Advances in three-dimensional fluorescence microscopy are limited by poor axial resolution. We introduce Fluorescence Optical Nanotomography (FONT) system that bypasses this limitation by integrating widefield fluorescence imaging with serial ultramicrotomy. FONT achieves an axial resolution directly defined by the physical section thickness (40-200 nm), effectively decoupling it from optical diffraction. We demonstrate FONT's capability by reconstructing the 3D architecture of hepatocytes in rat liver and astrocytic networks in a mouse model of Alzheimer's disease, achieving a axial resolution of similar to 100 nm/pixel. Furthermore, we present the design and theoretical validation of a dedicated platform that enables seamless correlation of FONT with in situ Scanning Probe Microscopy (SPM). This SPM-FONT platform is engineered to perform both modalities within a single cutting cycle, directly on the block-face, eliminating morphological artifacts. Our results establish FONT as a powerful standalone technique for nanoscale bioimaging and pave the way for a fully integrated correlative system to provide simultaneous topological, mechanical, and biochemical information from the same biological volume.

Improvement of the existing methods of ultrahigh-resolution microscopy and development of new ones, in particular, methods of three-dimensional (3D) analysis and imaging of the ultrastructure of biological objects and nanomaterials, are extremely important tasks in biomedicine, cell biology, and nanotechnology. Here, a method of 3D total internal reflection fluorescence (TIRF) microscopy is proposed that combines ultramicrotomy (successive ultrathin sections of the sample) and TIRF microscopy of the sample surface. The key feature of this combined technique is the use of a specialized diamond ultramicrotome knife both as a tool for making the sections and as a TIRF prism for obtaining layer-by-layer TIRF images within a single measurement procedure. This makes it possible to reconstruct the 3D ultrastructure of objects.

Предложен метод 3D-TIRF-микроскопии, основанный на объединении методик ультрамикротомии и флуоресцентной микроскопии полного внутреннего отражения поверхности образца после среза и позволяющий реконструировать трехмерную ультраструктуру объектов.

The effect of recombinant spidroin (RS) hydrogel (HG) on anterior epithelial cells and keratocytes of the human cornea was studied in vitro. Corneal injuries are highly prevalent in developing countries according to the World Health Organization. Various technologies have recently been proposed to restore the damaged surface of the cornea. Use of biodegradable silk-based materials, including recombinant analogs of the spider silk protein spidroin, is an important avenue of research in the field of wound healing and corneal regeneration. Spidroins are well known for their optimal balance of strength and elasticity. Given their biological compatibility, lack of immunogenicity, and biodegradability, spidroins provide a biomaterial for tissue engineering and regenerative medicine. HGs based on RS rS2/12-RGDS were therefore tested for cytotoxicity toward isolated corneal epithelial cells and keratocytes with regard to possible changes in cell phenotype and migratory activity. A promising outlook and therapeutic potential were demonstrated for RS-based HGs.

Myocardial remodeling is an inevitable risk factor for cardiac arrhythmias and can potentially be corrected with cell therapy. Although the generation of cardiac cells ex vivo is possible, specific approaches to cell replacement therapy remain unclear. On the one hand, adhesive myocyte cells must be viable and conjugated with the electromechanical syncytium of the recipient tissue, which is unattainable without an external scaffold substrate. On the other hand, the outer scaffold may hinder cell delivery, for example, making intramyocardial injection difficult. To resolve this contradiction, we developed molecular vehicles that combine a wrapped (rather than outer) polymer scaffold that is enveloped by the cell and provides excitability restoration (lost when cells were harvested) before engraftment. It also provides a coating with human fibronectin, which initiates the process of graft adhesion into the recipient tissue and can carry fluorescent markers for the external control of the non-invasive cell position. In this work, we used a type of scaffold that allowed us to use the advantages of a scaffold-free cell suspension for cell delivery. Fragmented nanofibers (0.85 µm ± 0.18 µm in diameter) with fluorescent labels were used, with solitary cells seeded on them. Cell implantation experiments were performed in vivo. The proposed molecular vehicles made it possible to establish rapid (30 min) electromechanical contact between excitable grafts and the recipient heart. Excitable grafts were visualized with optical mapping on a rat heart with Langendorff perfusion at a 0.72 ± 0.32 Hz heart rate. Thus, the pre-restored grafts' excitability (with the help of a wrapped polymer scaffold) allowed rapid electromechanical coupling with the recipient tissue. This information could provide a basis for the reduction of engraftment arrhythmias in the first days after cell therapy.

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.

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.

