Ultrastructural 3D Microscopy for Biomedicine: Principles, Applications, and Perspectives.

Modern biomedical research often requires a three-dimensional microscopic analysis of the ultrastructure of biological objects and materials. Conceptual technical and methodological solutions for three-dimensional structure reconstruction are needed to improve the conventional optical, electron, and probe microscopy methods, which to begin with allow one to obtain two-dimensional images and data. This review discusses the principles and potential applications of such techniques as serial section transmission electron microscopy; techniques based on scanning electron microscopy (SEM) (array tomography, focused ion beam SEM, and serial block-face SEM). 3D analysis techniques based on modern super-resolution optical microscopy methods are described (stochastic optical reconstruction microscopy and stimulated emission depletion microscopy), as well as ultrastructural 3D microscopy methods based on scanning probe microscopy and the feasibility of combining them with optical techniques. A comparative analysis of the advantages and shortcomings of the discussed approaches is performed.

Correlative Fluorescent Scanning Probe Nanotomography Used to Study the Intracellular Distribution of Doxorubicin in MCF-7 Human Breast Adenocarcinoma Cells.

Developing technologies for efficient targeted drug delivery for oncotherapy requires new methods to analyze the features of micro- and nanoscale distributions of antitumor drugs in cells and tissues. A new approach to three-dimensional analysis of the intracellular distribution of cytostatics was developed using fluorescence scanning optical-probe nanotomography. A correlative analysis of the nanostructure and distribution of injected doxorubicin in MCF-7 human breast adenocarcinoma cells revealed the features of drug penetration and accumulation in the cell. The technology is based on the principles of scanning optical probe nanotomography and is applicable to studying the distribution patterns of various fluorescent or fluorescence-labelled substances in cells and tissues.

Assessment of core-shell nanoparticles surface structure heterogeneity by SAXS contrast variation and ab initio modeling.

For the biomedical applications of nanoparticles, the study of their structure is a major step towards understanding the mechanisms of their interaction with biological environment. Detailed structural analysis of particles' surface is vital for rational design of drug delivery systems. In particular, for core-shell or surface-modified nanoparticles surface structure can be described in terms of shell coating uniformity and shell thickness uniformity around the nanoparticle core. Taken together, these terms can be used to indicate degree of heterogeneity of nanoparticle surface structure. However, characterization of nanoparticle surface structure under physiological conditions is challenging due to limitations of experimental techniques. In this paper, we apply SAXS contrast variation combined with ab initio bead modeling for this purpose. Approach is based on the fact that nanoparticles under study are produced by self-assembly of phospholipid-conjugated molecules that possess moieties with significantly different electron densities enabling SAXS technique to be used to distinguish nanoparticle shell and study its structure. Ab initio single phase and ab initio multiphase modeling based on SAXS curve of nanoparticles in phosphate buffer solution allowed to reconstruct nanoparticle shell coating and assess its uniformity, while serial nanoparticle reconstructions from solutions with gradually increased solvent electron densities revealed relative shell coating thickness around nanoparticle core. Nanoparticle shell structure representation was verified by molecular dynamics simulation and derived full-atom nanoparticle shell structure showed good agreement with SAXS-derived representation. Obtained data indicate that studied nanoparticles exhibit highly heterogeneous surface structure.

Identification of Ultrastructural Details of the Astrocyte Process System in Nervous Tissue of the Brain Using Correlative Scanning Probe and Transmission Electron Microscopy.

Nanoscale morphological features of branched processes of glial cells may be of decisive importance for neuron-astrocyte interactions in health and disease. The paper presents the results of a correlation analysis of images of thin processes of astrocytes in nervous tissue of the mouse brain, which were obtained by scanning probe microscopy (SPM) and transmission electron microscopy (TEM) with high spatial resolution. Samples were prepared and imaged using a unique hardware combination of ultramicrotomy and SPM. Astrocyte details with a thickness of several tens of nanometers were identifiable in the images, making it possible to reconstruct the three-dimensional structure of astrocytic processes by integrating a series of sequential images of ultrathin sections of nervous tissue in the future.

Investigation of the Distribution of Magnetic Nanoparticles in Tumor Tissues by the Method of Scanning Magnetic Force Nanotomography.

