For this, an aqueous colloidal solution of NPs was precipitated by centrifugation and dissolved in the same volume of DMSO. The kinetics of static fluorescence quenching of Nd3+ donors of doped NPs dispersed in two solvents was analyzed to determine and to compare the concentrations of OH- quenching acceptors uniformly distributed throughout the volume of the NPs. The dependences of the relative fluorescence quantum yield φ of colloidal solutions on the concentration of OH- groups in the NPs were calculated and were also used to determine concentration of acceptors in the volume of NPs in different solvents. It was found that the concentration of OH- groups in NPs dispersed in DMSO is almost two times lower than in NPs dispersed in water. This gives an almost two-fold increase in the relative fluorescence quantum yield φ for the former. The sizes of synthesized NPs were monitored by common TEM and by applying a rapid procedure based on optical visualization of the trajectories of the Brownian motion of NPs in solution using a laser ultramicroscope. The use of two different methods made it possible to obtain more detailed information about the studied NPs.An original method was proposed to reduce the quenching of the NIR fluorescence of colloidal solutions of 0.1 at. % Nd These ions have absorption and luminescence spectral lines in the first biological window in the spectral range of 750–950 nm, i.e., in the near infrared (NIR). At present, an urgent problem is the creation of efficient nontoxic colloidal solutions of nanosized phosphors based on dielectric crystals doped with REIs. Such luminescent NPs have a number of unique properties, such as narrow absorption and luminescence spectral lines, long lifetimes of excited states—which allow time detuning from biotissue autofluorescence, and high photo- and physico-chemical stability [Fundamental research on luminescence of colloidal nanoparticles (NPs) is important due to the prospects for their wide application in different fields of biological and medical applications, such as bioimaging, local controlled heating, local thermal destruction, and drug delivery. Doping the NPs with rare earth ions (REI), for example, with the Ndtability ,2,3,4,5.tability ,7, secontability  and thirtability ,13,14,153)2SO (DMSO) was chosen as a “drying agent”, a polar aprotic universal solvent for organic and inorganic compounds, resistant to oxidation and reduction processes. DMSO is widely used in chemistry as a universal aprotic solvent, and in medicine as an antiseptic and anti-inflammatory agent, and meets all of the listed mandatory criteria [One of the problems that arise when using nanoparticles synthesized by aqueous methods is the presence of structural OH- groups and water in mesopores, which leads to quenching of the luminescence of RE doping ions. The simplest way to remove water is to dry the NPs at an appropriate temperature; however, this method cannot be called universal and suitable for use for any purpose, since it is important for medical research that the NPs be in a stable nontoxic colloidal solution. In the case of aqueous colloidal solutions of the NPs, the maximum possible removal of water from NPs can be achieved using a hygroscopic liquid that will not affect the NPs in any way , will be miscible with water in any proportions, and will not be toxic. Based on the foregoing, dimethyl sulfoxide  without damaging them, increasing the transdermal transfer of substances dissolved in it, penetrating even through the endothelial coatings of the walls of blood vessels and the brain, i.e., they can even overcome the blood-brain  barrier , which is inaccessible to conventional drug therapy.DMSO exhibits transparency in the spectral range of 350–2200 nm (according to some sources from 260 nm).DMSO is a good blood and tissue preservation agent.Regarding the toxicity of DMSO, there are still conflicting opinions in the literature, but most of the claims made have no real evidence. For example, in 1965, studies of DMSO were banned due to the alleged appearance of changes in the lens of the eye of experimental animals, which was later refuted by preclinical and clinical studies in animals and humans ,18,19,203+: LaF3 NPs with the same NPs redispersed in DMSO.In this work, we study and compare the fluorescence quenching of aqueous colloidal solutions of NdAnother problem of using of NPs synthesized by colloidal synthesis in medical and other applications is their aggregation into clusters ,22,23. TThis paper reports the results of applying a simple and rapid control method based on the nanoparticle tracking analysis (NTA) of Brownian motion of single NPs in solutions. To visualize these trajectories, we have developed a highly sensitive laser ultramicroscope operating in the mode of recording scattering signals at an unshifted frequency. The essential advantage of this method is the visualization and analysis of individual trajectories of single particles that allows inherent problems associated with averaging over an ensemble of particles to be overcome. For example, such averaging is inherent for another method widely used for diagnostics of NPs in solutions: the dynamic light scattering (DLS). The latter is based on measurement of the total light scattering of ensemble NPs in solution and yields information averaged over an ensemble of particles, which is usually heavily weighted towards small values of large, usually contaminant, particles. The use of two different methods here made it possible to obtain more detailed information about the studied NPs.A convenient quantitative criterion for the degree of luminescence quenching is the relative luminescence quantum yield, which is determined from the measured luminescence kinetics of the excited electronic level as presented in .(1)φ=1τHere,  follows ,25.(2)NFor the dipole–dipole mechanism of donor–acceptor interaction,kinetics .(4)N0(tHere the macroparameter of static quenching is determined as followsSubstituting (3) into (1) and integrating, we obtain the concentration dependence of the relative luminescence quantum yield:Here function . As follWhen the argument  to zero . For smaNote that the asymptotic Formula (9) is obtained from (1) and (4) neglecting radiative decay against the background of rapidly decaying static kinetics . Its detailed characterization including X-ray phase analysis of the synthesized LaF3 NPs was carried out in Ref. [3+: LaF3 NPs (ACS), and one of 0.1 at. % Nd3+: LaF3 NPs in a DMSO dispersion medium (DMSO). The DMSO colloid was then centrifuged again and redispersed in water to obtain a reduced aqueous colloidal solution of LaF3 NPs (DMSO-ACS). Finally, the last sample was again subjected to the procedures described above and the nanoparticles were again transferred into a new DMSO solution, followed by another redispersion step in a fresh DMSO solution (we designate this sample as ACS-DMSOx2).A colloidal solution of 0.1 at. % Nd in Ref.  (see Sup3+: LaF3 NPs and their size distribution characterization were carried out at the Institute of Physics of the University of Tartu (Estonia) using transmission electron microscopy (TEM) analysis. The measurements were performed in the scanning mode (STEM) at 200 kV using a Cs-probe-corrected transmission electron microscope (FEI Titan Themis 200). Powders in solutions were diluted in ethanol and ultrasonicated. The colloid was placed on a TEM copper grid with carbon film and dried for several hours.Visualization of the morphology of synthesized 0.1 at. % Ndλ = 405 nm) was focused into the observed liquid volume in the form of a thin plate several microns thick (“light sheet”), located in the lateral direction relative to the microscopic objective. The solution of synthesized NPs in deionized water with NP concentration in the range of 10−6 ÷ 10−4 g/mL3 was placed in a transparent optical cuvette with transverse dimensions of 10 × 10 mm to a height of no more than 5–6 mm (to reduce the temperature induced convection of the liquid). Using a sensitive CCD-camera (PCO Sensicam em) operating in the continuous shooting mode, the microscopic images of single NPs and theirs aggregates entering the light sheet region due to random Brownian motion were continuously recorded as a sequence of video frames. The high sensitivity of the developed ultramicroscope made it possible to visualize single NPs as small as ~20 nm in size and to observe their individual trajectories of Brownian motion in aqueous solution. 3+: LaF3 NPs in an aqueous solution, which fall into the “light sheet” area.For rapid control of the size distributions of investigated NPs in aqueous solutions and their temporal stability, a dark-field laser ultramicroscope developed by us was used. Laser radiation . The formula is as follows (10)d¯23+: LaF3 NPs, the zeta potential was measured using a Zetasizer Nano ZS  at 25 °C based on laser Doppler velocimetry (λ = 633 nm). Prior to these measurements, a colloidal solution of investigated NPs with a concentration of ~20 mg/mL was preliminarily homogenized for 10 min using an ultrasonic bath.To determine the stability of the synthesized colloidal solutions of the 0.1 at. % Nd3+: LaF3 NPs in NIR spectral range at different concentrations of Nd3+ ions was carried out using a pulsed tunable Ti–Sapphire laser  for fluorescence excitation . An MDR-23 monochromator was used for the dispersion of fluorescence. To limit the incident laser radiation on the photodetector, an interference filter FEL0850  was placed in front of the entrance slit of the monochromator. A Hamamatsu R13456P photomultiplier tube (PMT) operating in the single-photon counting mode was used as a radiation detector. The luminescence kinetics was recorded by the time-gated method of single photon counting using a TimeHarp 260 Nano multichannel scaler  with a specified nanosecond time gate duration. For precise synchronization of the detection system with the laser pulse, an avalanche pin photodiode was used. The electronic modules of the NIM standard were used as constant-fraction discriminators for start and stop pulses.The study of the fluorescence kinetics of colloidal solutions of 0.1 at. % Nd3+: LaF3 NPs, deposited from an aqueous solution onto a TEM copper grid and dried for several hours, have shown that most single nanocrystals, which we will call primary NPs, are combined into clusters that can reach over 100 nanometers in size, having a complex fractal-like structure. TEM studies of synthesized 0.1 at. % NdDf is widely accepted, which quantifies how densely packed their constituent particles are. The number of primary particles N in a cluster (or the mass m of a cluster) increases with its effective size R (gyration radius gR) according to the