Scanning Probe Nanotomography Research Articles

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.

Scaffolds Based on Silk Fibroin with Decellularized Rat Liver Microparticles: Investigation of the Structure, Biological Properties and Regenerative Potential for Skin Wound Healing.

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.

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.

Silk Fibroin/Spidroin Electrospun Scaffolds for Full-Thickness Skin Wound Healing in Rats.

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.

3D analysis of the micro- and nanostructure of lung tissue by scanning probe nanotomography

Objective: to analyze the 3D micro- and nanostructure and quantitative morphological parameters of rat lung tissue. Materials and methods. Wistar rat lung tissue samples were obtained for the study. The 3D structure of the lung tissue was studied via scanning probe nanotomography using an experimental setup combining an ultramicrotome and a scanning probe microscope. Results. Nanoscale images and 3D nanotomographic reconstructions of the interalveolar septal sections of the rat lung were obtained. Morphological parameters (average roughness and specific effective area) of the interalveolar septal surface were determined. It was found that the average roughness of the reconstructed septal surface was 345.4 ± 24.5 nm, and the specific effective area was 2.7 ± 0.2 units. Conclusions. Results obtained demonstrate that scanning probe nanotomography allows to quantify lung morphology. The use of scanning probe nanotomography for 3D analysis of the structure and characteristics of lung tissue will increase the efficiency of future developments on creation of new criteria for diagnosing pathological conditions.

Наноразмерный корреляционный анализ морфологической, оптической и магнитной структуры полимерных микросфер для мультиплексной диагностики

A united analytical technology based on the integration of scanning probe nanotomography and optical microscopy (SPNT-OM) is presented and the potential of the technology is demonstrated by the example of multi-parameter analysis of encoded microspheres. The presented SPNT-OM technology can become a powerful tool for multimodal nanocharacterization of a wide range of microparticles, composite and hybrid polymers.

3D scanning probe nanotomography of tissue spheroid fibroblasts interacting with electrospun polyurethane scaffold

3D scanning probe nanotomography of tissue spheroid fibroblasts interacting with electrospun polyurethane scaffold

Three-dimensional analysis of micro- and nanostructure of biomaterials and cells by method of scanning probe nanotomography

Aim: to perform a three-dimensional analysis of micro- and nanosctucture and quantitative morphological parameters of alginate spherical microcarriers and porous regenerated silk macrocarriers modifi ed by microparticles of decellularized rat liver matrix and human hepatoma HepG2 cells adhered to micro- and macro carriers.Materials and methods. Three-dimensional porous matrices made from regenerated silk by salt leaching technique and alginate spherical microcarriers fabricated by encapsulation were vitalized by human hepatome HepG2 cells. Study of three-dimensional structure of cells and micro- and macro carriers was carried out at –120 °С by scanning probe cryonanotomography technique with use of experimental setup combining cryoultramicrotome and scanning probe microscope.Results. Three-dimensional nanotomographical reconstructions of HepG2 cells adhered to macropore wall of regenerated silk macrocarrier and to spherical alginate microcarrier are obtained. Morphological parameters (mean roughness, effective surface area and autocorrelation length) are determined for surfaces of macro and microcarriers and adhered cells. The determined mean roughness of alginate microcarrier surface is 76.4 ± 7.5 nm, while that of surface of macropore wall of regenerated silk macrocarrier is 133.8 ± 16.2 nm. At the same time mean roughness of cells adhered to micro- and macrocarriers are 118.5 ± 9.0 и 158.8 ± 21.6 nm correspondingly. Three-dimensional reconstructions of intracellular compartments with dimensions from 140 to 500 nm are also obtained.Conclusion. Obtained as a result of study quantitative morphology characteristics of surfaces of cell carriers and adhered cells show signifi cant degree of correlation of morphological parameters of cells and their carriers. Use of scanning probe cryonanotomography technique for three-dimensional analysis of structure and characteristics of biomaterials, cells and bio-artifi cial cellular systems enables to improve effi ciency of development of novel cell-engineered constructions with predicted morphological, physical, chemical and biological characteristics for tasks of tissue engineering and regenerative medicine.

High resolution 3D microscopy study of cardiomyocytes on polymer scaffold nanofibers reveals formation of unusual sheathed structure

Building functional and robust scaffolds for engineered biological tissue requires a nanoscale mechanistic understanding of how cells use the scaffold for their growth and development. A vast majority of the scaffolds used for cardiac tissue engineering are based on polymer materials, the matrices of nanofibers. Attempts to load the polymer fibers of the scaffold with additional sophisticated features, such as electrical conductivity and controlled release of the growth factors or other biologically active molecules, as well as trying to match the mechanical features of the scaffold to those of the extracellular matrix, cannot be efficient without a detailed knowledge of how the cells are attached and strategically positioned with respect to the scaffold nanofibers at micro and nanolevel. Studying single cell – single fiber interactions with the aid of confocal laser scanning microscopy (CLSM), scanning probe nanotomography (SPNT), and transmission electron microscopy (TEM), we found that cardiac cells actively interact with substrate nanofibers, but in different ways. While cardiomyocytes often create a remarkable “sheath” structure, enveloping fiber and, thus, substantially increasing contact zone, fibroblasts interact with nanofibers in the locations of focal adhesion clusters mainly without wrapping the fiber. Statements of SignificanceWe found that cardiomyocytes grown on electrospun polymer nanofibers often create a striking “sheath” structure, enveloping fiber with the formation of a very narrow (∼22 nm) membrane gap leading from the fiber to the extracellular space. This wrapping makes the entire fiber surface available for cell attachment. This finding gives a new prospective view on how scaffold nanofibers may interact with growing cells. It may play a significant role in effective design of novel nanofiber scaffolds for tissue engineering concerning mechanical and electrical properties of scaffolds as well as controlled drug release from “smart” biomaterials.

An instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography

In the past decade correlative microscopy, which combines the potentials of different types of high-resolution microscopies with a variety of optical microspectroscopy techniques, has been attracting increasing attention in material science and biological research. One of outstanding solutions in this area is the combination of scanning probe microscopy (SPM), which provides data on not only the topography, but also the spatial distribution of a wide range of physical properties (elasticity, conductivity, etc.), with ultramicrotomy, allowing 3D multiparametric examination of materials. The combination of SPM and ultramicrotomy (scanning probe nanotomography) is very appropriate for characterization of soft multicompound nanostructurized materials, such as polymer matrices and microstructures doped with different types of nanoparticles (magnetic nanoparticles, quantum dots, nanotubes, etc.), and biological materials. A serious problem of this technique is a lack of chemical and optical characterization tools, which may be solved by using optical microspectroscopy.Here, we report the development of an instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography in a single apparatus. This approach retains all the advantages of SPM and upright optical microspectroscopy and allows 3D multiparametric characterization using both techniques. As the first test of the system developed, we have performed correlative characterization of the morphology and the magnetic and fluorescent properties of fluorescent magnetic microspheres doped with a fluorescent dye and magnetic nanoparticles. The results of this study can be used to obtain 3D volume images of a specimen for most high-resolution near-field scanning probe microscopies: SNOM, TERS, AFM–IR, etc. This approach will result in development of unique techniques combining the advantages of SPM (nanoscale morphology and a wide range of physical parameters) and high-resolution optical microspectroscopy (nanoscale chemical mapping and optical properties) and allowing simultaneous 3D measurements.

Engineering of Optically Encoded Microbeads with FRET-Free Spatially Separated Quantum-Dot Layers for Multiplexed Assays.

Quantum dot (QD) encoded microbeads are emerging for multiplexed analysis of biological markers. The quantitative encoding of microbeads prepared with different concentrations of QDs of different colors suffers from resonance energy transfer from the QDs fluorescing at shorter wavelengths to the QDs fluorescing at longer wavelengths. Here, we used the layer-by-layer deposition technique to spatially separate QDs of different colors with several polymer layers so that the distance between them would be larger than the Förster energy transfer radius. We performed fluorescence lifetime measurements to investigate and determine the conditions excluding significant resonance energy transfer between QDs within QD-encoded microbeads. Additionally, the number of QDs adsorbed onto microbeads was systematically established and multilayer structures of the QD-encoded microbead shells were characterized by scanning probe nanotomography. Finally, we prepared eight populations of FRET-free microbeads encoded with QDs of three colors at two intensity levels and demonstrated that all the optical codes are excitable at a single wavelength and may be clearly identified in three channels of a flow cytometer. The developed approach for engineering QD-encoded microbeads that are free from optical artefacts related to inter-QD resonance energy transfer paves the way to quantitative QD-based multiplexed assays.

A novel design of a scanning probe microscope integrated with an ultramicrotome for serial block-face nanotomography.

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.

Cryo scanning probe nanotomography study of the structure of alginate microcarriers

Nanostructure of microparticles of decellularized rat liver ECM on spherical alginate hydrogel microcarriers is analyzed by cryo scanning probe nanotomography.

Инструментальное объединение конфокальной микроспектроскопии и 3D сканирующей зондовой нанотомографии

Инструментальное объединение конфокальной микроспектроскопии и 3D сканирующей зондовой нанотомографии

Investigating the micro- and nanostructure of microfibrous biocompatible polyurethane scaffold by scanning probe nanotomography

This paper presents a study of three-dimensional micro- and nanosctucture of polyurethane dual scale biocompatible scaffold made by three-dimensional printing and electrospinning. The three-dimensional structure of the scaffold was analyzed by scanning probe nanotomography with use of an experimental setup combining an ultramicrotome and a scanning probe microscope. We performed a quantitative analysis of microporosity, nanoroughness, and three-dimensional morphology parameters of the scaffold. The electrospun scaffold consists of a network of microfibers with diameter ranging from 1.7 to 6.0 μm. The measured mean microfiber diameter is 3.54 ± 1.23 μm. The volume porosity of the electrospun scaffold is 72.5%, while mean surface area to volume ration is 0.28 μm–1 and mean nanoroughness of microfiber surface is 22.1 ± 3.0 nm. The quantitative characteristics of the micro- and nanostructure of elecrospun polyurethane matrices secure the high efficacy of its usage for increasing the biocompatibility of dual-scale hybrid bioengineered scaffolds for regenerative medicine tasks. The use of scanning probe nanotomography for analyzing threedimensional morphology characteristics and the topology of electrospun microfiber systems enables us to improve the efficiency of development of new bioengineered products.

