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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., (Copyright ® 2024 National Research University Higher School of Economics.)

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., (© 2023. Pleiades Publishing, Ltd.)

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., (© 2023. Pleiades Publishing, Ltd.)

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., (© 2023. Pleiades Publishing, Ltd.)

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., (© 2022. Pleiades Publishing, Ltd.)

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., (© 2021. Pleiades Publishing, Ltd.)

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies. The aim of the work was to study the effect of biodegradable silk fibroin-based scaffolds containing GDNF on the corneal regeneration process. During cultivate the highest keratocytes proliferative activity was registered with scaffolds containing 250 ng/ml and 500 ng/ml GDNF. In mice with an experimental model of epithelial-stromal damage to the cornea, silk fibroin-based scaffolds containing GDNF in various concentrations were used (in groups 1, 2 and 3 silk fibroin-based scaffolds containing GDNF in a concentration of 50 ng/ml, 250 ng/ml and 500 ng/ml, respectively; in group 4 - silk fibroin-based scaffolds without GDNF; in group 5 - a solution of GDNF with concentration of 500 ng/ml; group 6- control). The area of the corneal epithelial defect in groups 2, 3, and 5 was less than in the other groups. The most pronounced positive immunohistochemical reaction with antibodies to Bcl2, Bax, phosphoERK1/2 and phospho-JNK1/2, Ki67, Gap43 was observed in groups 2 and 3. Thus, silk fibroin-based scaffolds with GDNF stimulate the epithelialization process, proliferative activity of epithelial cells and keratocytes, accelerate the formation of the stromal nerve plexus and exhibit anti-apoptotic activity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2021. Pleiades Publishing, Ltd.)

Organ decellularization is one of the promising technologies of regenerative medicine, which allows obtaining cell-free extracellular matrix (ECM), which provide preservation of the composition, architecture, vascular network and biological activity of the ECM. The method of decellularization opens up wide prospects for its practical application not only in the field of creating full-scale bioengineered structures, but also in the manufacture of vessels, microcarriers, hydrogels, and coatings. The main goal of our work was the investigation of structure and biological properties of lyophilized decellularized Wistar rat liver fragments (LDLFs), as well as we assessed the regenerative potential of the obtained ECM. We obtained decellularized liver of a Wistar rat, the vascular network and the main components of the ECM of tissue were preserved. H&E staining of histological sections confirmed the removal of cells. DNA content of ECM is equal to 0.7% of native tissue DNA content. Utilizing scanning probe nanotomogrphy method, we showed sinuous, rough topography and highly nanoporous structure of ECM, which provide high level of mouse 3T3 fibroblast and Hep-G 2 cells biocompatibility. Obtained LDLF had a high regenerative potential, which we studied in an experimental model of a full-thickness rat skin wound healing: we observed the acceleration of wound healing by 2.2 times in comparison with the control., (© 2021 IOP Publishing Ltd.)

The layer-by-layer (LbL) deposition approach allows combined incorporation of fluorescent, magnetic, and plasmonic nanoparticles into the shell of polyelectrolyte microcapsules to obtain stimulus-responsive systems whose imaging and drug release functions can be triggered by external stimuli. The combined use of fluorescent quantum dots (QDs) and magnetic nanoparticles (MNPs) yields magnetic-field-driven imaging tools that can be tracked and imaged even deep in tissue when the appropriate type of QDs and wavelength of their excitation are used. QDs are excellent photonic labels for microcapsule encoding due to their close-to-unity photoluminescence (PL) quantum yields, narrow PL emission bands, and tremendous one- and two-photon extinction coefficients. However, the presence of MNPs and electrically charged polyelectrolyte molecules used for the LbL fabrication of magneto-optical microcapsules provokes alterations of the QD optical properties because of the photoinduced charge and energy transfer resulting in QD photodarkening or photobrightening. These lead to variation of the microcapsule PL signal under illumination, which hampers their tracking and quantitative analysis in cells and tissues. Here, we have studied the effects of the structure and spatial arrangement of the nanoparticles within the microcapsule polyelectrolyte shell, the total shell thickness, and the shell surface charge on their PL properties under continuous illumination. The roles of the charge transfer and its main driving forces in the stability of the microcapsules PL signal have been established, and the design of the microcapsules dually encoded with QDs and MNPs providing the strongest and most stable PL has been determined. Controlling the energy transfer from the QDs and MNPs and the charge transfer from QDs to polyelectrolyte layers in the engineering of magneto-optical microcapsules with a bright and stable PL signal extends their applications to long-lasting quantitative fluorescence imaging.

We present a new concept of a combined scanning probe microscope (SPM)/ultramicrotome apparatus. It enables "slice-and-view" scanning probe nanotomography measurements and 3D reconstruction of the bulk sample nanostructure from series of SPM images after consecutive ultrathin sections. The sample is fixed on a flat XYZ scanning piezostage mounted on the ultramicrotome arm. The SPM measuring head with a cantilever tip and a laser-photodiode tip detection system approaches the sample for SPM measurements of the block-face surface immediately after the ultramicrotome sectioning is performed. The SPM head is moved along guides that are also fixed on the ultramicrotome arm. Thereby, relative dysfunctional displacements of the tip, the sample, and the ultramicrotome knife are minimized. The design of the SPM head enables open frontal optical access to the sample block-face adapted for high-resolution optical lenses for correlative SPM/optical microscopy applications. The new system can be used in a wide range of applications for the study of 3D nanostructures of biological objects, biomaterials, polymer nanocomposites, and nanohybrid materials in various SPM and optical microscopy measuring modes.

We fabricated bioresorbable microcarriers from water solution of Bombyx mori silk fi broin. The microcarriers are 3D structures with intricate surface and pores allowing penetration of culture medium, gas exchange, and cell adhesion. Fibroin molecules form hydrophobic structures and normally have a negative charge, which stimulates migration, but inhibits cell adhesion and makes it less effective. In order to improve adhesion efficiency and velocity, gelatin (hydrophilic biopolymer with integrin-recognizing RGD sequence) was added to the microcarrier composition. The resultant bioresorbable microcarriers support adhesion and proliferation of 3T3 murine fibroblasts.

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50-150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.