non-integer power law [fk is the scaling prefactor, having a value between 1 and 1.2, and r—the radius of primary particle.The effect of aggregation of NPs in solutions into fractal-like structures, particularly the aggregation of colloidal NPs, has been observed and studied in a number of works NfD, which can range between 1 and 3. When fD is close to 1, clusters look like filamentous structures. If, on the other hand, fD is close to 3, tightly packed compact clusters are formed [fD in the range from 1.75 to 1.85 [fD values between 2.0 and 2.1 are formed [In general, particles can aggregate into porous clusters with different e formed . The par to 1.85 . If, on e formed .gR/hR is often used [To describe the structure and packing density of primary particles in clusters, the ratio of gyration radius to hydrodynamic radius ten used . For cluten used . In the ten used ).N—number of primary NPs in cluster, and To characterize the sizes of the observed primary NPs and structure of clusters based on them, we calculated two distributions: the distribution of the effective radii for each primary NP, regardless of whether it is included in the cluster or not, and the distribution of gyration radii for each cluster. To determine the value of effective radius of a primary NP, the image of each NP was replaced by a disk of equal area, the radius of which was used as effective radius. To calculate radii of gyration, we used the standard formula of 1.24 .The resulting distributions are shown in Let us analyze the obtained distributions of the sizes of primary NPs and the clusters formed by them. As we have already pointed out, both parameters under consideration, gyration and hydrodynamic radius, can be considered as parameters characterizing the effective sizes of clusters, although they are based on their different properties. The gyration radius reflects the spatial distribution of masses of primary particles in the cluster, while the latter—their joint hydrodynamic resistance. It should also be taken into account that in the case of NTA studies, we observe particles in aquatic environment; whereas in the case of TEM images, we detect clusters smeared after drying on a flat surface, the geometric parameters of which could change during drying. Therefore, the distributions under discussion should not completely coincide. Indeed, the distributions of the hydrodynamic and gyration radii shown in The reason that the values of the hydrodynamic radii of the studied clusters exceed the values of the gyration radii requires an explanation. The ratio between the hydrodynamic and gyration radii of a cluster strongly depends on its structure. In the case of clusters consisting of approximately identical primary particles with a pronounced fractal structure, the values of the hydrodynamic radius, according to a number of theoretical studies , shouldLarge values of hydrodynamic radii compared to the values of their gyration radii, as determined in our case, were observed for clusters with a structure other than fractal . The maA more detailed understanding of this complex phenomenon is important for a more correct comparison of the results obtained in studies of colloidal NPs by electron microscopy and NTA. This makes relevant further research in this direction, which is accordingly planned by us in subsequent studies.Thus, measuring the ratio of the hydrodynamic radius to the gyration radius of colloidal clusters provides important information about their structure. In the case when the hydrodynamic radius is less than the gyration radius, we can assume that the clusters have a fractal-like structure when the primary particles are grouped into some loose lumps limited in space. If the hydrodynamic radius exceeds the gyration radius, we can assume that the clusters are better described by a more complex structure, characterized by the presence of branches.The main conclusion that can be drawn from the analysis of the data obtained by two fundamentally different methods, TEM and NTA, when studying colloidal nanoparticles in two different media, is that they provide information on the characteristic sizes and structure of the synthesized nanoparticles and clusters formed by them, as well as about the temporal stability of solutions of such particles. At the same time, the NTA method is simpler and much faster, which is very important for most applications.An important issue for applications of colloidal NPs is the stability of their solution parameters over time. One of the processes leading to undesirable changes in these parameters is the aggregation of NPs, which can cause coagulation of a colloidal solution. Stability of colloidal solutions, as is known, is largely determined by the value of the zeta potential. Our measurement of the zeta potential gave a value of +47 mV ± 5% that is rather high and means that the synthesized colloidal solution should be stable.3+: LaF3NPs colloidal solution and obtain additional information about the aggregation processes, we measured the size distribution in a freshly prepared solution (three days after preparation) and in a solution subjected to long-term storage (seven months) in a stationary state. The measurements were performed in two modes: (a) without preliminary preparation of the solution and (b) after ultrasonic treatment of the solution. Results of these measurements are shown in To control the temporal stability of the 0.1 at. % Nd3+: LaF3NPs leads to a shift of the distribution maximum towards larger sizes and an increase in the distribution width. This shows that during a long storage, some of the NPs stacked together. Sample homogenization in an ultrasonic bath made it possible to significantly eliminate this undesirable effect.It can be seen that long-term storage of a solution of colloidal 0.1 at. % NdThe measurements also showed that, after long-term storage of synthesized colloidal solutions in a vertical tube, the size distribution of NPs in the upper and lower parts of the tube began to differ . Under tThe slow process of NP agglomeration in the studied colloidal solutions during storage can be explained by the adhesion of clusters, when they come into contact during convection and Brownian motion. It is significant that in our case, this process could be partially reversed by using ultrasonic treatment of the solution. From this fact and the very slow coagulation of the solution during storage, it can be assumed that the main part of the primary NPs had already rapidly aggregated during the synthesis and isolation of the desired fraction. Note that a detailed analysis of the TEM data obtained with the high spatial resolution showed that the crystallization process in each primary nanocrystal occured independently of each other. Thus, further studies are required for a deeper understanding of the aggregation process of the studied NPs.4F3/2 level of the Nd3+ ion in the 0.1 at.% Nd3+: LaF3 NPs in aqueous and DMSO media was measured at the 4F3/2→4I9/2 transition of Nd3+ at λdet = 863 nm upon excitation to the 4F5/2 + 2H9/2 mixed state at λexc = 789 nm ) and the kinetics of spontaneous emissionSince the concentration of Nd4F3/2 excited state of the Nd3+ ion. The method to find the radiative lifetime for ACS colloid in the case of static fluorescence quenching was described in detail in [=To determine the relative fluorescence quantum yield etail in . The valAfter correctly selecting the  = 1.477  is higherding to , the radAccording to Equation (5) the ratio of macroparameters 6/ms [4F3/2 level, The dependences of relative fluorescence quantum yield  and (7) . For thi6 nm6/ms  was takeeen them . The exp6/ms [The experimental values of the relative fluorescence quantum yield cy of 2% . This me- groups  in the v6 nm6/ms , we alsocolloids .3+ ions and the contribution of upconversion. Due to the distortion of the initial stage of the luminescence decay kinetics, it is also impossible to properly normalize the decay curve to the luminescence maximum. At the same time, the determination of the macroparameter The concentration of OH- acceptors eter γOH  and the trations  obtained−3, 3+ acceptors (−2 nm−3). This result is consistent with the small contribution of Nd–Nd self-quenching and energy migration over the Nd3+ ions to the fluorescence quenching.It has been established that concentration of OH- acceptors  with weight analysis of the 0.1 at. % Nd3+: LaF3 powders obtained by drying at 80 °C an aqueous colloid and DMSO colloid of nanoparticles  were melted in a vacuum of 10−2 mm Hg and, according to the results of chemical analysis, 0.010–0.015 and 0.4–0.5 wt.% of the oxygen content were found, respectively.The decrease in the weight of the powder of particles obtained from an aqueous colloid of NPs without post-treatment upon heating to 800 °C . A possi data of ,39, in wnOH (in DMSO) = 0.69 mass%), determined from the analysis of the kinetics of impurity luminescence quenching in the DMSO colloid of these NPs (At the same time, the weight loss of the powder particles obtained from the colloid with post-treatment in DMSO at 800 °C is about 1.4 wt.% (hese NPs . The rea3+: LaF3 NPs colloidal solutions prepared by HTMW treatment were studied in different media, one in water and two in DMSO. The fluorescence static quenching kinetics of NPs In this research, three samples of 0.1 at. % Nd6/ms [nOH concentrations obtained by these two methods lies within 5% (−3, which is two orders of magnitude higher that the concentration of Nd3+ ions. Thus, the static mechanism of the fluorescence quenching was confirmed.At the same time, the concentration of acceptors can be determined much more accurately from the analysis of the kinetics of impurity fluorescence quenching. As shown in , for thi6 nm6/ms  and thusithin 5% . It shou3+: LaF3 NPs were studied according to TEM data and optical scattering ultramicroscopy, operating at an unshifted frequency. The combined application of two fundamentally different methods made it possible to conclude that the increased hydrodynamic viscosity of the formed clusters is better described by the presence of a loose structure with divergent side branches than by a fractal structure.The dimensions and structure of the synthesized colloidal clusters of 0.1 at. % NdLong-term studies of colloidal solutions by the NTA method have shown that NPs slowly aggregate during long-term storage, and under the action of ultrasound this process can be reversed to a significant extent, which indicates the weakness of the bonds formed. The irreversible fraction of observed clusters arises either during the synthesis or shortly after it. According to TEM data with high spatial resolution, the joint growth of nanocrystals during the synthesis is not observed.


