rus
DOI: https://doi.org/10.22184/1993-8578.2026.19.1.46.54
The paper examines the capabilities of modern ultramicroscopy – an optical method based on visualizing light scattering from individual nanoobjects. The principles of the method and its differences from classical microscopic and light-scattering techniques are discussed. Specific practical examples demonstrate the wide range of applied problems solved using ultramicroscopy: determining the size and numerical concentration of nanoparticles (including metallic, oxide, and organic ones); estimating density and porosity of nanoparticles; studying aggregation processes and determining stability threshold of colloidal systems (using asphaltenes as an example); detecting nanobubbles; and monitoring purity of liquids. It is demonstrated that this method, combining high sensitivity, the ability to work with native samples, and rapid measurement, is a powerful tool for nanoindustry, materials science, oil and gas industry, and analytical control.
The paper examines the capabilities of modern ultramicroscopy – an optical method based on visualizing light scattering from individual nanoobjects. The principles of the method and its differences from classical microscopic and light-scattering techniques are discussed. Specific practical examples demonstrate the wide range of applied problems solved using ultramicroscopy: determining the size and numerical concentration of nanoparticles (including metallic, oxide, and organic ones); estimating density and porosity of nanoparticles; studying aggregation processes and determining stability threshold of colloidal systems (using asphaltenes as an example); detecting nanobubbles; and monitoring purity of liquids. It is demonstrated that this method, combining high sensitivity, the ability to work with native samples, and rapid measurement, is a powerful tool for nanoindustry, materials science, oil and gas industry, and analytical control.
Received: 19.01.2026 | Accepted: 30.01.2026 | DOI: https://doi.org/10.22184/1993-8578.2026.19.1.46.54
Original paper
Ultramicroscopy: Potential and Applications in Science and Technology
V.N.Kuryakov1, Cand. of Sci. (Physics and Mathematics), Leading Researcher, ORCID: 0000-0002-1271-8082 / vladimir.kuryakov@ipng.ru
Abstract. The paper examines the capabilities of modern ultramicroscopy – an optical method based on visualizing light scattering from individual nanoobjects. The principles of the method and its differences from classical microscopic and light-scattering techniques are discussed. Specific practical examples demonstrate the wide range of applied problems solved using ultramicroscopy: determining the size and numerical concentration of nanoparticles (including metallic, oxide, and organic ones); estimating density and porosity of nanoparticles; studying aggregation processes and determining stability threshold of colloidal systems (using asphaltenes as an example); detecting nanobubbles; and monitoring purity of liquids. It is demonstrated that this method, combining high sensitivity, the ability to work with native samples, and rapid measurement, is a powerful tool for nanoindustry, materials science, oil and gas industry, and analytical control.
Keywords: ultramicroscopy, nanoparticles, numerical concentration, aggregation, nanobubbles, purity control, applied problems
For citation: V.N. Kuryakov. Ultramicroscopy: Potential and Applications in Science and Technology. NANOINDUSTRY. 2026. Vol. 19. No. 1. PP. 46–54. https://doi.org/10.22184/1993-8578.2026.19.1.46.54.
INTRODUCTION
The rapid development of nanotechnology imposes high demands on the methods for characterizing nano-objects. Precise knowledge of the size, concentration, and other parameters of nanoparticles (NPs) is critically important for both fundamental research and applied tasks in medicine, catalysis, electronics, the oil and gas industry, and environmental science [1, 2]. Existing experimental methods for studying nanoparticles (electron microscopy, atomic force microscopy, dynamic light scattering (DLS)) have their limitations: the need for vacuuming the sample for analysis, complexity in sample preparation, or averaging results over a particle ensemble.
In this context, interest is being revived in the method of ultramicroscopy, for development of which Richard Zsigmondy was awarded the Nobel Prize in Chemistry in 1925. Modern ultramicroscopy, which combines the classical principle of dark-field observation with laser illumination, high-sensitivity digital cameras, and powerful computational algorithms, has become a highly effective analytical tool for working with nano-objects in liquids [3]. In the market for analytical equipment, there are several devices operating on the principle of ultramicroscopy, such as the NanoSight NS300 from Malvern (UK) or the ZetaView from Particle Metrix (Germany). Since 2020, a Russian device, the NP Counter (LLC ‘NP VIZHN’), has been produced. In the mid-20th century, the USSR developed and widely used the Deryagin-Vlasenko particle counter, which operated on a similar principle, but no information could be found in scientific articles over the past 20 years regarding the use of this device in research.