The development of effective biomedical technologies using magnetic nanoparticles (MNPs) for the tasks of oncotherapy and nanodiagnostics requires the development and implementation of new methods for the analysis of micro- and nanoscale distributions of MNPs in the volume of cells and tissues. The paper presents a new approach to three-dimensional analysis of MNP distributions - scanning magnetic force nanotomography as applied to the study of tumor tissues. Correlative reconstruction of MNP distributions and nanostructure features of the studied tissues made it possible to quantitatively estimate the parameters of three-dimensional distributions of composite nanoparticles based on silicon and iron oxide obtained by femtosecond laser ablation and injected intravenously and intratumorally into tumor tissue samples of B16/F1 mouse melanoma. The developed technology based on the principles of scanning probe nanotomography is applicable for studying the features of three-dimensional micro- and nanoscale distributions of magnetic nanoparticles in biomaterials, cells and tissues of various types.

Nanohybrid Structures Based on Plasmonic or Fluorescent Nanoparticles and Retinal-Containing Proteins.

Rhodopsins are light-sensitive membrane proteins enabling transmembrane charge separation (proton pump) on absorption of a light quantum. Bacteriorhodopsin (BR) is a transmembrane protein from halophilic bacteria that belongs to the rhodopsin family. Potential applications of BR are considered so promising that the number of studies devoted to the use of BR itself, its mutant variants, as well as hybrid materials containing BR in various areas grows steadily. Formation of hybrid structures combining BR with nanoparticles is an essential step in promotion of BR-based devices. However, rapid progress, continuous emergence of new data, as well as challenges of analyzing the entire data require regular reviews of the achievements in this area. This review is devoted to the issues of formation of materials based on hybrids of BR with fluorescent semiconductor nanocrystals (quantum dots) and with noble metal (silver, gold) plasmonic nanoparticles. Recent data on formation of thin (mono-) and thick (multi-) layers from materials containing BR and BR/nanoparticle hybrids are presented.

[Silent myocardial ischemia in acute coronary syndrome].

The given review considers the pathogenesis, diagnosis, and clinical significance of silent myocardial ischemia (SMI) in individual patient groups. It discusses the problem of SMI in acute coronary syndrome (ACS), the possible causes of SMI and the specific features of its diagnosis. It also indicates that there is a need for 12-lead ECG telemonitoring in intensive care unit patients with ACS to intraoperatively correct patient management and treatment policy.

[Role of the determination of aortic pressure and rigidity in patients with cardiovascular diseases].

The paper reviews the literature on studies of the structural and functional properties of great arteries in patients with cardiovascular diseases. It analyses the pathophysiological components of the formation of central blood pressure (BP) and describes current registration procedures. The negative aspects of the impact of elevated central BP on target organs are considered. The prognostic and clinical values of the indicators of central arterial stiffness are demonstrated.

[The features of myocardial deformation of left ventricle in patients with ischemic heart disease defined by the two dimensional strain method].

Aim of this study was to assess qualitative and quantitative features of deformation parameters of left ventricular myocardium in patients with ischemic heart disease (IHD) with and without history of myocardial infarction (MI) using two-dimensional strain imaging. We examined 30 patients with clinical IHD with (group 1, n = 15) and without (group 2, n = 15) history of MI and 20 healthy volunteers. Compared with healthy subjects IHD patients of both groups had reduced longitudinal and circular myocardial deformation. There were no significant differences between patients with IHD and controls in parameters of radial, global, and regional deformation.

[Corrective effect of olmesartan on parameters of the local rigidity using the echo-tracking technology].

Purpose of this study was to determine clinical value of intima-media thickness (IMT) and indicators of local stiffness obtained by ultrasonography of the carotid arteries by echo-tracking technology in patients with hypertension and coronary artery disease (CAD), and to assess vasoprotective effect of 24-week treatment with olmesartan. We included into the study 22 patients with 1-2 degree hypertension, 25 patients with CAD combined with hypertension, and 44 healthy subjects. In patients with CAD and hypertension local stiffness was reassessed after 24-week treatment with olmesartan (20-40 mg/day). Patients with-hypertension compared with healthy subjects had higher values of loc P sys and loc P dia, P (T1), pulse wave propagation velocity (PWPV), stiffness index, as well as lower compliance and distensibility coefficients (CC and DC). In patients with CAD and hypertension all parameters of local stiffness and IMT were significantly higher, while CC and DC were significantly lower than in control group. Therapy with olmesartan for 24-weeks was associated with reductions of IMT (-4.8%, p < 0.05), loc P sys and loc P dia (-13.5 and -11%, respectively, p < 0.05), stiffness index ß (-19.1%, p < 0.05), loc PWPV (-17%) and P (T1) (-7%, p < 0.05), and increase of CC (3.3%, p < 0.05). Conclusion. Combination of CAD and hypertension worsened the local stiffness and increased IMT. Therapy with olmesartan in this group of patients was associated with indications of vasoprotective effect.