To reduce the risk of post-hepatectomy liver failure in patients with hepatic pathologies, it is necessary to develop an approach to express the intraoperative assessment of the liver’s regenerative potential. Traditional clinical methods do not enable the prediction of the function of the liver remnant. Modern label-free bioimaging, using multiphoton microscopy in combination with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), can both expand the possibilities for diagnosing liver pathologies and for assessing the regenerative potential of the liver. Using multiphoton and SHG microscopy, we assessed the structural state of liver tissue at different stages of induced steatosis and fibrosis before and after 70% partial hepatectomy in rats. Using FLIM, we also performed a detailed analysis of the metabolic state of the hepatocytes. We were able to determine criteria that can reveal a lack of regenerative potential in violated liver, such as the presence of zones with reduced NAD(P)H autofluorescence signals. Furthermore, for a liver with pathology, there was an absence of the jump in the fluorescence lifetime contributions of the bound form of NADH and NADPH the 3rd day after hepatectomy that is characteristic of normal liver regeneration. Such results are associated with decreased intensity of oxidative phosphorylation and of biosynthetic processes in pathological liver, which is the reason for the impaired liver recovery. This modern approach offers an effective tool that can be successfully translated into the clinic for express, intraoperative assessment of the regenerative potential of the pathological liver of a patient. Today, in worldwide clinical practice, about 1 million new cases of primary and secondary liver cancers are recorded annually. The only treatment option for such patients is liver resection. Generally, 13–30% of patients can undergo surgical treatment, but the 5-year survival rate is only 14–61% . DespiteAlthough there are already substantial data related to the regeneration processes of a healthy liver, in actual clinical practice, about 20% of patients undergoing liver resection, and up to 25% of donors for liver transplantation, have some degree of steatosis or fibrosis ,4,5. It Non-alcoholic fatty liver disease (NAFLD) is the most frequent pathology (in 30% of the adult population in developed countries) that reduces the regenerative potential of the liver. The most common form of NAFLD is steatosis, associated with an increased intensity of biosynthesis of fatty acids and glycerol and an accumulation of triglycerides .Another frequent liver disease is fibrosis, or its decompensated stage, cirrhosis. Fibrosis is accompanied by a disruption of tissue architecture, the development of portal hypertension, and of cell hypoxia. Among patients with underlying cirrhosis or severe fibrosis, the mortality rate after surgical treatment of hepatocellular carcinoma is 30% and is associated with an insufficient functional reserve and a reduced regenerative potential of the liver ,9.Despite continuing improvements in the methods for assessing the structure of liver tissue, histological and immunohistochemical analyses are still considered the gold standards. However, these methods are time-consuming and do not provide information about vital processes within the tissue. Indeed, the diagnosis of cirrhosis relies primarily on histopathological evidence of late-stage fibrosis , but does not include any analysis of the pathogenesis and functional capabilities of the liver tissue . Currently, fluorescence bioimaging methods are being actively used in biomedical research. These include multiphoton microscopy, second harmonic generation (SHG) microscopy, and fluorescence lifetime imaging microscopy (FLIM). The procedure for obtaining and processing the data resulting from such methods is not time-consuming, so this is an advantage for enabling express, intraoperative assessment of the state of the liver tissue. The main additional benefits of these methods are that they are minimally invasive, do not require additional contrast agents, and allow the intravital visualization of tissue structure at cellular resolution. Additionally, based on data on the fluorescence lifetimes of various forms of the intracellular cofactors NAD(P)H and their relative contributions, we can assess changes in the metabolic state of the hepatocytes, which is a sensitive indicator of the state of the liver tissue. Previously, we were able to show that metabolic bioimaging methods are effective for assessing the metabolic state of hepatocytes in the presence of pathologies and in the process of normal regeneration ,12,13. WThus, the purpose of this current work was to identify new criteria related to the reduced regenerative potential of liver with underlying pathologies by using the label-free, minimally invasive methods of multiphoton microscopy with SHG and FLIM.4) diluted in oil twice a week for 8 weeks [A series of experiments were carried out using 30 male Wistar rats with an average weight of 300–400 g. Induction of toxic fibrosis was carried out by the intraperitoneal injection of a 33% solution of carbon tetrachloride /0.7. The percentage of liver weight recovery was carried out according to the following formula: weight of remnant (g)/initial liver weight (g)—this is the absolute percentage of liver weight recovery, i.e., “absolute weight”. To assess the percentage of recovery of liver tissue with induced pathologies relative to the corresponding day of normal regeneration, we calculated the relative percentage of liver weight recovery, i.e., “relative weight”. For this purpose, the values of “absolute weight” for normal liver regeneration were taken as 100% (absolute weight norm) and divided into “absolute weight” for the liver with pathology (absolute weight path) at the corresponding time point of regeneration, i.e., the 3rd or 7th day after PH: path(g)/absolute weightnorm(g) × 100%absolute weight.To determine the effectiveness of liver recovery, the liver weight was measured before the induction of regeneration and at different stages of the recovery process. The initial liver weight was calculated according to the following formula: Morphometric analysis was performed with the following indicators being evaluated: the number of tetraploid hepatocytes  and the numbers of binucleate cells and of mitotic cells. The average values of all these parameters were calculated in proportion to 100 normal cells . The morBlood samples (1.5 mL) were obtained from the tail vein of each rat. The blood was then centrifuged at 3000 rpm for 15 min to obtain the serum. The following parameters were evaluated: the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), total protein, creatinine, urea, high-density lipoproteins (HDL), low-density lipoproteins (LDL), and triglycerides (TG) . The anaInvestigation of all the fresh liver samples was performed using an LSM 880  equipped with a Ti:Sapphire femtosecond laser  and a time-correlated single photon counting (TCSPC) system . The average laser power used was about 10 mW. A C Plan-Apochromat 40×/1.3 oil immersion objective was used to collect the fluorescence signal. From 10 fields of view for each sample, both the NAD(P)H fluorescence intensity images and the FLIM data were acquired. NAD(P)H: λex = 750 nm, λem = 450–490 nm. The SHG signal was generated at 800 nm and recorded in the 371–421 nm range. We performed a quantitative assessment of the NAD(P)H autofluorescence intensity in the cytoplasm of the hepatocytes by manual selection of ~40 × 40 pixel zones as regions of interest (ROIs) using ImageJ software .The FLIM analysis was performed using SPCImage software  with a tri-exponential decay model. To maintain a minimum of 10,000 counts per pixel, the binning parameter was set at 3. The goodness-of-fit model was assessed by the χ2 value (it should be at 1). The following parameters were analyzed in 20–30 regions of cell cytoplasm for each field of view: tm (ps), the amplitude weighted mean fluorescence lifetimes; t1 (ps) were fixed at 400; t2 (ps), fluorescence lifetimes of the bound form of NADH; t3 (ps), fluorescence lifetimes of NADPH; the relative contributions of the free, a1 (%), and the bound, a2 (%), forms of NADH; and the relative contribution of NADPH, a3 (%).For the histological studies, the liver was fixed in a 10% solution of buffered formalin, passed through isopropyl alcohol and embedded in paraffin. Deparaffinized 7 μm sections were stained with hematoxylin/eosin and picrofuxin by van Gieson according to the standard protocol . For eacp-value ≤ 0.05. To determine the statistical significance of changes in the degree of tissue regenerative capacity of the liver with induced pathology, the nonparametric Mann–Whitney U test was used. Comparative analyses were performed between recovered liver weight with induced pathology on the 3rd and 7th days after 70% PH with the corresponding time point of normal liver regeneration; p-value ≤ 0.05.For each time point of the experiment, we obtained 8–10 NAD(P)H autofluorescence and FLIM images. For each image, we performed a quantitative assessment of the NAD(P)H autofluorescence intensity in the cytoplasm of 30 hepatocytes (in zones with a high NAD(P)H autofluorescence intensity) and for 30 hepatocytes (in zones with a low NAD(P)H autofluorescence intensity), excluding the nucleus. For each image, we determined the FLIM parameters in the cytoplasm of 30 hepatocytes (in zones with a high NAD(P)H autofluorescence intensity). R-language was used for the statistical calculations. Statistical differences between different groups were analyzed using the pairwise multiple comparison procedure. Differences in mean values between groups were considered significant when they were larger than might be expected by chance. After the tests for normality (Shapiro–Wilkes) and equal variance (F-test) showed a normal distribution of data, the pairwise t-test method was used, for pairwise comparison, using the Bonferroni correction; The design of this study is depicted in We analyzed the restoration of liver weight at different stages during the induced regeneration process of liver with steatosis and fibrosis using standard methods ,21,22. TFor steatosis at the 3rd week, we observed a dramatic and significant increase in the absolute and relative weight of the recovered liver on the 3rd and 7th days, followed by a decrease in these parameters up to 12th week. At the 12th week of steatosis, the values of the absolute and relative weight were almost equal to the corresponding values for normal regeneration. Such high values are probably associated not only with the restoration of the mass of the liver parenchyma but also with the accumulation