This paper summarizes the published information on the use of this method for solving various practical problems.
RESEARCH METHODS
The principle of the ultramicroscopy method is based on special lateral illumination of a small sample volume (usually 0.4–1 µL) with a powerful focused laser beam and observing the light scattered from individual nano-objects at a 90° angle using an optical microscope with 10–20x magnification. In the field of view, individual glowing points can be seen on a dark background (Fig.1), each corresponding to scattering from a single nanoparticle. The particles themselves are not visible; what is observed is the light scattered by the particles, but their presence and movement are clearly detected. With this approach, observation is not limited by visualization constraints of nanoparticles associated with the diffraction limit.
Thus, the ultramicroscopy method allows measuring numerical concentration of nanoparticles from 105 particles/mL for the standard version of the device and down to lower concentrations when using a flow cuvette. At higher nanoparticle concentrations in the tested sample (more than 108 particles/mL), preliminary controlled dilution of the sample with a clean dispersing medium is required, followed by measuring concentration in the diluted sample and calculation of concentration in the original sample taking the dilution factor into account. The minimum particle size detectable by the ultramicroscopy method is a few nanometers and depends on sensitivity of the digital camera used in the device, the power of the laser, and scattering ability of the particles being studied. No more than 0.5 ml of sample is required for the studies. The observation volume is about 1.2 · 10⁻⁷ mL, which allows for statistically accurate determination of concentration by counting particles in a series of video frames. Each frame of the recorded video showing scattering from the studied sample is a separate measurement; averaging over multiple frames is performed to determine the average particle concentration in a unit volume of liquid.
Observing Brownian motion and analyzing it allows one to determine the mean square displacement of each individual particle, calculate the diffusion coefficient, and, using the Stokes-Einstein relation, estimate the hydrodynamic radius of nanoparticles. This provides information on particles size distribution in the sample. It is important to note the advantages of this method: ability to work with native samples in a liquid medium without the need for drying; direct measurement of numerical concentration; high sensitivity to appearance of initial aggregates; ability to analyze polydisperse systems; and quick measurement times.
RESULTS AND DISCUSSION
Measurement of the numerical concentration of nano- and submicron objects in liquid media
Direct observation of nanoparticles in a liquid using the ultramicroscopy method allows for measurement of their numerical concentration. Most of the currently used methods for measuring concentration of nanoparticles in a liquid are indirect, i.e., they measure a certain characteristic of the sample that is related to nanoparticles concentration in it. With this approach, either a pre-established calibration curve of such a parameter versus NP concentration is required, or information about optical properties of the NPs, which makes such indirect methods non-universal. Measuring numerical concentration of NPs using ultramicroscopy does not require prior calibration curves or information about the optical properties of the NPs. It is necessary for scattering intensities from individual nanoparticles to be sufficient for detection by the digital camera used in the device. The shape of the nanoparticles does not significantly affect possibility of measuring their concentration. In the optical scheme of ultramicroscopy, particles of different shapes (spheres, rods, stars, etc.) appear in the field of view almost the same, as bright glowing dots.
Measurement of Nanoparticle Sizes through Mass and Number Concentration
In [5], an original approach is proposed for estimating the average radius of spherical nanoparticles using the ultramicroscopy method. If the mass concentration of nanoparticles in the studied sample is known, or can be determined by the dry residue method (or is known from the sample preparation information), and the numerical concentration of particles in the sample is measured (using ultramicroscopy), and density of the particle material is known (tabulated data for the specific material), then the average nanoparticle size can be determined, assuming a spherical particle shape:
,
where M1 is the average particle mass; M is the mass of a sphere with radius R and density ρ; Cm and CN are the mass and numerical concentrations of particles in the sample, respectively; ρнч is density of the nanoparticle material, and R1 is the average particle radius. This approach allows determining the mass-averaged size of the nanoparticles. The method was successfully tested on monodisperse gold nanoparticles, where the results (colloidal gold nanoparticle radius 17±2 nm) were in good agreement with TEM and DLS data.
