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.

Objective: Modern neurobiology focuses on understanding and preventing neurodegeneration by detecting cellular changes early. Changes in astrocyte ultramorphology may indicate early neurodegeneration, associated with decreased synaptic plasticity and astrocyte aging. While high-resolution techniques like EM and SPM can study these structures, they can’t use fluorescence microscopy for complete 3D analysis. We suggest using fluorescent dye-conjugated nanoantibodies for astrocyte labeling, allowing detailed ultramorphological research with electron, optical, and scanning probe microscopy, plus optical probe nanotomography. Methods: Hippocampi from male C57BL/6 mice were stained using commercial or GFAP-specific VHH-E9 nanoantibodies. The VHH-E9 antibodies were created by synthesizing a nucleotide sequence with added histidines and cysteine, then cloning it into the pET22 plasmid at XbaI and BamHI sites. Escherichia coli Rosetta-gami (DE3) cells were transformed to produce the pET22-VHHE9 strain. The nanoantibodies were produced via autoinduction and conjugated with a fluorophore. Astrocyte morphology was analyzed using the Sholl test with FiJi software. Results and Discussion: Traditional immunoglobulins can cause visualization inaccuracies due to the distance between the label and target protein. Nanoantibody conjugates, being smaller, reduce these errors. Immunostainings with both types showed similar efficiency, as Sholl profiles from different samples aligned within error margins. This supports the effectiveness of the sample preparation method and VHH-E9 nanoantibodies. It also suggests potential for developing similar procedures with fluorescent contrast agents, like semiconductor nanocrystals. Conclusions: To study astrocyte changes in early neurodegenerative stages, we propose combining high-resolution microscopy, optical microspectroscopy, and 3D ultrastructure restoration. Our method, OPNT, uses fluorescence microscopy for localization data, and SPM/UMT for ultrastructure. This involves creating UMT sections for simultaneous OM/SPM analysis and reconstructing them into a 3D array. We efficiently reconstruct 3D astrocyte ultramorphology and developed an immunostaining procedure using camel VHH-E9 nanoantibodies specific to GFAP, highlighting its efficiency and potential with fluorescent semiconductor nanocrystals.

Optical microscopy has undergone significant changes in recent decades due to the improved diffraction limit of optical resolution and the development of high-resolution imaging techniques referred to under the common name fluorescence nanoscopy. These techniques allow researchers to observe in detail biological structures and processes at a nanoscale level to reveal previously hidden features and answer basic biological questions. The advanced methods of fluorescent nanoscopy include STED (Stimulated Emission Depletion) Microscopy, STORM (STochastic Optical Reconstruction Microscopy), PALM (Photo-activated Localization Microscopy), TIRF (Total Internal Reflection Fluorescence) microscopy, SIM (Structured Illumination Microscopy), MINFLUX (Minimal Photon Fluxes) microscopy, PAINT (Points Accumulation for Imaging in Nanoscale Topography), and RESOLFT (REversible Saturable Optical Fluorescence Transitions) microscopy, and others. Most these methods make it possible to obtain 3D images of the objects under study. In this review, we consider the principles of these methods, their advantages and disadvantages, and their application in biological research.

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.

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.

Multiple studies have demonstrated that various nanoparticles (NPs) stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibit adipogenic ones. The mechanisms of these effects are not determined. The aim of this paper was to estimate Wharton’s Jelly MSCs phenotype and humoral factor production during tri-lineage differentiation per se and in the presence of silicon–gold NPs. Silicon (SiNPs), gold (AuNPs), and 10% Au-doped Si nanoparticles (SiAuNPs) were synthesized by laser ablation, characterized, and studied in MSC cultures before and during differentiation. Humoral factor production (n = 41) was analyzed by Luminex technology. NPs were nontoxic, did not induce ROS production, and stimulated G-CSF, GM-CSF, VEGF, CXCL1 (GRO) production in four day MSC cultures. During MSC differentiation, all NPs stimulated CD13 and CD90 expression in osteogenic cultures. MSC differentiation resulted in a decrease in multiple humoral factor production to day 14 of incubation. NPs did not significantly affect the production in chondrogenic cultures and stimulated it in both osteogenic and adipogenic ones. The major difference in the protein production between osteogenic and adipogenic MSC cultures in the presence of NPs was VEGF level, which was unaffected in osteogenic cells and 4–9 times increased in adipogenic ones. The effects of NPs decreased in a row AuNPs > SiAuNPs > SiNPs. Taken collectively, high expression of CD13 and CD90 by MSCs and critical level of VEGF production can, at least, partially explain the stimulatory effect of NPs on MSC osteogenic differentiation.

We have developed, manufactured, and tested a system of low-profile scanning probe microscopy (SPM) deflectometer capable of increasing the numerical aperture of objectives to a record high value of NA = 0.75. Implementation of this system will significantly improve performance characteristics of the optical microspectroscopy (OM) methods realized in combined SPM/OM systems.