of lipid droplets and the progression of fibrosis.For induced fibrosis, we could observe a sharp drop in both the absolute and the relative liver weights on the 3rd and 7th day after PH by the 2nd week of fibrosis induction. However, the values of the liver weight on the 3rd day after PH increased gradually from the 4th week, and by the 8th week even exceeded the corresponding values for normal regeneration. However, despite the high values of liver weight on the 3rd day after PH, on the 7th day (termination of the regeneration process) for all stages of the pathology, except for the 7th day at the 4th week, the percentage of liver recovery was already falling.It is worth considering that our histological analysis and analysis of the structural and functional state of the liver tissue using multiphoton microscopy showed obvious pathological changes.We observed that during the development of hepatic steatosis, there was a significant decrease in the hepatocyte proliferative activity on the 3rd day of regeneration in comparison with the model of normal regeneration . In the The results of a biochemical blood test are presented in the Histological analysis of the liver tissue with induced steatosis showed a pronounced lipid infiltration of the hepatocytes by the 6th week of the pathology. Starting in the 6th week, we identified portal fibrosis without septa (METAVIR fibrosis score of F1). By the 12th week, an advanced stage of steatosis was observed, characterized by macrovesicular steatosis a. HoweveIn the model of regeneration induction after steatosis, the results of SHG analysis are consistent with the data of morphological analysis, where we could observe single foci of accumulation of collagen fibers a. In theFor induced fibroses, we observed typical pathological changes that were most pronounced at the 6th and 8th weeks of the pathology. In the 6th week, we identified the presence of fibrosis with single septa (METAVIR fibrosis score of F2 or greater) and the accumulation of lipid droplets in the hepatocytes. In the 8th week, histological analysis showed foci of necrosis, the accumulation of lipid droplets in the hepatocytes, and severe fibrosis (METAVIR fibrosis score of F3 or greater) a. FurtheThe regeneration process at different stages of fibrosis was characterized by pronounced microvesicular steatosis being seen on both the 3rd and 7th day after PH. We also observed a violation of tissue architecture on both the 3rd and 7th day after PH for each pathological stage. This was primarily due to the rate of hepatocyte proliferation being higher than the rate of synthesis of intercellular substance ,25. The We performed quantitative assessments of the NAD(P)H autofluorescence intensity for both pathologies in two identified zones—with high (I1) and low (I2) NAD(P)H auto-fluorescence intensities. We showed a statistically significant decrease in the NAD(P)H autofluorescence intensities in zones I2, associated with damaged hepatocytes, lipid infiltration, and fibrous tissue. Furthermore, in the case of hepatic steatosis, at the 3rd week of pathology, we observed a sharp, statically significant decrease in the intensity of the NAD(P)H autofluorescence in zones I1, in comparison with corresponding zones I1 for normal regeneration. Further, the intensity of NAD(P)H autofluorescence in such I1 zones gradually increased. By the 12th week, the values of the intensity of NAD(P)H autofluorescence on the 3rd and 7th days after PH were almost equal to the corresponding values in zones I1 for normal regeneration. The results are presented in At the same time, in the case of fibrosis, the intensity values of NAD(P)H autofluorescence in zones I1 were statically significantly lower compared to the corresponding values in the I1 zones for normal regeneration at all experimental time points. The results are presented in Thus, we found that the main criterion indicating steatosis and fibrosis was the presence of zones with a reduced NAD(P)H autofluorescence signal intensity, these being associated with foci of infiltration or foci of fibrosis.Using FLIM, we analyzed the metabolic state of the hepatocytes in liver tissue with induced pathologies before and after the induction of the regeneration process.In the case of liver steatosis, the fluorescence lifetime values of the bound form of NADH  and of NADPH  did not change significantly at any of the monitoring time points. In the case of liver fibrosis, we revealed statistically significant drops in the values of the fluorescence lifetime of the bound form of NADH in the later stages (6th and 8th weeks) that are probably associated with hypoxia in the hepatocytes ,27. The An analysis of the relative contributions of various forms of the NAD(P)H showed that the values of the lifetime fluorescence contribution of the bound form of NADH  sharply decreased in the 3rd week of hepatic steatosis. However, with further development of the pathology (by the 6th week) this parameter increased again, and by the 9th week, the values of a2 almost reached the control values  b,c. HoweDuring the regenerative process at different stages of steatosis, it was shown that by the 3rd week, on the 3rd day after PH, there was a sharp increase in the fluorescence lifetime contribution of the bound form of NADH  and the contribution of NADPH , followed by a decrease on the 7th day b,c. Such4 administration  by the 2nd and 4th weeks after the beginning of CClstration b,c. DespAs in the case of steatosis, we found a similar trend in the regeneration process with fibrosis. It was shown that during the early stages (in the 2nd and 4th weeks) the values of a2 and a3 before and after PH (both on 3rd and 7th days) were similar b,c. By tThus, using FLIM, we have identified several criteria related to the reduced regenerative potential of livers with pathologies. In the early stages of the pathology, decreased values of a2 and a3 were a typical change. For the later stages of the pathologies, an important aspect was the absence of a sharp increase in the contributions of a2 on the 3rd day after PH. However, the absence of a corresponding sharp jump in a3 on the 3rd day after PH was observed only in the case of steatosis. Such observations are consistent with the fact that effective regeneration requires a significant increase in the intensity of OXPHOS  to drive ATP synthesis and also requires an increase in the biosynthetic processes  that meet the energy needs of hepatocytes.Previously, we have shown that in a healthy liver, OXPHOS is the most active metabolic pathway in the hepatocytes. Our results showed that in the process of physiological  regeneration, on the 3rd day after 70% PH, there is a sharp jump in the contribution of the bound form of NADH and NADPH that indicates an even greater increase in the intensity of OXPHOS and synthetic processes due to the high energy demands of the proliferating hepatocytes . HoweverIt is widely known that hepatic fibrosis and cirrhosis appear to be significant risk factors for post-operative complications, disrupting hepatic regeneration ,29. The In our work, we confirmed that morphological methods are not sufficiently specific and informative; therefore, we tried to determine specific features of the metabolic state of hepatocytes during steatosis and fibrosis undergoing induced regeneration. The metabolic state of the hepatocytes is a more sensitive marker of the presence of pathological processes at the level of individual cells, while also being associated with liver function even at the level of the whole organ . Thus, tUsing multiphoton microscopy, we have shown that the existence of pathology can be identified by the presence of zones with reduced NAD(P)H autofluorescence signals, these being associated with zones of lipid infiltration and with the foci of fibrosis. However, such structural analysis does not provide detailed information about the metabolic processes occurring in the cells. To obtain more detailed data on the metabolic state of the hepatocytes, we performed FLIM analysis. FLIM allows us to distinguish the short- and long-lifetime component forms of NAD(P)H that reflect whether this cofactor is in its free or protein-bound state, respectively. The free form of NADH drives ATP production in the cytosol by glycolysis, while the bound form of NADH is localized in the mitochondria and is involved in OXPHOS . The redDespite the fact that NADPH is involved in the process of lipogenesis, in our work, we did not observe an increase in NADPH contribution in the hepatocytes, either in the case of hepatic steatosis or during the liver regeneration process, even in the later stages of steatosis. These results indicate damage to the hepatocytes, leading to a general decrease in their synthetic activity. Since it is known that mitochondrial dysfunction impairs fat homeostasis in the liver and leads to an overproduction of reactive oxygen species (ROS), this could explain why increased oxidative stress and lipid peroxidation have been identified in the literature as the most prominent pathogenic features of injury in steatosis . Eventua4 enters an animal’s body, it is activated by the liver cytochrome P450 to generate the free radicals CCl3·and CCl3O2· [For induced fibrosis, FLIM analysis showed a sharp decrease in the fluorescence lifetime contributions of the bound forms of NADH and NADPH in the early stages of pathology. The sharp drop in the bound form of NADH during the early stages is probably associated with mitochondrial dysfunction, which is common for fibrosis ,41. It i CCl3O2· , causing CCl3O2· . Additio CCl3O2· . Moreove CCl3O2·  and acti CCl3O2· ,36. Our  CCl3O2· ,12. Our  CCl3O2· . Any incUsing an approach based on label-free methods of multiphoton microscopy in combination with SHG and FLIM, we have identified criteria for indicating reduced liver regenerative potential in the presence of steatosis and fibrosis. These criteria are as follows: 1. the presence of zones with reduced NAD(P)H autofluorescence signals, corresponding to zones of lipid infiltration and to foci of fibrosis; 2. low values of the contributions of the fluorescence lifetimes of the bound form of NADH and NADPH, due to the development of mitochondrial dysfunction and damage to the hepatocytes; 3. the absence of sharp increases in the contributions of the fluorescence lifetimes of the bound forms of NADH and NADPH in proliferating hepatocytes, associated with the depletion of energy resources in the pathological liver, a situation that significantly reduces the efficiency of liver restoration. Thus, determining the state of the above criteria will expand the possibilities for assessing the regenerative potential of a liver remnant subject to the presence of various pathologies.