With this approach, it is important to note that if the size of nanoparticles being studied can be measured by another method, then in the formulas given above, the desired parameter can be density of the nanoparticle material. This allows for estimation of porosity of the nanoparticles themselves.
Study of aggregation and determination of threshold stability
In the oil and gas industry, it is critically important to determine the onset point of asphaltene aggregation [6]. In work [7], the ultramicroscopy method was applied for the first time for this purpose in a model system of toluene-asphaltenes-heptane. The method showed higher sensitivity compared to DRS, detecting appearance of the first asphaltene aggregates at earlier stages of solution instability (at 60 vol.% heptane), while DRS registered the appearance of aggregates only at 76 vol.% heptane. This allows the method to be used for express monitoring of oil systems stability at the earliest stages. Modifying the ultramicroscope with an infrared laser will make it possible to apply this approach to the study of less transparent, more concentrated oil systems.
Detection of Nanobubbles and Control of Liquid Purity
Nanobubbles (NBs) are gas-filled cavities with a diameter typically less than 500 nm, possessing unique physicochemical properties due to their size. Unlike ordinary microbubbles, NBs exhibit exceptional stability in liquid media, remaining in liquid volume for weeks or even months. According to the Epstein – Plesset theory [8], a bubble with the radius of 100 nm would have an internal pressure approximately 14.4 times higher than atmospheric, and its lifetime should not exceed 1 millisecond, which contradicts experiments where the lifetime of NBs is significantly longer, up to several days. The paradox between the short lifetime predicted by the Epstein–Plesset theory and the experimentally observed long lifetime of NPs in water has not yet been resolved. There are several theories explaining possibility of the existence of long-lived NPs.
Nanobubbles can be produced by various methods, including ultrasonic cavitation, gas diffusion through a membrane into a liquid, and electrochemical processes [9, 10]. The concentrations of nanobubbles are usually low, rarely exceeding 108 per milliliter, which makes their detection by commonly used experimental methods (DLS, spectroscopy, etc.) difficult. The applications of nanoparticles cover a wide range of areas: biomedicine (contrast enhancement for imaging, drug delivery, tissue oxygenation); water treatment (pollutant degradation, disinfection); agriculture (improving plant growth, precision delivery of agrochemicals); mining and processing industries (enhancing froth flotation, surface cleaning, water treatment, and others). The field of nanobubble research is relatively young. Due to difficulties of producing and detecting nanobubbles, there are few papers on this topic, but their number is growing every year. In the ScienceDirect database, a search for "Nanobubbles" yielded only 4 articles in 2002, and by 2024 there were already 829.
Ultramicroscopy is one of the few methods that allows direct visualization of nanobubbles (NB) in a liquid. In [11], a new method for generating air NBs by filtering water through a dry membrane was presented, and their formation and changes in concentration over time were successfully tracked using a combination of DLS and ultramicroscopy.
Monitoring of liquids for the presence of nanoscale mechanical impurities
An important practical task is monitoring purity of ultra-pure liquids (water, solvents). Traditional conductometric methods are not sensitive to nanoscale mechanical impurities. Widely used laboratory water purification systems, such as Milli-Q from Merck Millipore and others, allow obtaining Type I ultra-pure water using the reverse osmosis method. The only parameter characterizing water purity in such purification systems is its specific conductivity (or specific resistance). There is usually no control over the content of nanoscale impurities in these systems. An analysis of a series of water samples with a resistance of 18.2 MΩ·cm, obtained from several commercial laboratory reverse osmosis systems, revealed presence of nanoparticles in such water at concentrations of up to 10⁷ particles/ml, which can be critical for microelectronics and pharmaceuticals.