Resonance energy transfer in self-organized organic/inorganic dendrite structures.

Hybrid materials formed by semiconductor quantum dots and J-aggregates of cyanine dyes provide a unique combination of enhanced absorption in inorganic constituents with large oscillator strength and extremely narrow exciton bands of the organic component. The optical properties of dendrite structures with fractal dimension 1.7-1.8, formed from J-aggregates integrated with CdTe quantum dots (QDs), have been investigated by photoluminescence spectroscopy and fluorescence lifetime imaging microscopy. Our results demonstrate that (i) J-aggregates are coupled to QDs by Förster-type resonant energy transfer and (ii) there are energy fluxes from the periphery to the centre of the structure, where the QD density is higher than in the periphery of the dendrite. Such an anisotropic energy transport can be only observed when dendrites are formed from QDs integrated with J-aggregates. These QD/J-aggregate hybrid systems can have applications in light harvesting systems and optical sensors with extended absorption spectra.

[Carbohydrate metabolic disturbances and obstructive sleep apnea syndrome].

The paper analyzes an association between obstructive sleep apnea syndrome (OSAS) and carbohydrate metabolic disturbances. It presents the data of clinical trials that consider OSAS as a risk factor of type 2 diabetes mellitus (T2DM). The most important factors influencing the progression of carbohydrate metabolic disturbances in patients with OSAS are analyzed. Whether OSAS can be corrected as a modifiable risk factor in patients with T2DM is considered.

[CPAP therapy for obstructive sleep apnea syndrome in patients with carbohydrate metabolic disturbances and type 2 diabetes mellitus].

The review presents the data of clinical trials assessing the association between type 2 diabetes mellitus (DM) and obstructive sleep apnea syndrome (OSAS). The factors influencing the development of OSAS are analyzed in patients with DM; and disturbed sleep patterns are described in detail in patients with OSAS. The relationship of OSAS, diabetic autonomic neuropathy, and insulin resistance is analyzed. Whether CPAP therapy may be used to correct metabolic disturbances in patients with DM is considered.

[Assessment of characteristics of myocardial deformation in healthy subjects of various age groups by the X-STRAIN echocardiography].

Aim of the study was to master XStrain software and to obtain values of left ventricular (LV) myocardial velocity, longitudinal, circumferential, radial strain, and strain rate in a population of healthy subjects from various age groups. Sixty healthy volunteers were divided into 2 groups: aged 19-30 years (n = 35, mean age 24.6 +/- 3.1 years) and 30-58 years (n = 25, mean age 43.2 +/- 9.1 years). Values of all types of myocardial strain and circumferential strain rate did not differ substantially between groups at all LV levels: basal, median, and apical. Radial strain rate in the studied groups diminished from basal to apical LV segments. Longitudinal strain rate differed between LV sections in subjects older than 30 years. Myocardial velocity decreased from basal to apical myocardial segments.

[Semiconductor fluorescent nanocrystals (quantum dots) in biological biochips].