To analyze mortality trends in Switzerland in 2021, the second year of the COVID-19 pandemic.Using data from the Swiss Federal Statistical Office, we compared mortality in Switzerland in 2021 with that of previous years in terms of standardized weekly deaths, standardized  mortality rates  and life expectancy. The latter is a well-known demographic concept defining the average lifespan of a hypothetical cohort living and dying according to the mortality rates of a given year.After a favorable first half of the year and a fairly standard second half in terms of mortality in Switzerland, the year 2021 ended with a wave of deaths of moderate intensity related to the 5th wave of COVID-19. Overall, and after a notable increase in mortality in 2020 , the pre-pandemic mortality level was approximately recovered in 2021 . Life expectancy, after declining by 10 months for men and 6 months for women in 2020 (i.e. men would have lost 10 months and women 6 months had they lived their entire lives with COVID-19 as it was in 2020), returned in 2021 to 2019 levels for women (85.6 years) and regained 2018 levels for men . The age group responsible for the small remaining loss for men was the 50–70 age group, which had similar mortality in 2020 and 2021.The second year of the COVID-19 pandemic in Switzerland was characterized by an approximate return to pre-pandemic mortality levels, with a faster recovery for women than for men with respect to 2020. COVID Live - Coronavirus Statistics - Worldometer (worldometers.info)). A key indicator to assess the impact of the pandemic is all-cause mortality [The years 2020 and 2021 have seen the global spread of the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the time of writing, more than 500 million cases and more than 6 million deaths due to COVID-19 have been reported worldwide. In Switzerland, the pandemic has caused about 14’000 deaths to date, representing 1600 deaths per million inhabitants ) and with those of a large study on the loss of life expectancy in 29 countries [There is now a large literature studying the impact of the pandemic on all-cause mortality during the year 2020 –8. The lountries .On the other hand, while a few studies have been published concerning the excess death of (part of) the year 2021 , 13, or The objective of this paper was therefore to analyze the change of mortality and life expectancy in Switzerland in 2021 in comparison with the year 2020 and the previous years. We also aimed to compare the mortality and life expectancy changes in men and women and identify the age groups mainly responsible for the observed evolutions and possibly more affected by this second year of pandemic.Décès selon l’âge et le sexe, 1970-2020 - 1970-2020 | Tableau | Office fédéral de la statistique (admin.ch)), while it was available by 5-years age groups (with a last open class of 90+) for each of the 52 weeks of 2021 ), and by 3 large age groups  for the entire 2021 (January 1—December 31) ). By convention, the year 2021 is divided into 52 weeks, the first of which begins on January 4, 2021 and the last of which ends on January 2, 2022. Therefore, for the annual analysis, we adjusted these weekly deaths to obtain the same reported annual totals for men and women as well as in each of the three age groups . To facilitate comparisons across all years, we finally excluded 1/366 of deaths in the case of leap years (2000-04-08-12-16-20). Weekly death data were also available for years 2015–21 with a partition in five large age classes  ) which have been used for the weekly analysis 2015–21 described below.We used official data on deaths in Switzerland published by the Swiss FSO for 2000–2021 . The annual number of deaths, separately for men and women, was available by 1-year age groups (with a last open class of 110+) until year 2020 ). The size of the Swiss population as of January 1, 2000–21, stratified by sex and one-year age groups (with a last open class of 110+), was also found in the Human Mortality Database (HMD) [The size of the Swiss population as of January 1, 2011–21, stratified by sex and one-year age groups (with a last open class of 105+) was available in the FSO database (se (HMD) . Since tse (HMD) . It was w  and year y  standardized at January 1 of year s (s = 2021) was calculated as follows:Weekly mortality for the years 2015–21 was analyzed using Standardized Weekly Deaths (SWD), with the 5 available age classes  and considering population as of January 1, 2021 as the reference. The SWD for a given week and year represents the number of deaths that would have occurred if the age specific mortality rates observed that week and year were applied to the age structure of the reference population. The SWD of week y, week w and age class k, and k as of January 1 of year t.In this formula y  standardized at January 1 of year s (s = 2021) was obtained as follows:To compare mortality of 2021 with that of previous years we used one-year of age and sex (directly) Standardized Mortality Rates (SMR) , 18. Thei and sex j in year y, y, for age i and sex j, i and sex j of the year t, and Ps the total population of the reference year. Note that the SMR or the reference year y = s =2021 corresponds to the Crude Mortality Rate (CMR) in that year: In this formula, SMRs were calculated on the entire population and by sex and 10-year age groups (with a last open class of 90+). Mortality between two years  was compared  using the relative change of SMR expressed in %. The 95% confidence intervals around any relative change of SMR were obtained as in [i  and sex j  in year y , i belongs to. LE at age x (x = 0 and 65) for sex j and year y was then obtained as follows (Life expectancy (LE) at birth and at 65 years of age was obtained applying a piecewise exponential model  on death follows :exjy=1For validation we compared LEs at birth for years 2000–20 obtained with our method and data with the ones published by the HMD. We found a mean relative difference of 0.05% with no difference exceeding 0.1%. Life expectancies between two years were then compared using absolute differences expressed in months.ED20 and ED21, as well as for the whole period 2020–21 (ED20+ED21), by comparing the number of deaths in 2020 and 2021 to that in 2019, all three standardized to the population as of January 1, 2021:Finally, we calculated an excess death for each of the two years of pandemic, Grippe saisonnière (influenza) (admin.ch)). In 2020, we clearly observe two waves of mortality attributable to COVID-19, where the second wave  was much higher and broader than the first (in spring 2020). In 2021, after the end of the second wave of COVID-19, we observed low mortality during the first half of the year, due to the absence of an influenza-related mortality wave that year and because the third and fourth waves of COVID-19 in Switzerland (spring and summer waves) had almost no impact on overall mortality. The second half of the year was characterized by a fairly standard mortality level with another moderate wave of mortality at the end of the year, corresponding to the 5th wave of COVID-19 in Switzerland. COVID-19 Suisse | Coronavirus | Dashboard (admin.ch)).The evolution of the number of deaths, crude mortality rates (CMR), and standardized mortality rates (SMR) between 2000 and 2021 is shown in Comparing the SMRs between 2019, 2020 and 2021 separately for both sexes and for 10-year age classes (last open class of 90+), we observe that the increased mortality in 2020 (compared to 2019) in age classes over 70 years [Life expectancy at birth, which reached 81.9 years for men and 85.6 years for women in 2019  after deThe year 2021 was characterized in Switzerland by a first half of the year with unusually low mortality, due to the absence of any COVID-19 or influenza wave, followed by a second half of the year that was quite standard until an unusual end-autumn wave of mortality, corresponding to the 5th wave of COVID-19. Taken as a whole, 2021 marked an approximate return to 2019 mortality levels. After a 9.2% excess mortality in 2020, standardized mortality rates decreased by 7.7% in 2021, bringing the rate approximately back to the pre-pandemic level. Life expectancy, meanwhile, after declining by 10 months for men and 6 months for women in 2020, recovered in 2021 the 2019 levels for women (85.6 years) while returning to 2018 levels for men (81.6 years). In all age groups, mortality was not significantly different in 2021 and 2019, with the exception of men aged 60–70 , who still had an excess mortality in 2021 compared to 2019, and who were indeed responsible for the slightly higher mortality observed for men in 2021 compared to 2019.We also calculated an estimate of excess deaths based on standardized mortality rates. This indicator captures changes in mortality at different ages adjusting for modifications that occur each year in the size and age structure of the population , 18. Com• Deaths in Germany 1991-2021 | Statista) or Sweden (• Sweden: death rate 2010-2020 | Statista), where mortality in 2019 was notably low, well below the trend. Second, and more importantly, the downward trend in the pre-pandemic years was so strong in Switzerland , life expectancy in 1919 (53.7 years for men and 55.9 for women) had largely returned to that of 1917 (54.1 years for men and 57.4 years for women), before the pandemic of Spanish flu [Regarding the choice of scale to compare mortality across years, we believe that life expectancy has several advantages over excess mortality (or excess deaths). First, being measured in months, a loss of life expectancy is easier to interpret than an excess mortality (in %) or an excess deaths . Secondly, by giving more weight to the (excess) deaths of the young than to those of the elderly, it recognizes that the former are indeed more dramatic than the latter, an essential point when it comes to assess the severity of a pandemic. The conclusions that can be drawn by using one indicator rather than another can be quite different. For example , estimatactual population living in Switzerland in that year will be indeed much smaller [It may also be useful to recall that the concept of life expectancy calculated in a given year in a given country, as used here and in –11, refe smaller .One limitation of this study is that it uses mortality data that are not yet totally finalized. Indeed, while the total number of deaths in 2021 is now supposed to be definitive, the age distribution that we have been able to adopt in our analyses is not the most refined possible. Moreover, as the population at the end of 2021 is not yet available, we have calculated the rates by dividing the number of deaths by the population at the beginning of the year, rather than considering the average population at the beginning and end of the year, which would better correspond to the notion of rate. However, as already mentioned, similar calculations for years prior to 2021 have produced almost the same results as those published in HMD. In addition, calculations made early 2021 for 2020  proved tIn summary and conclusion, in Switzerland, after being notably high in 2020, mortality in 2021 returned to 2019 levels for women and to 2018 levels for men. Thus, while the first year of the COVID-19 pandemic led to a significant increase in mortality, the second year was characterized by an approximate return to pre-pandemic mortality levels, with a faster recovery for women than for men.S1 Fig(TIF)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


