because of the complexities of calculating the spatial distribution of the optical fluence. In the paper error in the determination of blood oxygen saturation, associated with the use of approximate methods of optical fluence evaluation within the blood vessel, was investigated for optoacoustic measurements at two wavelengths. The method takes into account both acoustic pressure noise and the error in determined values of the optical scattering and absorption coefficients used for the calculation of the fluence. It is shown that, in conditions of an unknown (or partially known) spatial distribution of fluence at depths of 2 to 8 mm, minimal error in the determination of blood oxygen saturation is achieved at wavelengths of 658 ± 40 nm and 1069 ± 40 nm.

Trans-illumination fluorescence imaging of deep-seated tumors in small animals

Abstract Background: Fluorescence diffuse tomography (FDT) is the most accurate technique for the imaging of labeled tumors in the small animal body. However, the procedure for reconstruction of the spatial distribution of the fluorophore requires a high signal-to-noise ratio due to the ill-condition of the inverse problem. Therefore, the FDT technique is ineffective for imaging tumors of small size or with dim fluorophores because of the low intensity of their fluorescence compared with the high level of tissue autofluorescence. In these cases, the size and position of a marked tumor in the animal body can be estimated from two-dimensional fluorescence images obtained using trans- or epi-illumination techniques. Material and methods: A versatile system for small animal fluorescence imaging which combines planar epi- and trans-illumination geometries of the light source and of the fluorescence receiver was created and tested. For epi-illumination imaging, light-emitting diode sources were used to provide homogeneous and stable illumination of the experimental animal, in combination with a cooled CCD camera which covers the entire illuminated area. For trans-illumination imaging, mechanical raster-scanning devices modulated at a low frequency were used for the laser source, together with a cooled photomultiplier tube, which provided outstanding sensitivity. Results: Monitoring the orthotopic tumor growth in animal bodies has demonstrated the efficacy of trans-illumination imaging in comparison with the epi-illumination technique. The results obtained also showed that the effective use of the trans-illumination technique requires Born normalization of the fluorescence signal and the exclusion of lateral illumination by surrounding the animal with additional light absorption material using light-absorption pads on both sides of the body.

Fluorescence diffuse tomography technique with autofluorescence removal based on dispersion of biotissue optical properties

The dispersion of biotissue optical properties results in distortion of the spectrum of radiation during propagation through biotissues. This phenomenon can be used for solving the inverse problem in fluorescence diffuse tomography. We have developed the spectrally resolved fluorescence diffuse tomography (SFDT) technique, which allows reconstruction of the spatial distribution of the fluorophore in biotissue even in the presence of unknown autofluorescence. The experimental setup combining epi-illumination and trans-illumination imaging geometries with spectral resolution has been created. Experimental studies on tissue phantoms and small animals in vivo with the proposed SFDT technique have been conducted. The obtained results have shown high accuracy of the inverse problem solution for spatial distribution of fluorophore concentration.

Method of measuring blood oxygenation based on spectroscopy of diffusely scattered light

A new approach to the measurement of blood oxygenation is developed and implemented, based on an original two-step algorithm reconstructing the relative concentration of biological chromophores (haemoglobin, water, lipids) from the measured spectra of diffusely scattered light at different distances from the radiation source. The numerical experiments and approbation of the proposed approach using a biological phantom have shown the high accuracy of the reconstruction of optical properties of the object in question, as well as the possibility of correct calculation of the haemoglobin oxygenation in the presence of additive noises without calibration of the measuring device. The results of the experimental studies in animals agree with the previously published results obtained by other research groups and demonstrate the possibility of applying the developed method to the monitoring of blood oxygenation in tumour tissues.

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.

A technique for measuring oxygen saturation in biological tissues based on diffuse optical spectroscopy

A new approach to optical measuring blood oxygen saturation was developed and implemented. This technique is based on an original three-stage algorithm for reconstructing the relative concentration of biological chromophores (hemoglobin, water, lipids) from the measured spectra of diffusely scattered light at different distances from the probing radiation source. The numerical experiments and approbation of the proposed technique on a biological phantom have shown the high reconstruction accuracy and the possibility of correct calculation of hemoglobin oxygenation in the presence of additive noise and calibration errors. The obtained results of animal studies have agreed with the previously published results of other research groups and demonstrated the possibility to apply the developed technique to monitor oxygen saturation in tumor tissue.

Fluorescence imaging for photodynamic therapy of non-melanoma skin malignancies – A retrospective clinical study

Abstract Background and objective: 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. Study design and methods: 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 (Fotoditazin®); the tumors were irradiated with a 662 nm continuous wave diode laser with fiber delivery system and total fluence of up to 300 J/cm2. The fluorescence imaging was performed using a commercially available system with a camera and bandpass filter in the range of 710–800 nm. Fluorescence contrast (FC) of the tumor (the ratio of the average fluorescence intensities in the tumor and the surrounding tissues) and its change during the PDT treatment (photobleaching, dFC) was measured. Then the correlation between the clinical outcome (tumor response and recurrence rate) and measured fluorescence parameters was evaluated. The follow-up period was 6–53 months (median, 28 months). Results: FC or dFC below their median values independently correspond to a significant increase in tumor recurrence rate (p<0.05), and slight increase of partial or no tumor response cases. Tumor response is better correlated with the value of FC, and not correlated with the photobleaching. Conclusion: Baseline FC and its change after PDT treatment may serve as a predictor of recurrence. This finding is a step towards individualized PDT cancer treatment.

Reconstruction in fluorescence diffuse tomography based on non-negativity condition

We propose a novel method for solving a system of linear equations based on non-negativity condition. This method was applied for reconstruction in fluorescence diffuse tomography and was compared with other well-known methods.