Comprehension of biological processes in cells, tissues and organisms requires identification and analysis of numerous biological objects, mechanisms of their action and regulation. Microarray (biochips) technology is a rare tool to solve this problem. It is based on high-throughput recognition of a target to the probe and has the potential to measure simultaneously the presence of numerous molecules in multiplexed testes, all contained in a small drop of test fluid. Biochips allow the parallel analysis of genomic or proteomic content in healthy versus disease-affected or altered tissues or cells. The signals read-out from the biochips is done with organic dyes which often suffer from photobleaching, low brightness and background fluorescence. Recent data show that the use of fluorescent nanocrystals "quantum dots" (QDs) allows push away these restrictions. The QDs are sufficiently bright to be detected as individual particles, extremely resistant to photobleaching and provide unique possibilities for multiplexing thus supplying the microarray technology with the novel read-out option enabling the sensitivity of detection reaching the single molecule level. This paper is aimed at the development of the approaches to the QDs application in microarray-based detection. Possibilities of QDs application both in solid state (planar) biochips as well as intensively developing technique of suspension biochips (bead-based assays or liquid biochips) are demonstrated. The latter are more and more applied for simultaneous identification of very large numbers of molecules in proteomics, genomics, drug screening and clinical diagnostics. This assays base on spectral encoded elements (as a rule polymer microbeads). The benefits of using optically encoded microbeads (instead of the solid-state two-dimensional arrays) are derived from the freedom of bead to move in three dimensions. Polymeric beads optically encoded with organic dyes allow for a limited number of unique codes, whereas the use of semiconductor nanocrystals as fluorescent tags improves the beads multiplexed imaging capabilities, photostability and sensitivity of the biological objects detection. Additionally, an employment in suspension biochips of Frster resonance energy transfer (FRET) allows improving detection specificity. The absence of fluorescent background from non-interacting with the beads dye-labelled antibodies additionally increases the sensitivity of detection and further facilitates the multiplexing capabilities of nanocrystals-based detection and diagnostics. So the combination of the biochips and QDs techniques allow increasing detection sensitivity and significantly raising the number of detected objects (multiplexing capacities). Such combination should provide the breakthrough in proteomics, particularly in new drugs development, clinical diagnostics, new disease markers identification, better understanding of intracellular mechanisms.

[The diagnostic value and clinical significance of a method for estimating the arterial stiffness by cardio-ankle vascular index].

The review presents the data of clinical studies of a new method for estimating the arterial stiffness by cardio-ankle vascular index (CAVI). The most significant factors influencing the vascular wall are analyzed and a CAVI measuring procedure and a calculation algorithm are described in detail. An association of the estimation of CAVI with other procedures determining the rigidity of the arterial wall is analyzed. A possibility for the practical application of this index to the study of the elastic properties of the vascular wall and for its use as a surrogate criterion to evaluate the vasoprotective effect of antihypertensive agents is considered.

[Provision of complex cardioprotection with the help of carvedilol in 1-2 degree arterial hypertension combined with metabolic syndrome].

In an open prospective 16-week study we carried out assessment of clinical efficacy, vaso- and cardioprotective properties of nonselective alpha and beta-adrenoblocker carvedilol used as monotherapy and in combination with nifedipine or as combination of these drugs with metformin and simvastatin. We have shown that the use of carvedilol both as monotherapy and in combination with nifedipine normalizes arterial pressure irrespective of the number of risk factors. We also revealed pronounced cardio- and vasoprotective action without negative effect on carbohydrate and lipid metabolism.

[Clinical study of arterial rigidity. Part II. Remodeling of vessels in arterial hypertension and metabolic syndrome. possibilities of correction with drugs].

In the second part of the review we present data on vascular remodeling in arterial hypertension and metabolic syndrome with analysis of factors and mechanisms influencing pulse wave propagation velocity in these diseases. We also consider possibilities of correction of remodeling of vessels with contemporary antihypertensive drugs.

[Comparative assessment of a calcium antagonist with controlled release of nifedipine and enalapril in arterial hypertension in subjects older than 60 years].

We compared antihypertensive effects and organoprotective action of a calcium antagonist nifedipine RD and angiotensin converting enzyme (ACE) inhibitor enalapril in subjects older than 60 years with systolic diastolic (group 1) and isolated (group 2) I-II degree arterial hypertension. Subgroups of patients receiving nifedipine RD, enalapril, or combination therapy were distinguished. We used ultrasound methods for measurement of parameters of hypertrophy, systolic and diastolic function of the heart, increments of diameter and blood flow velocity in brachial artery during test with reactive hyperemia. Nifedipine RD appeared to be the drug of choice in elderly persons with isolated systolic form of arterial hypertension. Combination of calcium antagonists and ACE inhibitors in resistant hypertension in the elderly lead to achievement of target values of blood pressure and potentiation of organoprotective action of drugs.

[Clinical value of the study of stiffness of arterial wall. Part I].

In this review we present concise historical background of formation of the interest to investigation of properties of arterial wall in this review. We consider in detail interrelation of arterial functions with their structure, main mechanisms of action of elastic properties of arteries on systemic hemodynamics. Definitions of terms describing structural-functional properties of arteries are also given. Attention is accentuated on velocity of pulse wave propagation -- an independent predictor of cardiovascular morbidity and mortality and generally accepted parameter characterizing stiffness of arterial segment. Analysis of most valuable factors affecting pulse wave propagation velocity and modern methods and devices for the study of arterial wall stiffness is presented as well.