In [12], the ultramicroscopy method was used for quantitative assessment of solid nanoscale impurities in reaction-pure, specially prepared solvents: deionized water and isopropanol. It was shown that even in such highly purified media there is a significant number of nanoparticles larger than 20 nm: about 106 particles/ml in water and 105 particles/ml in isopropanol. Extrapolation of these data to particles larger than 1 nm allowed estimating the total concentration of nanoscale impurities at the level of 1011 particles/ml for water and 108 particles/ml for isopropanol.
Measurement of the concentration of biological submicron objects
For biological objects at the submicron scale, the issue of measuring their concentration and size is also relevant. The method of ultramicroscopy with nanoparticle tracking analysis (NTA) is in demand for studying the extracellular vesicles properties [13, 14]. Extracellular vesicles are lipid spherical structures ranging in size from tens of nanometers to several micrometers, which are released by cells from various tissues or organs into their surrounding environment. They have been found in various body fluids, including blood plasma, urine, saliva, breast milk, and others.
The NTA method is considered optimal for studying extracellular vesicles, as it allows for the simultaneous determination of two key parameters: concentration and particle size distribution in their native state. Unlike averaging methods such as dynamic light scattering (DLS), NTA tracks the Brownian motion of each individual particle in real time, building a profile of a heterogeneous population in the range of 10–1000 nm. This is critically important because vesicles are heterogeneous in size. The analysis is conducted in the original medium, which preserves the natural biological properties of the sample. An additional advantage is possibility of fluorescent detection (NTA-Fluorescence), which allows for identification and characterization of specific vesicle subpopulations.
The main limitations of the method are necessity for thorough sample preparation to eliminate impurities (protein aggregates, lipoproteins), manual parameter adjustment affecting reproducibility, and relatively low throughput. Nevertheless, NTA serves as the "gold standard" for the physical characterization of vesicles, providing a unique set of data (size, concentration, phenotype) that complements, but does not replace, molecular and morphological analysis by other methods.
CONCLUSIONS
Modern ultramicroscopy, once recognized with a Nobel Prize, has become a unique and powerful analytical tool for the nanotechnology industry. Its key advantages – ability to directly measure numerical concentration of nano-objects in their natural environment, high sensitivity to the initial stages of aggregation, and capability to work with a wide range of materials (from metals and oxides to organic compounds and gas bubbles) – open up broad prospects for application. The review of published data showed that the method is effective for solving a wide range of tasks, such as: controlling the size and concentration of synthesized nanoparticles; studying porosity and internal structure of nanoparticles; monitoring stability and investigating aggregation kinetics in colloidal systems (oil systems, biopharmaceuticals, etc.); detecting nanobubbles and studying their properties; controlling purity of process liquids and solvents in microelectronics, pharmaceuticals, and other high-tech industries. The conducted review of the use of ultramicroscopy in scientific and applied tasks highlights relevance and promise of further developing instrumentation and methodological support of the ultramicroscopy method for the needs of Russian science and industry.
ACKNOWLEDGMENTS
The research was carried out as part of the state assignment of Institute of Oil and Gas Problems of the RAS No. 125020501404-4.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Original paper
Ultramicroscopy: Potential and Applications in Science and Technology
V.N.Kuryakov1, Cand. of Sci. (Physics and Mathematics), Leading Researcher, ORCID: 0000-0002-1271-8082 / vladimir.kuryakov@ipng.ru
Abstract. The paper examines the capabilities of modern ultramicroscopy – an optical method based on visualizing light scattering from individual nanoobjects. The principles of the method and its differences from classical microscopic and light-scattering techniques are discussed. Specific practical examples demonstrate the wide range of applied problems solved using ultramicroscopy: determining the size and numerical concentration of nanoparticles (including metallic, oxide, and organic ones); estimating density and porosity of nanoparticles; studying aggregation processes and determining stability threshold of colloidal systems (using asphaltenes as an example); detecting nanobubbles; and monitoring purity of liquids. It is demonstrated that this method, combining high sensitivity, the ability to work with native samples, and rapid measurement, is a powerful tool for nanoindustry, materials science, oil and gas industry, and analytical control.
Keywords: ultramicroscopy, nanoparticles, numerical concentration, aggregation, nanobubbles, purity control, applied problems
For citation: V.N. Kuryakov. Ultramicroscopy: Potential and Applications in Science and Technology. NANOINDUSTRY. 2026. Vol. 19. No. 1. PP. 46–54. https://doi.org/10.22184/1993-8578.2026.19.1.46.54.
