The paper presents an algorithm based on low order statistics for the informative feature extraction for Raman spectroscopy data. The proposed method was tested on mouse preimplantation embryos Raman spectra. Both supervised and unsupervised machine learning methods were applied to selected the most informative features to test the separability of the processed data.
The paper presents an analysis of the Raman spectra of mouse preimplantation embryos using machine learning for visualization, assessing the separability of classes, and highlighting informative areas of the spectrum. Separation of lipid reach areas and nucleus spectra was shown by principal component analysis coupled with a linear support vector machine.
As a promising drug delivery system, itself or coupled with red blood cells (RBC), nanoparticles (NP) should be studied in frames of their interaction at the cellular level. Experiments were performed on RBC in autologous blood plasma incubated with different NP – TiO2, ZnO, nanodiamonds and polymeric nanocapsules. RBC aggregates formation in RBC suspension was observed with conventional microscopy, while quantitative interaction force measurements between individual RBC was assessed with optical tweezers. Scanning electron microscopy (SEM) imaging demonstrated NP localization and RBC membrane modifications upon binding with NP. Among tested NP, nanodiamonds caused increasing the size of aggregates in RBC suspensions, RBC interaction force increase and strong membrane surface modifications, comparing to other tested NP and control sample. Nanocapsules do not cause any adverse effects on RBC properties, confirming biocompatibility and applicability for drug delivery purposes. Optical tweezers combined with SEM imaging serves as fast informative assessment of NP effects on RBC.
The research and development for biomedical applications are recently focused on multifunctional nanoparticles. To integrate various functionalities, different methods of modifying the particle’s physical properties are developed. Among the considered, nanodiamond (ND) is a promising candidate for the development of multifunctional complex due to its variable features in size, structure, surface chemistry, physical properties, and biocompatibility. In addition to its well-studied structural, surface, electrochemical and photonic properties, strong magnetism of ND can be observed. In the present work, magnetically modified ND is introduced in terms of its bioapplications. Along with the soft ferromagnetism of ND, the increased fluorescence at one- and two-photon excitation is realized. Utilizing the combined magnetic and fluorescence properties of the magnetically modified ND, fluorescence imaging, fluorescence lifetime imaging and manipulation of cells by magnetic field are demonstrated. The perspectives to use the magnetic ND for drug delivery, cells magnetic separation and filtration, in bioengineering to control the cell distribution combined with imaging and treatment are discussed.
We present and discuss several modern optical methods based on elastic light scattering (ELS), along with their technical features and applications in biomedicine and life sciences. In particular, we review some ELS experiments at the single-cell level and explore new directions of applications. Due to recent developments in experimental systems (as shown in the literature), ELS lends itself to useful applications in the life sciences. Of the developed methods, we cover elastic scattering spectroscopy, optical tweezer-assisted measurement, goniometers, Fourier transform light scattering (FTLS), and microscopic methods. FTLS significantly extends the potential analysis of single cells by allowing monitoring of dynamical changes at the single-cell level. The main aim of our review is to demonstrate developments in the experimental investigation of ELS in single cells including issues related to theoretical “representations” and modeling of biological systems (cells, cellular systems, tissues, and so on). Goniometric measurements of ELS from optically trapped single cells are shown and the importance of the experimental verification of theoretical models of ELS in the context of biomedical applications is discussed.
Optical methods are widely used in biophotonic applications. They can be used for imaging cellular structures and living tissues. They also provide a tool to analyse cell cultures and cell suspensions. For example fluorescence, optical absorption or optical scattering can account for the contrast mechanism. Luminescence has also found various application areas. Luminescence from modified gene reporters can be measured to quantify biological phenomena and dynamic processes. In this paper the principles of phase sensitive detection and photon counting instrumentation systems to detect low-intensity light are shortly reviewed. They are typically using a photomultiplier tube as a detecting element. We discuss the experimental approach and the potential application areas in the context of elastic light scattering measurements of single particles and cells as well as in characterization of tissue-mimicking phantoms. Moreover, we describe a photon counting measurement system for measuring luminescence and show some results of monitoring luminescence in supernatant samples from cell cultures. The same instrument is capable to measure elastic light scattering from single cells and tissue-mimicking phantoms by using a phase sensitive detection with small modifications.