After the fourteenth day post the end of the first lockdown (18 May 2020), the SPR increased daily at a rate of 0.024 ). In addition, the SPR appeared to increase significantly every day prior to 17 November 2020 by 0.024 ). After the fourteenth day post the start of the second lockdown (17 November 2020), the SPR decreased daily at a rate of 0.039 ). These data demonstrate that, in our Institution, the lockdowns helped to both protect healthcare workers and maintain adequate standards of care for COVID and non-COVID patients for the duration of the state of emergency in Italy.At the onset of the SARS-CoV-2 pandemic, individual and social measures were strengthened through restrictive non-pharmaceutical interventions, labelled with the term “lockdown”. In Italy, there were two lockdowns (9 March 2020–3 May 2020 and 3 November 2020–27 March 2021). As part of preventive measures, healthcare workers and the administrative staff population of Policlinico A. Gemelli underwent nasopharyngeal swab tests from 1 March 2020 to 9 February 2022, a long time interval that includes the two aforementioned lockdowns. The population included 8958 people from 1 March 2020 to 31 December 2020; 8981 people from 1 January 2021 to 31 December 2021; and 8981 people from 1 January 2022 to 9 February 2022. We then analysed pseudo-anonymized data, using a retrospective observational approach to evaluate the impact of the lockdown on the incidence of SARS-CoV-2 infections within the population. Given the 14 day contagious period, the swab positivity rate (SPR) among the staff decreased significantly at the end of the first lockdown, every day prior to 18 May 2020, by 0.093 ( There is much evidence in the literature supporting the effectiveness of individual and social measures, such as wearing masks ,2,3,4, hOther than this, there is little evidence supporting the validity of more restrictive non-pharmaceutical interventions (NPIs) that have been labeled with the term “lockdown”, such as the strict control of country borders and the closure of schools, workplaces, and other public institutions ,11. SomeIn Italy, on 31 January 2020, the declaration of national emergency due to the COVID-19 pandemic became effective, and urgent measures to safeguard human health were implemented. On 23 February 2020, the so-called red zone (suspension of work and school activities and travel prohibition) was established in ten cities in Northern Italy. A lockdown was introduced for the first time on 9 March 2020, and subsequently extended until 3 May. Later, although in a less restrictive form, a second lockdown was declared on November 2020, which ended on 27 March 2021 .The aim of this retrospective observational study is to assess the impact of the lockdown measures on the incidence of SARS-CoV-2 infections within a well-defined population, that is, the staff of a large hospital that was also a reference hub for the management of COVID-19 patients throughout the pandemic.Policlinico A. Gemelli Hospital is both a welfare and educational institution. Eight clinical and research departments include 113 medical care units, including 86 complex operating units, 27 simple operating areas, and 1536 beds. In one year, 215 organ transplantations were performed, 94,509 patients were discharged, and 83,419 people accessed the emergency roomOn 25 February 2020, at Policlinico Gemelli Hospital, the Crisis Unit had its first meeting, determining all strategies for both health and administrative areas. On 28 February 2020, the red zone was extended to the Lombardy region. The following day, the first autochthonous case of COVID-19 in the Lazio region was diagnosed in our Institution. Within the hospital, several containment measures have been promptly introduced since March 2020, while our Institution was becoming a COVID center.In order to manage both COVID and non-COVID hospitalized patients, the following two types of clinical pathways were created: the traditional one, reserved for negative patients, and one dedicated to confirmed cases of infection with SARS-CoV-2.The staff population , administrative and support staff, and a heterogeneous group including maintenance workers, food service workers, cleaners, etc.  in the staff population during the following months. Data relating to admissions for infection with SARS-CoV-2 registered in the staff population in the period 1 March 2020–9 February 2022 and their outcome  were also extracted.Given the contagious period was calculated as 14 days in March 2020, the daily swab positivity ratio (SPR) was calculated and used as data points in the time series analysis using 23 March 2020 (start of first lockdown + 14 days), 18 May 2020 (end of first lockdown + 14 days), 17 November 2020 (start of second lockdown + 14 days) and 11 April 2021 (end of second lockdown + 14 days) as the intervention points.itsa syntax in Stata®. The level and trajectory of change following the intervention, obtained via ordinary least-squares regression estimates, were evaluated yielding β-coefficients and Newey–West standard errors.Data were modelled as a single group interrupted time series analysis without a comparator for both scenarios using the Statistical analysis was performed using Stata software .From 1 March 2020 to 31 December 2020, the staff population consisted of 8958 people and a total of 36,437 swabs were performed, 22,148 molecular (M) and 14,289 antigenic (A). From 1 January 2021 to 31 December 2021, the staff population consisted of 8981 people, and a total of 105,783 swabs were performed . Finally, from 1 January 2022 to 9 February 2022, the staff population consisted of 8981 people and 25,937 swabs were performed .In the three above reported periods, the number of patients accepted were 113,842, 117,834, and 12,684, respectively. They underwent a total of 111,486 swabs  in the first period; 200,073 swabs  in 2021; and 29,895 swabs  until 9 February 2022.In the staff group, there were 40 reinfections between March 2020 and February 2022, with an average interval of 11 months between the first and second episode. The minimum interval between two newly acquired cases was 45 days and was observed from the beginning of January 2022; these re-infections were caused by the Omicron variant in previously Delta-infected subjects during November 2021. In the same group, a single fatality was recorded in a radiology technician in his fifties, who died in October 2020.The trend of infections and positivity in the staff group and the patient group admitted to our Institution in the period 1 March 2020–9 February 2022 is shown in p < 0.0001, CI = (−0.138–−0.047)). In the first days of the intervention, there is no decrease, followed by a significant increase in the daily trend of SPR (relative to the preintervention trend) of 0.116 per day ). We also see, from the lincom estimate produced by specifying posttrend, that after the fourteenth day post the end of first lockdown (18 May 2020), the SPR increased daily at a rate of 0.024 ).As shown in the regression p < 0.0001, CI = (0.013–0.034)). In the first day of the intervention, there appeared to be a significant increase of 2.130 ) in SPR per day, followed by a significant decrease in the daily trend of SPR (relative to the preintervention trend) of 0.062 ). We also see, from the lincom estimate produced by specifying posttrend, that after the fourteenth day post the start of second lockdown (17 November 2020), the SPR decreased daily at a rate of 0.039 ).As shown in the regression p < 0.0001, CI = (−0.050–−0.027)). In the initial days of the intervention, there was no decrease, followed by a significant increase in the daily trend of SPR (relative to the preintervention trend) of 0.046 ). We also see, from the lincom estimate produced by specifying posttrend, that after the fourteenth day post the end of second lockdown (11 April 2021), the SPR increased daily at a rate of 0.007 ).As shown in the regression Most of the literature describing the effectiveness of restrictive measures during the pandemic emergency reflects the experience of countries that have been able to strengthen the strategic importance of their geographical position , or who Previous studies are based on estimation of the effectiveness of NPIs by the use of a statistical model to estimate the impact of each intervention on the incidence of COVID-19 ; other cMoreover, several studies have shown that restrictive measures can lose their efficacy because of many factors in the medium and long term ,25. NeveDuring the first lockdown, the Italian government tried to limit the spread of the virus to Northern Italy and adopted a 24/7 curfew.For the duration of the second lockdown, the curfew, ranging from 10 PM to 5 AM, was complementary to measures such as distance learning for high schools, closure of shopping malls on weekends, a reduction of 50% in the capacity of public transport, and the semi-closing of bars and restaurants from 6PM. The measures were then tightened during the Christmas holidays (from 21 