Scanning Probe Nanotomograph: Features of Engineering Solutions for Low-Temperature Analysis of Biomedical Materials

The design of a nanotechnological complex for scanning probe nanotomography based on a combination of cryoultramicrotomy and scanning probe microscopy (SPM) is presented. Features of the structural and technical solutions of the SPM module that are required for effective functioning in the cryochamber at low temperatures and coordinated operation with the ultramicrotome for three-dimensional tomographic reconstructions of the samples are reviewed. The complex reveals unique information about the native micro- and nanostructure of the objects. The article discusses opportunities and use of the complex for biomedical research.

COMPARATIVE ANALYSIS OF THREE-DIMENSIONAL NANOSTRUCTURE OF POROUS BIOCOMPATIBLE SCAFFOLDS MADE OF RECOMBINANT SPIDROIN AND SILK FIBROIN FOR REGENERATIVE MEDICINE

Aim.To perform a comparison of three-dimensional nanostructure of porous biocompatible scaffolds made of fibroinBombix moriand recombinant spidroin rS1/9.Materials and methods.Three-dimensional porous scaffolds were produced by salt leaching technique. The comparison of biological characteristics of the scaffolds shows that adhesion and proliferation of mouse fibroblastsin vitroon these two types of scaffolds do not differ significantly. Comparative experimentsin vivoshow that regeneration of bone tissue of rats is faster with implantation of recombinant spidroin scaffolds. Three-dimensional nanostructure of scaffolds and interconnectivity of nanopores were studied with scanning probe nanotomography (SPNT) to explain higher regenerative activity of spidroin-based scaffolds.Results.Significant differences were detected in the integral density and volume of pores: the integral density of nanopores detected on 2D AFM images is 46 μm–2 and calculated volume porosity is 24% in rS1/9-based scaffolds; in fibroin-based three-dimensional structures density of nanopores and calculated volume porosity were 2.4 μm–2 and 0.5%, respectively. Three-dimensional reconstruction system of nanopores and clusters of interconnected nanopores in rS1/9-based scaffolds showed that volume fraction of pores interconnected in percolation clusters is 35.3% of the total pore volume or 8.4% of the total scaffold volume.Conclusion.Scanning probe nanotomography method allows obtaining unique information about topology of micro – and nanopore systems of artificial biostructures. High regenerative activity of rS1/9-based scaffolds can be explained by higher nanoporosity of the scaffolds.

Three-dimensional Analysis of Nanomaterials by Scanning Probe Nanotomography

Micro and nanostructure of scaffolds made from fibroin of Bombyx mori silkworm by salt leaching technique was studied by scanning probe nanotomography. Nanopores with dimensions in range from 30 to 180nm are observed in the scaffold volume. Three – dimensional analysis of obtained data shows that degree of scaffold nanoporosity is 0.5% and nanopores are not interconnected with each other. Usage of scanning probe nanotomography technique enables to obtain unique nanoscale information of 3D structure of biopolymer nanomaterials.

3D nanostructural analysis of silk fibroin and recombinant spidroin 1 scaffolds by scanning probe nanotomography

We use serial section scanning probe nanotomography to study the 3D structure, nanoporosity and pore interconnectivity of biocompatible scaffolds made of fibroin and recombinant spidroin 1 (rS1/9). Significant nanoporosity (24%) and pore percolation is detected for rS1/9 scaffolds. It is assumed to contribute to higher in vivo tissue regeneration efficiency of rS1/9 scaffolds.

Investigation of micro- and nanostructure of biocompatible scaffolds from regenerated fibroin of Bombix mori by scanning probe nanotomography

This paper presents a study of three-dimensional micro- and nanosctuctures of porous biocompatible matrices from regenerated fibroin and a quantitative analysis of their microporosity parameters. An analysis of the three-dimensional structure of matrices has been carried out by scanning probe nanotomography with the use of an experimental setup combining an ultramicrotome and scanning probe microscope. The formation of a three-dimensional network of interconnected pores with characteristic dimensions ranging from 1.7 to 6.0 μm is observed in the bulk volume of studied matrices. The measured mean pore diameter is 3.54 ± 1.23 μm; the mean pore wall thickness is 672 ± 282 nm. The volume porosity of macropore walls is 65.7%, while the volume fraction of pores interconnected in large pore clusters is more than 80% of the whole pore volume. Quantitative characteristics of porous micro- and nanostructures of matrices obtained as a result of the study show a significant degree of porosity and percolation of micropores, which correlates with the reported high efficiency of tissue regeneration on such matrices. The use of scanning probe nanotomography to analyze three-dimensional morphology characteristics and the topology of micro- and nanopore systems enables us to improve the efficiency of developing new biomaterials.