Strong light scattering in tissues and blood reduces the usability of many optical techniques. By reducing scattering, optical clearing enables deeper light penetration and improves resolution in several optical imaging applications. We demonstrate the usage of optical tweezers and elastic light scattering to study optical clearing [one of the major mechanisms—matching of refractive indices (RIs)] at the single particle and cell level. We used polystyrene spheres and human red blood cells (RBCs) as samples and glycerol or glucose water solutions as clearing agents. Optical tweezers kept single microspheres and RBCs in place during the measurement of light scattering patterns. The results show that optical clearing reduces the scattering cross section and increases g . Glucose also decreased light scattering from a RBC. Optical clearing affected the anisotropy factor g of 23.25-μm polystyrene spheres, increasing it by 0.5% for an RI change of 2.2% (20% glycerol) and 0.3% for an RI change of 1.1% (13% glucose).
Optical tweezers have been used in biophysical studies for over twenty years. Typical application areas are force
measurements of subcellular structures and cell biomechanics. Optical tweezers can also be used to manipulate the
orientation of objects. Moreover, using various beam shapes, optical tweezers allow measuring light scattering from
single and multiple objects by keeping particles and cells in place during the measurement. At single cell level, light
scattering yields important information about the object being studied, including its size, shape and refractive index. Also
dependent scattering can be studied. In this paper, we review experimental work conducted in this area by our group and
show new results relating to optical clearing phenomena at single microparticle level.
We report on the high-resolution deep-tissue imaging using novel water-dispersible upconversion nanoparticles (UCNPs)
β-NaYF4:Yb3+:Tm3+. Luminescence from the UCNP embedded into tissue-mimicking phantoms at the depth of 4 mm epi-illuminated with 975-nm laser radiation was detected. Fiber-optic detection shows 2-times better resolution compared with that obtained using CCD-based imaging modality. The conversion efficiency of upconversion particles and their cytotoxicity to HeLa cells were also investigated and reported.
Different theoretical models have been developed to understand light propagation in biological media and to facilitate the
analysis of experimental data, both at the cellular level and in bulk tissues. Optical tweezers, combined with a light
scattering measurement facility, enable the measurement of elastic light scattering distributions from single particles and
cells. The aim of this paper is to present elastic light scattering measurement results from several red blood cells (RBCs)
held in elliptical optical tweezers and to compare these results with theoretical predictions found in literature. Both faceon
and rim-on incidence of He-Ne laser light (vertical polarization) in relation to the measured RBCs was considered. In
the face-on case, light scattering intensity was larger from two RBCs than from one, but almost no difference was found
when using three RBCs instead of two. In rim-on incidence, clear changes in the shape of the scattering light intensity
field were found when the number of RBCs was increased from one to two. These results are supported by modelling results from literature.
We have developed an optical tweezers setup combined with a laser light scattering measurement system to measure the
elastic light scattering from trapped particles. The setup consists of a near infrared laser (λ=1064nm), a water immersion
objective for trapping, a single or double structure sample cuvette, and a HeNe-laser for illuminating the trapped
particles. The light is detected with an amplified photomultiplier and a lock-in amplifier. Light scattering images from
the trapped particles are also shown with a CCD camera. An optical trap keeps the particle stable during the
measurement. The measured scattering patterns from 23.25 μm diameter polystyrene spheres were shown to have good
comparability with theoretical modelling. 6.0 μm particles were also measured. The light scattering from trapped red
blood cells was much weaker than that from 23.25 μm and 6.0 μm polystyrene spheres, almost at the detection limit of
our current detection system configuration. The stability of the polystyrene sphere was much better during the
measurements than that of the red blood cell.
