Ilya I. Fiks

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.

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.

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.

Quantitative evaluation of atherosclerotic plaques using cross-polarization optical coherence tomography, nonlinear, and atomic force microscopy

Abstract. 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.

Three-dimensional in vivo imaging of tumors expressing red fluorescent proteins.

3D imaging of genetically-engineered fluorescent tumors enables quantitative monitoring of tumor growth/regression, metastatic processes, including during anticancer therapy in real-time.Fluorescent tumor models for 3D imaging require stable expression of genetically encoded fluorescent proteins and maintenance of the properties of tumor cell line including growth rate, morphology, and immunophenotype.In this chapter, the protocol for 3D imaging of tumors expressing red fluorescent protein are described in detail.

Tissue sensing by structured illumination in optical diffuse reflectometry

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.

Quantitative analysis of the polarization characteristics of atherosclerotic plaques

In this study we demonstrate the capability of cross-polarization optical coherence tomography (CP OCT) to assess collagen and elastin fibers condition in atherosclerotic plaques basing on ratio of the OCT signal levels in cross- and со- polarizations. We consider the depolarization factor (DF) and the effective birefringence (Δn) as quantitative characteristics of CP OCT images. We revealed that calculation of both DF and Δn in the region of interest (fibrous cap) yields a statistically significant difference between stable and unstable plaques (0.46±0.21 vs 0.09±0.04 for IDF; (4.7±1.0)•10–4 vs (2.5±0.7)•10–4 for Δn; p<0.05). In parallel with CP OCT we used the nonlinear microscopy for analysis of thin cross-section of atherosclerotic plaque, revealing the different average isotropy index of collagen and elastin fibers for stable and unstable plaques (0.30 ± 0.10 vs 0.70 ± 0.08; р<0.001). The proposed approach for quantitative assessment of CP OCT images allows cross-scattering and birefringence characterization of stable and unstable atherosclerotic plaques.

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.

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.

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.