INTRODUCTION
The rapid development of nanotechnology imposes high demands on the methods for characterizing nano-objects. Precise knowledge of the size, concentration, and other parameters of nanoparticles (NPs) is critically important for both fundamental research and applied tasks in medicine, catalysis, electronics, the oil and gas industry, and environmental science [1, 2]. Existing experimental methods for studying nanoparticles (electron microscopy, atomic force microscopy, dynamic light scattering (DLS)) have their limitations: the need for vacuuming the sample for analysis, complexity in sample preparation, or averaging results over a particle ensemble.
In this context, interest is being revived in the method of ultramicroscopy, for development of which Richard Zsigmondy was awarded the Nobel Prize in Chemistry in 1925. Modern ultramicroscopy, which combines the classical principle of dark-field observation with laser illumination, high-sensitivity digital cameras, and powerful computational algorithms, has become a highly effective analytical tool for working with nano-objects in liquids [3]. In the market for analytical equipment, there are several devices operating on the principle of ultramicroscopy, such as the NanoSight NS300 from Malvern (UK) or the ZetaView from Particle Metrix (Germany). Since 2020, a Russian device, the NP Counter (LLC ‘NP VIZHN’), has been produced. In the mid-20th century, the USSR developed and widely used the Deryagin-Vlasenko particle counter, which operated on a similar principle, but no information could be found in scientific articles over the past 20 years regarding the use of this device in research.
This paper summarizes the published information on the use of this method for solving various practical problems.
RESEARCH METHODS
The principle of the ultramicroscopy method is based on special lateral illumination of a small sample volume (usually 0.4–1 µL) with a powerful focused laser beam and observing the light scattered from individual nano-objects at a 90° angle using an optical microscope with 10–20x magnification. In the field of view, individual glowing points can be seen on a dark background (Fig.1), each corresponding to scattering from a single nanoparticle. The particles themselves are not visible; what is observed is the light scattered by the particles, but their presence and movement are clearly detected. With this approach, observation is not limited by visualization constraints of nanoparticles associated with the diffraction limit.
Thus, the ultramicroscopy method allows measuring numerical concentration of nanoparticles from 105 particles/mL for the standard version of the device and down to lower concentrations when using a flow cuvette. At higher nanoparticle concentrations in the tested sample (more than 108 particles/mL), preliminary controlled dilution of the sample with a clean dispersing medium is required, followed by measuring concentration in the diluted sample and calculation of concentration in the original sample taking the dilution factor into account. The minimum particle size detectable by the ultramicroscopy method is a few nanometers and depends on sensitivity of the digital camera used in the device, the power of the laser, and scattering ability of the particles being studied. No more than 0.5 ml of sample is required for the studies. The observation volume is about 1.2 · 10⁻⁷ mL, which allows for statistically accurate determination of concentration by counting particles in a series of video frames. Each frame of the recorded video showing scattering from the studied sample is a separate measurement; averaging over multiple frames is performed to determine the average particle concentration in a unit volume of liquid.
Observing Brownian motion and analyzing it allows one to determine the mean square displacement of each individual particle, calculate the diffusion coefficient, and, using the Stokes-Einstein relation, estimate the hydrodynamic radius of nanoparticles. This provides information on particles size distribution in the sample. It is important to note the advantages of this method: ability to work with native samples in a liquid medium without the need for drying; direct measurement of numerical concentration; high sensitivity to appearance of initial aggregates; ability to analyze polydisperse systems; and quick measurement times.