In this paper, we briefly review earlier approaches for optical stretching of red blood cells (RBCs) and introduce a novel
approach based on oscillatory optical tweezers. Preliminary experimental data for optical trap-and-stretch of RBCs by
two approaches, namely the counter-propagating dual-beam trap-and-stretch and the oscillatory optical tweezers, are
presented and discussed.
In this paper, we begin with a brief overview of optical trapping of micro- and nano- particles and of various techniques for the measurement of optical force constants in the linear spring model. We then move on to introduce two complimentary approaches to implement optical forced oscillation of the trapped particle, one by an oscillatory optical tweezers, and the other by chopping (i.e., switching on-and-off) one of the beams in a twin set of optical tweezers. In each implementation, we have measured the steady state amplitude and phase of the oscillating particle as a function of frequency (from ~ 10Hz to 600Hz) with the aid of a quadrant photo-diode in conjunction with a lock-in amplifier. For the case of optical forced oscillation of a "free" particle involving only the optical force and the viscous drag, the experimental data fit fairly well the theoretical curve obtained from the simple linear spring model; both the optical force constant and the viscosity of the surrounding fluid can be deduced with fairly high precision as the fitting parameters from the best fit of the experimental data to the theoretical curves.
When one or more external forces, in addition to the optical force and the drag force, were applied to the oscillating particle via mechanisms such as protein-protein interaction or DNA stretching, the oscillating amplitude and phase varied in response to the external forces. Preliminary data showing the change in oscillating amplitude and phase as a function of time in response to external forces will be presented, and potential biomedical applications of this approach will be discussed.
The combination of fluorescence spectroscopy and optical trapping under an integrated laser scanning microscopy platform has presented to be a powerful tool in manipulating and positioning the cells as well as standardizing the conditions of measurements. This platform was applied to investigate the autofluorescence of PC12 cells. Under 2-p excitation, the PC12 cells exhibit fluorescence in the range of 500-530 nm. It was shown that intensity of 2-p autofluorescence from PC12 cells is very sensitive to the cells' condition. Therefore autofluorescence from the cells may be a convenient and noninvasive method for monitoring the cell's or another biological object's condition, metabolism, and/or vitality under various physical and chemical influences.
Lipopolysaccharide (LPS) is one of the cell wall components of Gram-positive bacteria recognized by
and interacted with receptor proteins such as CD14 on macrophage cells. Such a process plays an
important role in our innate immune system. In this paper, we report the application of optical tweezers
(λ = 1064nm Gaussian beam focused by a water-immersed objective lens with N.A. = 1.0) to the study
of the dynamics of the binding of a LPS-coated polystyrene particle (diameter = 1.5μm) onto the
plasma membrane of a macrophage cell. We demonstrated that the binding rate increased significantly
when the macrophage cell was pre-treated with the extract of Reishi polysaccharides (EORP) which
has been shown to enhance the cell surface expression of CD14 (receptor of LPS) on macrophage cells.
We report the first direct experimental mapping of three-dimensional optical force field on a silica micro-particle trapped in a counter-propagating dual-beam trap. We tracked the three-dimensional Brownian motion of the trapped particle (by optical position sensing) and analyzed the particle position distribution to obtain the force constant of the optical force field on the particle along each direction. The trapping beams scattered by the trapped particle along two directions (mutually orthogonal to each other and also to the trapping beams) were projected on a pair of quadrant photo-detectors (QPDs) to facilitate high-speed (20 KHz) three-dimensional position tracking. Position tracking over two mutually orthogonal planes intrinsically provides one set of redundant data for a self-consistency check. At optical wavelength λ = 532nm, the force constants of the three-dimensional optical force field on a silica micro-particle (diameter = 2.58μm) were determined to be kx = 1.61×10-1 pN/μm, ky = 1.49×10-1 pN/μm, and kz = 4.43×10-2 pN/μm when the total trapping power was about 21mW and the distance between the two fiber end-faces was 125μm. The set of force constants (kx, ky, and kz) completely defines the optical force field E(x, y, z) = [kxx2 + kyy2 + kzz2]/2 (in the parabolic potential approximation) on the trapped particle.