Here, we propose and study several types of quartz surface coatings designed for the high-performance sorption of biomolecules and their subsequent detection by a photonic crystal surface mode (PC SM) biosensor. The deposition and sorption of biomolecules are revealed by analyzing changes in the propagation parameters of optical modes on the surface of a photonic crystal (PC). The method makes it possible to measure molecular and cellular affinity interactions in real time by independently recording the values of the angle of total internal reflection and the angle of excitation of the surface wave on the surface of the PC. A series of dextrans with various anchor groups (aldehyde, carboxy, epoxy) suitable for binding with bioligands have been studied. We have carried out comparative experiments with dextrans with other molecular weights. The results confirmed that dextran with a Mw of 500 kDa and anchor epoxy groups have a promising potential as a matrix for the detection of proteins in optical biosensors. The proposed approach would make it possible to enhance the sensitivity of the PC SM biosensor and also permit studying the binding process of low molecular weight molecules in real time.

Competitive schemes for constructing an optical system for a combination of a scanning probe microscope and an optical microspectrometer are examined, which make it possible to study opaque samples by the method of tip-enhanced Raman scattering. Taking into account the presence of a scanning microscope probe, optimal lenses are selected for implementing each scheme. The efficiency of using each optical scheme both for exciting a Raman signal and for collecting secondary radiation is quantitatively evaluated. As a result, the most effective optical system is revealed for achieving the optimal excitation/collection parameter.

Functional organic polymer materials with an ability to change their surface topography in response to external contactless stimuli, like light irradiation, have attracted considerable attention. This work is devoted to the study of contactless control of the surface topography and the formation of the surface features in the amorphousized and liquid crystalline films of two azobenzene-containing polymers. The investigated polymers are side-chain polymethacrylates containing azobenzene chromophores with two lateral methyl substituents in ortho-positions and differing in the length of flexible spacer with six and ten methylene units. Two lateral methyl substituents at the azobenzene chromophore ensure high photoresponses of these polymeric samples in the whole visible spectral range. Irradiation of the polymethacrylate films by focused polarized light of green (532 nm) and red (633 nm) lasers induces a specific photodeformation of the film surface. In the case of the green light formation of circular "craters" with anisotropic borders was found, whereas for the red light highly asymmetric "hills" were observed. The possible mechanisms of the surface topography formation and their features are discussed.

The synthetic function-spacer-lipid (FSL) amphiphile biotin-CMG-DOPE is widely used for delicate ligation of living cells with biotin residues under physiological conditions. Since this molecule has an "apolar-polar-hydrophobic" gemini structure, the supramolecular organization is expected to differ significantly from the classical micelle. Its organization is investigated with experimental methods and molecular dynamics simulations (MDS). Although the linear length of a single biotin-CMG-DOPE molecule is 9.5 nm, the size of the dominant supramer globule is only 14.6 nm. Investigations found that while the DOPE tails form a hydrophobic core, the polar CMG spacer folds back upon itself and predominantly places the biotin reside inside the globule or planar layer. MDS demonstrates that <10 % of biotin residues on the highly water dispersible globules and only 1 % of biotin residues in layer coatings are in an linear conformation and exposing biotin into the aqueous medium. This explains why in biotin-CMG-DOPE apolar biotin residues both in water dispersible globules and coatings on solid surfaces are still capable of interacting with streptavidin.

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.

A new experimental method for nanoscale measurements of the absorption spectra of single nanoobjects has been developed based on scanning near-field optical microspectroscopy (SNOM) and nanospectrophotometry (NSP). The main distinctive feature of the proposed SNOM-NSP technique consists in depositing a sample onto a coverglass followed by its probing in the total internal reflection spectroscopy mode. This approach allows the number of analyzed samples to be significantly increased and provides the possibility of combining measurements with other optical techniques. The proposed SNOM-NSP method has been successfully used for studying single plasmonic nanoparticles and their complexes with Rhodamine 6G dye.

The use of the achievements attained in colloid chemistry and other fields of chemical sciences allows us to develop new nanoscale systems for chemosensorics. Mixed monolayers consisting of a new chemosensory amphiphilic dithia-18-crown-6, which contains a dye of 4-alkylpyridine series with polyvinyl stearate at the water–air interface, are obtained and studied. The influence of various concentrations of silver ions on these mixed monolayers is studied. A promising method for the detection of silver ions in water using the given nanoscale systems based on chemosensory amphiphilic dithia-18-crown-6 is proposed.

The effect of silver nanoparticles (AgNPs) that are adsorbed on the surface of the purple membranes of Halobacterium salinarium bacteria on the optical properties and functional peculiarities of the lightsensitive protein bacteriorhodopsin (BR) has been demonstrated for the first time. Two mechanisms of the effect of AgNPs on the protein photocycle have been demonstrated using Raman scattering, giant Raman scattering, flash photolysis, and atomic force microscopy. It has been shown that the nanoparticles in the immediate spatial vicinity of BR fix its photocycle at the stage where it was at the moment of interaction with the nanoparticles. At greater distances, which reach three radii of an AgNPs, they have a weaker effect on BR, under which it retains the ability to be involved in the photocycle, however, has its parameters significantly changed. Thus, in the case of wild-type BR the photocycle accelerates and for the BR-D96N mutant it becomes slower. The data that are obtained could be of significance for creation of such optoelectronic hybrid systems with BR, where the parameters of its photocycle can be controlled using NPs. The results of the study may also be used in the field of nanobioengineering research, which is directed to creation of unique materials with controlled properties for recording and storage of information, energy transformation, and identification and characterization of trace amounts of analytes.