December to 6 January).While the first lockdown had an impact on a population of patients and healthcare workers who were less exposed to the virus because it was concentrated in Northern Italy, the second lockdown involved the same staff population and a different set of patients, at a time when the virus was widespread throughout the country.Given that the contagious period calculated as 14 days in March 2020 ,27, it wWe accordingly segmented the period of observation into segments of 28 days, starting from the first day of the first lockdown, to compare the rates of positivity recorded in the population that did not change during the period (the staff). We wanted to verify whether the lockdown measures have had a positive effect, in addition to the other protective measures adopted during the period and described in the Materials and Methods.This verification has been provided during the following four periods: (1) the first lockdown (9 March–3 May 2020) and the 14 day period immediately following it, which represents the interval of the lockdown’s assumed effectiveness; (2) the period between the first and the second lockdown; (3) the second lockdown (2 November 2020–27 March 2021) which was different from the first, and influenced by the vaccination campaign; (4) the period without lockdown (14 April 2021–9 February 2022), when the proportion of the population who were vaccinated increased noticeably.Regarding vaccinated healthcare workers, the protection period has been considered from 9 February 2021, as the majority of second doses were administered from 21 January 2021 to 2 February 2021, and the protective effect is expressed best after at least seven days from the date of administration of the second dose .This effect can be observed in In the patient group, the number of positive cases recorded on average fluctuated from 30.71 (in the two weeks from 9 March to 22 March 2020) to 24.61 (in the following four weeks from 23 March to 19 April 2020). This decreased even more significantly to the value of 3.68 in the next four weeks (from 20 April to 17 May 2020).In the staff group, after a paradoxical increase recorded in the weeks from 23 March to 19 April 2020 (0.79 vs. 0.43 recorded in the previous weeks 9 March–22 March), we observed a decrease to 0.29 in the weeks from 20 April to 17 May 2020.The trend is confirmed by data pertaining to the second lockdown, which is weaker, but in a population more exposed to the virus. Among patients, it went from a value of 50.93 in the period from 21 October to 17 November 2020, to the value of 25.36 in the period from 18 November to 15 December 2020. Among the staff population, it is interesting to compare the value of 11.32 in the period 21 October to 17 November 2020 to the value of 4.86 in the following period (18 November to 15 December 2020). Moreover, in January 2021 we observed that the two populations showed different patterns b, where The effect seemed to persist over time, not only in the periods from 16 December 2020 to 12 January 2021, and from 13 January 2021 to 9 February 2021 , but even in the same period of the following year (16 December 2021 to 9 February 2022).In particular, considering that in our Institution we completed the administration of the second dose to most healthcare workers between 21 January and 2 February 2021 , there was a marked reduction of the positivity rate due to a synergistic effect of both the second lockdown and vaccination, together with a lower prevalence in the following months. We then registered a new increase in positivity rate, as a consequence of the following three factors: (1) a progressive decrease of the protective effect of the second dose, as has already been reported in literature ,30, (2) Moreover, in the staff population there was an average positivity rate of 11.89 during the lockdown period (16 December 2020–12 January 2021); this reflects a possible influence of both Christmas holidays and the spread of the Alpha variant. A value of 13.32 was recorded in the same period of the following year (16 December 2021–12 January 2022) in the absence of a lockdown. This trend is reinforced by the comparison between the period 13 January 2021–9 February 2021 (3.96) and the period 13 January 2022–9 February 2022 (14.79) in almost the same population.The comparison between the data recorded during the first and second lockdown versus the remaining periods demonstrates that in our Institution these restrictive measures, together with the other classical preventive actions and, subsequently, with the synergistic effect of vaccination, helped not only to protect healthcare workers, but also to maintain adequate standards of care for COVID and non-COVID patients during the state of emergency in Italy.Furthermore, our results could help to answer the question of what would have happened in Italy in the first period, if the lockdown had not been adopted, considering the limited percentage of vaccinated people.The limitation of this study lies in the long time interval covered, affected by the spread of the SARS-CoV-2 variants . Regarding the vaccination plan, while all vaccinable healthcare workers received their second dose by 2 February 2021, the vaccination for administrative staff began on 5 February 2021.Another limitation is related to the accuracy of laboratory tests: since the beginning of the pandemic, the phenomenon of false negative responses even for molecular tests has been highlighted, especially in the period prior to the onset of symptoms . This phMoreover, during the early part of the pandemic, most healthcare workers were less exposed to the virus, due to its greater concentration in Northern Italy  and adopFinally, the approach to pandemics has evolved from the first phase, characterized by the availability of molecular and serological swabs, although the latter proved to be poorly sensitive , to the


The experimental results show that the H2 heat treatment on the as-grown MQWs may lead to the decomposition of InGaN and the formation of inhomogeneous In clusters. The atomic force microscope (AFM) study indicates that although the surface roughness of the uncapped samples increases after H2 treatment, the V-defects are suppressed. Moreover, the luminescence efficiency of the MQWs can be effectively improved by growing a GaN cap layer with an appropriate thickness on the top of the MQWs, which can reduce the effects of the H2 atmosphere and high temperature on the MQWs. In addition, a morphologic transformation from step bunching to shallow steps occurs and a much smoother surface can be obtained when a thicker cap layer is adopted.In this paper, the photoluminescence (PL) properties and surface morphology of InGaN/GaN multiple quantum well (MQW) structures with the hydrogen (H The presence of hydrogen (H2) in the growth environment is usually forbidden because an abundance of hydrogen can lead to the desorption of indium [The wide luminous range 0.7–6.2 eV) of GaN-based multiple quantum well (MQW) heterostructures, which can cover the whole visible range, makes them suitable for light emission in the short to long wavelength part of the visible spectrum ,2,3. InG eV of Gairection ) limits irection  and mainf indium . Howeverf indium , which mf indium  and leadf indium . Thus, i2 can also play a role in eliminating In clusters, and thus, inhibiting the thermal decomposition of MQWs [Several articles have reported the effects of hydrogen on InGaN quantum wells ,16,17,18 of MQWs ,20. Moon of MQWs ,22 repor of MQWs  also use2 growth atmosphere of the p-type cladding layer, which works on the as-grown MQWs directly. Additionally, it is found that exposure to the hydrogen atmosphere leads to the decomposition of the InGaN QWs, as well as to the formation of inhomogeneous In clusters and the degradation of the luminescence property. The experimental results show that a GaN cap layer with an appropriate thickness growing over the MQWs can effectively maintain the integrity of the MQW structure during the hydrogen treatment process and improve the MQW luminescence intensity at room temperature, which may be even higher in comparison to the as-grown sample. The evolution of the surface morphology is also studied using an atomic force microscope (AFM).In this paper, we investigate the luminescence characteristics and surface topography of InGaN/GaN MQWs in the presence of hydrogen by simulating the H2 acted as the carrier gas for the growth of InGaN and GaN epitaxial layers in all of the above-mentioned preparation processes. The above growth conditions of the three samples were the same. Then, different heat treatments were applied to them. As shown in 2 and H2 atmosphere were introduced during the heat treatment process, respectively. The treatment time and temperature were 230 s at 1020 °C and then 120 s at 950 °C. Such a two-temperature process was set up to simulate the growth condition of the p-type cladding layer and ohmic-contact layer grown during the fabrication of the GaN-based LED structure. In addition, in order to check the protective effect of the GaN cap layer on the MQW structure during the hydrogen treatment, two other samples named H2 and H3 were prepared in which a GaN cap layer was grown on the top of the upmost QB layer of MQWs at 750 °C. The growth time of the GaN cap layer was 1100 s for sample H2 and 3300 s for sample H3. Here, we assumed that the thickness of the GaN layer is approximately proportional to the growth time. It indicates that the cap layer thickness of sample H3 is three times thicker than sample H2. Subsequent hydrogen treatment conditions for H2 and H3 were the same as used for sample H1. The specific growth and heat treatment conditions are shown in Three InGaN/GaN MQW samples, named A, N1, and H1, were grown on c-plane sapphire substrates through metal–organic chemical vapor deposition (MOCVD). Trimethylgallium (TMGa), trimethylindium (TMIn), and ammonia (NH3) were used as precursors in the epitaxial growth process to provide Ga, In, and N sources, respectively. All the samples were prepared with a two-step growth technology ,24, in wHigh-resolution X-ray diffraction (HRXRD) was performed to measure the indium content and thickness of the InGaN/GaN MQW structure. Temperature-dependent