Andrei Reshetnickov, Stefan Spaniol, N. Neugodova, G. Sapozhnikova, Olga Abakumova, Tatyana Tsvetkova, Igor Zalevsky, S. Goncharov, Artashes Karmenyan, Gelii Ponomarev
Five novel photosensitizers for photodynamic tumor therapy have been offered as potential drug substances for the prospective clinical usage. A complex of pre-clinic biological studies has been performed, unambiguously demonstrating low systemic toxicity and high photodynamic potential of the substances proposed. The one named 'Photodithazine/Photochlorin I' has already successfully passed a number of clinical test on patients having immergent vital indications.
Chlorine6 has been known to be attractive as photosensitizer (PS) for PDT for long. Its usefulness as a photosensitizing part of photoimmunotoxines for targeted PDT, as a synton for further chemical modification and as a promising PS has been widely recognized. There is a patented procedure at our disposal now allowing for preparation of a stable, well soluble and filtrating form of chlorine6- 'Photodithazine'. The elaborated production technology includes 4 steps. 'Photodithazine's 1-octanol/Phosphate buffer, pH 7.4 partition coefficient has been found to be 1.4, indicating that it must be able to localize in the plasma membrane. This may be a partial explanation of its higher photodynamic efficacy in vitro, comparing to 'Photosense' and its closeness to 'Photogem'.
O. Abakumova, Rudolf Baum, Natalia Ermakova, A. Gradyushko, T. Guseva-Donskaya, Artashes Karmenyan, U. Koraboyev, V. Laptev, V. Mechkov, L. Mikhailova, N. Panferova, Aleksei Rebeko, Andrei Reshetnickov, M. Ryabov, Eugeny Stranadko, Tatyana Tsvetkova, O. Zhukova
A novel stable water-soluble form of well known photosensitizer chlorin e6 named `Photodithazine' has been obtained from Spirulina Platensis cyanobacteria as a noncovalent complex with N-methyl-D-glucosamine, and its biological characteristics evaluate, which proved to be as follows: in vitro photocytotoxicity was 1 (mu) M (EC50) as determined by the extent of DNA synthesis inhibition in CaOv cells after irradiation with 650 - 900 nm light, and 5 (mu) M (EC65) as determined using MTT test on PC12 cells after irradiation with 670 nm laser light at the doses of 15 and 20 J/cm2, respectively, with Al-sulfophthalocyanine `Photosense' (Russia) and oligomerized hematoporphyrin-IX mixture `Photogen' (Russia) being used as permitted reference drugs.
For using in the complex for diagnostics and therapy tumors with the aid photodynamic therapy the special fiberscope was developed. Ergonomic principles are incorporated in the design of these compact and lightweight control heads. The efficiency of phototreatment could be improved by reducing the traumatism of normal tissues during the phototherapeutic procedures with achieving the homogeneous distribution of laser emission intensity on the tumor surface and with decreasing the labor intensiveness of phototreatment. The method forming and delivering the image of phototreatment to tumor with the aid special fiberscope was described.
The complex for tumor diagnostics and therapy on the basis of spatial-temporary light modulators (STLM) conjugated with laser, gastroscopy, videocamera on the base of CCD- matrix and computer is assembled. The choice of modulator operating mode permits us to achieve many effects, in particular, to make a contour radiating area, to provide a more intensive effect on the border of the center of pathology and to redistribute the emission intensity on the surface of pathologic tissue. The work of STLM and dependencies its characteristics from different parameters in full is described.
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