Ilya V. Turchin

Complementary use of cross-polarization and standard OCT for differential diagnosis of pathological tissues

An experimental standard optical coherence tomography (OCT) setup that can be easily modified for cross-polarization OCT (CP OCT) operation has been developed to perform differential diagnosis of pathological tissues. The complementary use of CP OCT, a technique that provides a map of cross-polarization backscattering properties of an object being studied by means of low-coherence interferometry, and standard OCT imaging improves the specificity of diagnostics of pathological changes occurring in tissues. It is shown that healthy, neoplastic and scar tissues of the esophagus have different cross-polarization backscattering properties. A comparative analysis of CP OCT, OCT and histological images from one and the same tissue area has been made. A close correlation between the location of collagen fibers in biological tissue and signal intensity in CP OCT images is found.

Novel algorithm of processing optical coherence tomography images for differentiation of biological tissue pathologies

A numerical algorithm based on a small-angle approximation of the radiative transfer equation (RTE) is developed to reconstruct scattering characteristics of biological tissues from optical coherence tomography (OCT) images. According to the algorithm, biological tissue is considered to be a layered random medium with a set of scattering parameters in each layer: total scattering coefficient, variance of a small-angle scattering phase function, and probability of backscattering, which fully describe the OCT signal behavior versus probing depth. The reconstruction of the scattering parameters is performed by their variation to fit the experimental OCT signal by the theoretical one using a time-saving genetic algorithm. The proposed reconstruction procedure is tested on model media with known scattering parameters. The possibility to estimate scattering parameters from OCT images is studied for various regimes of OCT signal decay. The developed algorithm is applied to reconstruct optical characteristics of epithelium and stroma for normal cervical tissue and its pathologies, and the potential to distinguish between the types of pathological changes in epithelial tissue by its OCT images is demonstrated.

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer.

Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells

BACKGROUND Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells. METHODS HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo. RESULTS Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG. CONCLUSIONS miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor. GENERAL SIGNIFICANCE This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.

Optical coherence tomography for in situ monitoring of laser corneal ablation

OBJECTIVE To improve the precision of refractive surgery, a new approach for determination of the removed corneal thickness profile in situ with laser ablation by optical coherence tomography (OCT) is developed. STUDY DESIGN/MATERIALS AND METHODS The traditional method for precision (less than 10 microm) measurements of intraocular distances is based on the use of the reflected component of probing radiation. This component is characterized by a small range of operating angles between a probing beam and a normal to the surface under study. To enhance this range of operating angles we suggest using a light component backscattered from a biological object. This will enable precision measurements over the entire surface of the cornea without any changes in the orientation between a probing beam and the eye, a necessary condition for in situ monitoring of laser refraction correction in the eye. We suggest a specially developed algorithm of OCT signal processing to measure the corneal thickness by the backscattered light component for a single longitudinal scan (A scan). The corneal thickness profile is obtained by a series of such A scans acquired by successively scanning a probing beam along the corneal surface. The thickness profile of removed layer is determined by changes in the corneal thickness profile in the process of ablation. When the cornea is ablated by a beam with a fixed transverse profile, we propose using integral characteristics of the ablated layer profile, for example, the maximum ablation depth, as criteria of changes in refractive power of the eye. The measurement precision by these characteristics is considerably higher than by a single A scan. Since the cornea is a poorly scattering medium, the Fourier filtering is employed to increase reliability and precision of the method. Model experiments on monitoring the ablation process in a lavsan film and ex vivo human cornea are described. Preliminary experiments on in vivo measurements of human corneal thickness are performed. RESULTS In model experiments the precision of measurement of laser ablation depth by one A scan was 5-20 microm, depending on the signal-to-noise ratio (SNR), whereas the precision of measurement of laser ablation depth as the integral characteristic of the ablated layer profile was 0.3-5 microm. The experimental results showed that at small SNR Fourier filtering might considerably increase reliability and precision of measurements. When SNR is high, the measurement precision does not change. The precision of measurements of the corneal thickness in preliminary in vivo experiments was higher than in ex vivo experiments. This factor is very promising for application of the method suggested herein in refractive surgery.

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.

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

Lifetime imaging of FRET between red fluorescent proteins

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.

Simultaneous photoacoustic and optically mediated ultrasound microscopy: phantom study.

An experimental setup for combined photoacoustic (PA) and optically mediated ultrasound (US) microscopy is presented. A spherically focused 35 MHz polyvinylidene fluoride (PVDF) ultrasonic detector with a numerical aperture of 0.28, a focal distance of 9 mm, and a bandwidth (-6 dB level) of 24 MHz was used to obtain PA and US data with a 3 mm imaging depth. A fiber-optic system was employed to deliver laser excitation pulses from a tunable laser to the studied medium. A single optical pulse was used to form both PA and US A-scans. The probing US pulses were generated thermoelastically due to absorption of backscattered laser radiation by the metalized surface of a PVDF film.

Simultaneous photoacoustic and optically mediated ultrasound microscopy: an in vivo study.

We propose the use of thermoelastic (TE) excitation of an ultrasonic (US) detector by backscattered laser radiation as a means of upgrading a single-modality photoacoustic (PA) microscope to dual-modality PA/US imaging at minimal cost. The upgraded scanning head of our dual-modality microscope consists of a fiber bundle with 14 output arms and a 32MHz polyvinylidene difluoride (PVDF) detector with a 34 MHz bandwidth (-6 dB level), 12.7 mm focal length, and a 0.25 numerical aperture. A single optical pulse delivered through the fiber bundle to the biotissue being investigated, in combination with a metalized surface on the PVDF detector allows us to obtain both PA and US A-scans. To demonstrate the in vivo capabilities of the proposed method we present the results of bimodal imaging of the brain of a newborn rat, a mouse tail and a mouse tumor.