Cardiac arrhythmias are a major cause of cardiovascular mortality worldwide. Many arrhythmias are caused by reentry, a phenomenon where excitation waves circulate in the heart. Optical mapping techniques have revealed the role of reentry in arrhythmia initiation and fibrillation transition, but the underlying biophysical mechanisms are still difficult to investigate in intact hearts. Tissue engineering models of cardiac tissue can mimic the structure and function of native cardiac tissue and enable interactive observation of reentry formation and wave propagation. This review will present various approaches to constructing cardiac tissue models for reentry studies, using the authors' work as examples. The review will highlight the evolution of tissue engineering designs based on different substrates, cell types, and structural parameters. A new approach using polymer materials and cellular reprogramming to create biomimetic cardiac tissues will be introduced. The review will also show how computational modeling of cardiac tissue can complement experimental data and how such models can be applied in the biomimetics of cardiac tissue.

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.

The development of advanced biomaterials and constructs for accelerated recovery of damaged tissues is a key direction in regenerative medicine. Biocompatible scaffolds based on natural biopolymers are widely used for these tasks. Organ decellularization enables obtaining a cell-free extracellular matrix (ECM) with preserved composition and biological activity. The objectives of the present work were combining these two approaches for the development of a composite scaffold based on silk fibroin and ECM microparticles and assessing its structure, biological properties, and regenerative potential. ECM microparticles were obtained by grinding the decellularized matrix of Wistar rat liver in liquid nitrogen. Scaffolds in the form of films were prepared by the casting method. The sinuous and rough topography of the scaffold surface was assessed by the scanning probe nanotomography (SPNT) technique. The inclusion of ECM microparticles in the composition did not affect the elasticity and tensile strength of the scaffolds. The obtained scaffold was non-toxic to cells, maintained high levels of adhesion and proliferation of mouse 3T3 fibroblast and Hep-G2 cells, and showed high regenerative potential, which was studied in the experimental model of full-thickness rat skin wound healing. The wound healing was accelerated by 1.74 times in comparison with the control.

Induced pluripotent stem cells (iPSCs) constitute a potential source of patient-specific human cardiomyocytes for a cardiac cell replacement therapy via intramyocardial injections, providing a major benefit over other cell sources in terms of immune rejection. However, intramyocardial injection of the cardiomyocytes has substantial challenges related to cell survival and electrophysiological coupling with recipient tissue. Current methods of manipulating cell suspensions do not allow one to control the processes of adhesion of injected cells to the tissue and electrophysiological coupling with surrounding cells. In this article, we documented the possibility of influencing these processes using polymer kernels: biocompatible fiber fragments of subcellular size that can be adsorbed to a cell, thereby creating the minimum necessary adhesion foci to shape the cell and provide support for the organization of the cytoskeleton and the contractile apparatus prior to adhesion to the recipient tissue. Using optical excitation markers, the restoration of the excitability of cardiomyocytes in suspension upon adsorption of polymer kernels was shown. It increased the likelihood of the formation of a stable electrophysiological coupling in vitro. The obtained results may be considered as a proof of concept that the stochastic engraftment process of injected suspension cells can be controlled by smart biomaterials.

На сегодня известно, что биодеградируемые конструкции на основе фиброина шелка могут использоваться в качестве безопасного носителя биологически активных веществ и лекарственных препаратов. При этом последние исследования показали, что глиальный нейротрофический фактор является одним из необходимых факторов роста, участвующих в восстановлении роговичной ткани. Однако не изучен способ восстановления роговичных повреждений при помощи биодеградируемых конструкций на основе фиброина шелка, содержащих глиальный нейротрофический фактор, что объяснило цель данного исследования. Цель: изучить особенности восстановления роговичных повреждений при помощи биодеградируемых конструкций на основе фиброина шелка, содержащих глиальный нейротрофический фактор. Материалы и методы: Исследование проводилось на половозрелых самцах мышей линии C57BL/6. У животных формировали модель повреждения роговицы. В ходе эксперимента у мышей на 1-е, 3-и и 5-е сутки после повреждения проводили анализ площади эпителиального дефекта роговицы для оценки интенсивности регенеративного процесса. Результаты: установлено, что интенсивность регенеративного процесса была выше в группах мышей с применением биодеградируемых конструкций на основе фиброина шелка, содержащих глиальный нейротрофический фактор, по сравнению с контрольной группой. Выводы: Применение биодеградируемых конструкций на основе фиброина шелка, содержащих глиальный нейротрофический фактор, стимулирует процесс восстановления роговичной ткани после моделированного повреждения. Background: Today it is well known that biodegradable materials based on silk fibroin can be used as safe carriers of biologically active substances and drugs. At the same time, recent studies have shown that glial neurotrophic factor is one of the essential growth factors involved in corneal tissue regeneration. However, the method of restoring corneal damage with biodegradable materials based on silk fibroin containing glial neurotrophic factor has not been developed. Aim: To study pathogenetic features of corneal damage repair with biodegradable structures based on silk fibroin containing glial neurotrophic factor. Materials and Methods: The study was conducted on mature male C57BL/6 mice, in which corneal damage was modeled. During the experiment, the area of the epithelial corneal defect was assessed for the intensity of the regenerative process on days 1, 3, and 5 after injury. Results: In the groups of mice where biodegradable structures based on silk fibroin containing glial neurotrophic factor were used, the intensity of the regenerative process was higher compared to the control group (p < 0.05). Conclusion: The use of biodegradable structures based on silk fibroin containing glial neurotrophic factor stimulates the process of corneal tissue regeneration after corneal damage.