RESULTS AND DISCUSSION
Measurement of the numerical concentration of nano- and submicron objects in liquid media
Direct observation of nanoparticles in a liquid using the ultramicroscopy method allows for measurement of their numerical concentration. Most of the currently used methods for measuring concentration of nanoparticles in a liquid are indirect, i.e., they measure a certain characteristic of the sample that is related to nanoparticles concentration in it. With this approach, either a pre-established calibration curve of such a parameter versus NP concentration is required, or information about optical properties of the NPs, which makes such indirect methods non-universal. Measuring numerical concentration of NPs using ultramicroscopy does not require prior calibration curves or information about the optical properties of the NPs. It is necessary for scattering intensities from individual nanoparticles to be sufficient for detection by the digital camera used in the device. The shape of the nanoparticles does not significantly affect possibility of measuring their concentration. In the optical scheme of ultramicroscopy, particles of different shapes (spheres, rods, stars, etc.) appear in the field of view almost the same, as bright glowing dots.
Measurement of Nanoparticle Sizes through Mass and Number Concentration
In [5], an original approach is proposed for estimating the average radius of spherical nanoparticles using the ultramicroscopy method. If the mass concentration of nanoparticles in the studied sample is known, or can be determined by the dry residue method (or is known from the sample preparation information), and the numerical concentration of particles in the sample is measured (using ultramicroscopy), and density of the particle material is known (tabulated data for the specific material), then the average nanoparticle size can be determined, assuming a spherical particle shape:
,
where M1 is the average particle mass; M is the mass of a sphere with radius R and density ρ; Cm and CN are the mass and numerical concentrations of particles in the sample, respectively; ρнч is density of the nanoparticle material, and R1 is the average particle radius. This approach allows determining the mass-averaged size of the nanoparticles. The method was successfully tested on monodisperse gold nanoparticles, where the results (colloidal gold nanoparticle radius 17±2 nm) were in good agreement with TEM and DLS data.
With this approach, it is important to note that if the size of nanoparticles being studied can be measured by another method, then in the formulas given above, the desired parameter can be density of the nanoparticle material. This allows for estimation of porosity of the nanoparticles themselves.
Study of aggregation and determination of threshold stability
In the oil and gas industry, it is critically important to determine the onset point of asphaltene aggregation [6]. In work [7], the ultramicroscopy method was applied for the first time for this purpose in a model system of toluene-asphaltenes-heptane. The method showed higher sensitivity compared to DRS, detecting appearance of the first asphaltene aggregates at earlier stages of solution instability (at 60 vol.% heptane), while DRS registered the appearance of aggregates only at 76 vol.% heptane. This allows the method to be used for express monitoring of oil systems stability at the earliest stages. Modifying the ultramicroscope with an infrared laser will make it possible to apply this approach to the study of less transparent, more concentrated oil systems.
Detection of Nanobubbles and Control of Liquid Purity
Nanobubbles (NBs) are gas-filled cavities with a diameter typically less than 500 nm, possessing unique physicochemical properties due to their size. Unlike ordinary microbubbles, NBs exhibit exceptional stability in liquid media, remaining in liquid volume for weeks or even months. According to the Epstein – Plesset theory [8], a bubble with the radius of 100 nm would have an internal pressure approximately 14.4 times higher than atmospheric, and its lifetime should not exceed 1 millisecond, which contradicts experiments where the lifetime of NBs is significantly longer, up to several days. The paradox between the short lifetime predicted by the Epstein–Plesset theory and the experimentally observed long lifetime of NPs in water has not yet been resolved. There are several theories explaining possibility of the existence of long-lived NPs.
Nanobubbles can be produced by various methods, including ultrasonic cavitation, gas diffusion through a membrane into a liquid, and electrochemical processes [9, 10]. The concentrations of nanobubbles are usually low, rarely exceeding 108 per milliliter, which makes their detection by commonly used experimental methods (DLS, spectroscopy, etc.) difficult. The applications of nanoparticles cover a wide range of areas: biomedicine (contrast enhancement for imaging, drug delivery, tissue oxygenation); water treatment (pollutant degradation, disinfection); agriculture (improving plant growth, precision delivery of agrochemicals); mining and processing industries (enhancing froth flotation, surface cleaning, water treatment, and others). The field of nanobubble research is relatively young. Due to difficulties of producing and detecting nanobubbles, there are few papers on this topic, but their number is growing every year. In the ScienceDirect database, a search for "Nanobubbles" yielded only 4 articles in 2002, and by 2024 there were already 829.