The main goal of our research was to fabricate electrospun scaffolds from three different silk proteins—silk fibroin from Bombyx mori silkworm cocoons and two recombinant spidroins, rS2/12 and rS2/12-RGDS—and to perform a comparative analysis of the structure, biological properties, and regenerative potential of the scaffolds in a full-thickness rat skin wound model. The surface and internal structures were investigated using scanning electron microscopy and scanning probe nanotomography. The structures of the scaffolds were similar. The average fiber diameter of the scaffolds was 315 ± 26 nm, the volume porosity was 94.5 ± 1.4%, the surface-to-volume ratio of the scaffolds was 25.4 ± 4.2 μm−1 and the fiber surface roughness was 3.8 ± 0.6 nm. The scaffolds were characterized by a non-cytotoxicity effect and a high level of cytocompatibility with cells. The scaffolds also had high regenerative potential—the healing of the skin wound was accelerated by 19 days compared with the control. A histological analysis did not reveal any fragments of the experimental constructions or areas of inflammation. Thus, novel data on the structure and biological properties of the silk fibroin/spidroin electrospun scaffolds were obtained.

Objective: to analyze the 3D micro- and nano-structure and quantitative morphological parameters of liver cells cultured on biodegradable silk fibroin-based film scaffolds. Materials and methods . Samples of biodegradable silk fibroin-based scaffolds with cultured Wistar rat liver cells were obtained for the study. The 3D structure of liver cells cultivated on the scaffolds was studied by scanning probe optical nanotomography using an experimental setup combining an ultramicrotome and a scanning probe  microscope in correlation with fluorescence microscopy. Results . Nanoscale images and 3D nanotomographic reconstructions of rat liver cells cultured on scaffold were obtained. The morphological parameters of liver cells (average roughness, specific effective area) were determined. The average surface roughness of the liver cells R a was found to be 124.8 ± 8.2 nm, while the effective surface area σ was 1.13 ± 0.02. Analysis of the volume distribution of lipid droplets showed that they occupy 28% of the cell volume . Conclusion. Scanning probe optical nanotomography can successfully analyze the nanostructure and quantify the nanomorphology of liver cells cultured on biodegradable scaffolds.

The obtaining of microcarriers for the cell culture and delivery is an urgent task of tissue engineering and regenerative medicine. The novel method of surface modification of alginate microcarriers in the form of microspheres with a diameter of 200–300 μm was developed. The described method consists in covalent crosslinking between collagen and surface of alginate microcarriers. It was shown that the method makes it possible to completely modify the surface of the alginate microcarrier, which can be used to improve the biological properties of the microcarrier. Such microcarriers with improved biological properties can be considered as effective systems for cell delivery and culture.

A comparative analysis of the structure and biological properties of silk fibroin constructions was performed. Three groups of constructions were obtained: films obtained by casting an aqueous solution of silk fibroin and electrospun microfibrous scaffolds based on silk fibroin, with the addition of 30% gelatin per total protein weight. The internal structures of the films and single fibers of the microfibrous scaffolds consisted of densely packed globule structures; the surface area to volume ratios and volume porosities of the microfibrous scaffolds were calculated. All constructions were non-toxic for cells and provide high levels of adhesion and proliferation. The high regenerative potential of the constructions was demonstrated in a rat full-thickness skin wound healing model. The constructions accelerated healing by an average of 15 days and can be considered to be promising constructions for various tasks of tissue engineering and regenerative medicine.

Creation of new effective bio-artificial structures for tissue engineering and regenerative medicine requires development and implementation of new technological approaches for analysis of micro- and nanostructural features of constructs based on biomaterials and their interaction with cells. A new method of three-dimensional multiparametric analysis of nanostructure, scanning optical probe nanotomography, is presented in this paper, applied to the analysis of cells and biomaterials. Correlative reconstruction of fluorescent marker distributions and nanostructure features allows quantitative evaluation of a number of parameters of three-dimensional nanomorphology of fibroblasts and human hepatocarcinoma cells Hep-G2, adhered to biodegradable scaffolds based on silk fibroin. The developed technology with use of scanning optical probe nanotomography is applicable to investigation of three-dimensional micro- and nanostructure features of biomaterials and cells of different types.