Ultramicroscopy is one of the few methods that allows direct visualization of nanobubbles (NB) in a liquid. In [11], a new method for generating air NBs by filtering water through a dry membrane was presented, and their formation and changes in concentration over time were successfully tracked using a combination of DLS and ultramicroscopy.
Monitoring of liquids for the presence of nanoscale mechanical impurities
An important practical task is monitoring purity of ultra-pure liquids (water, solvents). Traditional conductometric methods are not sensitive to nanoscale mechanical impurities. Widely used laboratory water purification systems, such as Milli-Q from Merck Millipore and others, allow obtaining Type I ultra-pure water using the reverse osmosis method. The only parameter characterizing water purity in such purification systems is its specific conductivity (or specific resistance). There is usually no control over the content of nanoscale impurities in these systems. An analysis of a series of water samples with a resistance of 18.2 MΩ·cm, obtained from several commercial laboratory reverse osmosis systems, revealed presence of nanoparticles in such water at concentrations of up to 10⁷ particles/ml, which can be critical for microelectronics and pharmaceuticals.
In [12], the ultramicroscopy method was used for quantitative assessment of solid nanoscale impurities in reaction-pure, specially prepared solvents: deionized water and isopropanol. It was shown that even in such highly purified media there is a significant number of nanoparticles larger than 20 nm: about 106 particles/ml in water and 105 particles/ml in isopropanol. Extrapolation of these data to particles larger than 1 nm allowed estimating the total concentration of nanoscale impurities at the level of 1011 particles/ml for water and 108 particles/ml for isopropanol.
Measurement of the concentration of biological submicron objects
For biological objects at the submicron scale, the issue of measuring their concentration and size is also relevant. The method of ultramicroscopy with nanoparticle tracking analysis (NTA) is in demand for studying the extracellular vesicles properties [13, 14]. Extracellular vesicles are lipid spherical structures ranging in size from tens of nanometers to several micrometers, which are released by cells from various tissues or organs into their surrounding environment. They have been found in various body fluids, including blood plasma, urine, saliva, breast milk, and others.
The NTA method is considered optimal for studying extracellular vesicles, as it allows for the simultaneous determination of two key parameters: concentration and particle size distribution in their native state. Unlike averaging methods such as dynamic light scattering (DLS), NTA tracks the Brownian motion of each individual particle in real time, building a profile of a heterogeneous population in the range of 10–1000 nm. This is critically important because vesicles are heterogeneous in size. The analysis is conducted in the original medium, which preserves the natural biological properties of the sample. An additional advantage is possibility of fluorescent detection (NTA-Fluorescence), which allows for identification and characterization of specific vesicle subpopulations.
The main limitations of the method are necessity for thorough sample preparation to eliminate impurities (protein aggregates, lipoproteins), manual parameter adjustment affecting reproducibility, and relatively low throughput. Nevertheless, NTA serves as the "gold standard" for the physical characterization of vesicles, providing a unique set of data (size, concentration, phenotype) that complements, but does not replace, molecular and morphological analysis by other methods.
CONCLUSIONS
Modern ultramicroscopy, once recognized with a Nobel Prize, has become a unique and powerful analytical tool for the nanotechnology industry. Its key advantages – ability to directly measure numerical concentration of nano-objects in their natural environment, high sensitivity to the initial stages of aggregation, and capability to work with a wide range of materials (from metals and oxides to organic compounds and gas bubbles) – open up broad prospects for application. The review of published data showed that the method is effective for solving a wide range of tasks, such as: controlling the size and concentration of synthesized nanoparticles; studying porosity and internal structure of nanoparticles; monitoring stability and investigating aggregation kinetics in colloidal systems (oil systems, biopharmaceuticals, etc.); detecting nanobubbles and studying their properties; controlling purity of process liquids and solvents in microelectronics, pharmaceuticals, and other high-tech industries. The conducted review of the use of ultramicroscopy in scientific and applied tasks highlights relevance and promise of further developing instrumentation and methodological support of the ultramicroscopy method for the needs of Russian science and industry.
ACKNOWLEDGMENTS
The research was carried out as part of the state assignment of Institute of Oil and Gas Problems of the RAS No. 125020501404-4.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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