photoluminescence (TDPL) spectra and room-temperature photoluminescence (RTPL) spectra were measured using a 325 nm He-Cd laser in a closed-cycle helium refrigerator from CTI-Cryogenics. Meanwhile, microscopic photoluminescence (u-PL) with a high spatial resolution was performed by using a Nikon A1 confocal optical system excited with a 405 nm laser. The morphology of the samples was measured via an atomic force microscope (AFM).2 and H2 atmosphere, respectively. By fitting the experimental data with a global fit program, we can obtain the structural parameters of sample N1 and sample A as shown in 2 treatment, it is possible for In atoms to escape from their lattice site and move closer together, forming In clusters [clusters , which mTo further investigate the hydrogen heat treatment on the optical and structural properties of InGaN MQWs, temperature-dependent PL (TDPL) spectra of the two samples N1 and H1 were measured and the variations in the PL integral intensity with temperature are shown in 2 treatment. The resulting stress accumulation is considered to be the main reason for the increase in the surface roughness of sample H1. We can also see from The morphology of samples H1 and N1 is shown in 2 on the internal structure and surface morphology of InGaN/GaN MQWs were studied where the MQW structure was directly exposed to the hydrogen atmosphere. Now, we consider what can we do to decrease the decomposition of InGaN QWs if the hydrogen atmosphere is used in the practical device growth process. Could we even effectively use the hydrogen atmosphere to improve the luminescence property of MQW samples? We speculate that a thicker GaN cap layer grown on the top of the MQW layers may have two effects. The first effect is to reduce the diffusion of hydrogen into the sample; thus, only relatively less hydrogen can penetrate into the InGaN QWs. The second one is to reduce the diffusion of In atoms to the surface, which may improve the surface morphology. In the next several sections, we study two additional hydrogen-treated samples H2 and H3, which have a thicker GaN cap layer grown on the surface, to investigate their luminescence properties and morphology.In the above results, the influence of HThe structural parameters of samples H2 and H3 were obtained by HRXRD measurement, as shown in To further investigate the luminescence efficiency of samples with different cap layer thicknesses, room-temperature photoluminescence spectra were measured under the same laser power excitation and measurement conditions, as shown in Micro-PL images were measured at room temperature to investigate the uniformity of luminescence, as shown in 2 and the scan depth is −2.8 nm to +3.2 nm for samples H1 and H2 and −2.0 nm to +2.0 nm for samples H3 and A. The (100) plane is parallel to the bottom lines of the images. It is worth noting that all surfaces we measured here are GaN surfaces. The square mean root roughness (Rq) of samples H1, H2, H3, and A are 0.837 nm, 0.904 nm, 0.283 nm, and 0.499 nm, respectively. Compared with sample H1, the surface of sample H2 shows a larger step width and depth. As we discussed above, the stress accumulation increases the surface roughness. Despite the fact that a GaN cap layer is adopted to protect InGaN QWs from decomposition in sample H2, the MQW structure is still damaged and the defects and dislocations are produced. A thick GaN cap layer in sample H2 would make the stress accumulation more serious; therefore, the roughness of sample H2 is even larger than that of sample H1. It also indicates that the GaN cap layer of sample H2 cannot resist the penetration of hydrogen well. An obvious morphologic transformation from step bunching to shallower steps [er steps  has occuIn this work, the effect of the hydrogen heat treatment on the luminescence and morphology of MOCVD-grown InGaN/GaN MQWs were studied by simulating the growth conditions of p-type cladding layers in the LED device structure. It was found that the hydrogen heat treatment may lead to the decomposition of InGaN QWs and the formation of inhomogeneous In-rich clusters, which act as radiative recombination centers and cause a red shift of the luminescence peak. The defects and stress accumulation generated in the film lead to the degradation of the luminescence efficiency and an increase in surface roughness. It was also found that the hydrogen treatment can effectively reduce the number of V-defects on the surface. By growing a GaN cap layer with an appropriate thickness on MQWs, the luminescence efficiency of samples after hydrogen heat treatment can be improved strongly, indicating that the blocking effect of the cap layer on hydrogen diffusion and high temperature can effectively maintain the integrity of the MQW structure. Additionally, a morphologic transformation from step bunching to shallower steps can be obtained in the sample with a thicker cap layer.


The role of plasma functionalization of multi-walled carbon nanotubes (MWCNTs) for room-temperature ammonia gas sensors was investigated. Plasma functionalization of MWCNTs with maleic anhydride was carried out at various durations. The active material of the gas sensor was investigated by scanning electron microscopy, transmission electron microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. It was shown that the formation of functional groups on the surface of carbon nanotubes led to an increase in the ammonia sensor response by two to four times. The increase in functionalization duration induced the rise of O/C from 0.28 to 0.335, an increase in sensor resistance, and the distortion of the shape of the I-V curves. Carbon nanotubes (CNTs) have attracted interest within the last few decades and research into their synthesis, modification, and applications is still being carried out throughout the world. Multi-walled carbon nanotubes (MWCNTs) are a promising material for various applications due to their lower cost and simplicity of synthesis and scale-up compared to single-walled carbon nanotubes. MWCNTs can be used in polymer composites ,2, hydro3). Kim et al. ,,2 substr−1), which can be attributed to the disordered structure, and G mode (1589–1592 cm−1), relating to the graphitic structure of the material [2 hybridization. It can be noted that any treatment of MWCNTs can produce many defects on its surface, but the results on I(D)/I(G) confirm the relatively mild conditions of plasma functionalization compared to other types of treatment, such as chemical functionalization, as reported in [x formed as a result of the oxidation of carbon.Raman spectra of plasma-treated MWCNTs are shown in material ,25. The orted in . Plasma orted in  since thI–V curves of the active layer of ammonia gas sensors are presented in The resistance of sensors is rising with the increasing duration of treatment due to the formation of a plasma coating on the surfaces of MWCNTs. This complements the data on Raman spectroscopy and XPS. The resistance of the initial, non-treated MWCNTs was only 18 kΩ, and the plasma treatment led to an increase in resistance, to 52.7–110 kΩ. This was caused by the formation of a relatively thin film of plasma polymer on the surfaces of MWCNTs. This film possesses a higher resistance compared to carbon nanotubes and acts as an additional contact resistance between nanotubes, changing the paths for the transport of charge carriers.3 is an electron-donor compound, which donates electrons during adsorption on the surface of carbons. These electrons decrease the concentration of charge carriers in MWCNTs and the resistance of the latter increases [0 value changes.Typical curves of the sensor response are shown in ncreases ,29. This3. The plasma treatment led to the growth of the sensor response to 1.7–2.9% (2 min). The maximum response was observed at 5 min of treatment, and further growth of the duration of treatment led to a decrease in response, to 0.7–1.3% (100–500 ppm). This is in agreement with the O/C determined by XPS, which was 0.28, 0.35, and 0.335 for 2, 5, and 7 min, respectively. XPS shows no nickel on the spectra, confirming the preservation of carbon shells on the iron nanoparticles, which is a considerable difference compared to liquid-phase chemical treatment [The response of gas sensors was within the range of 0.3–0.6% for 100–500 ppm NHreatment . For fur3 sensor response. Further growth of the duration of treatment led to a decrease in the concentration of carboxyls and an increase of phenolic groups (carbon single-bonded to oxygen). This fact indicates the dominant role of carboxylic groups in the formation of the response of CNTs to NH3.All XPS spectra of the treated samples are presented in 3. The dynamics of the formation of this coating can be estimated by the values of resistance shown in Some increase in the response time may be attributed to the creation of an additional functional coating on the surface of carbon nanotubes, preventing the transportation of charge carriers from the surface of materials under contact with NHConsidering the response time of sensors, it can be noted that the response time  slightly changes after functionalization of MWCNTs . For exaThe presented results show that plasma functionalization of MWCNTs can be effective in the enhancement of ammonia detection using room-temperature gas sensors. Plasma functionalization made it possible to increase the sensor response towards ammonia by 3–4 times compared to pristine MWCNTs. The increase in the duration of plasma functionalization induced the increase of O/C from 0.28 to 0.335, an increase in sensor resistance, and distortion of the shape of the I-V curves. Plasma treatment of MWCNTs can be considered as among the most intensive types of treatment (zero-loss treatment), which maintains the material and makes it possible to achieve almost 100% yield of the material treated.


Cast austenitic chromium-nickel steel is commonly used for the manufacture of machine parts and components, which are exposed to the attack of corrosive media and abrasive wear during operation. The most commonly used grades include GX2CrNi18-9 and X10CrNi18-8 as well as GX2CrNiMo17-12-2 and X6CrNiMoNb17-12-2. To improve the abrasion resistance of cast chromium-nickel steel, primary niobium carbides were produced in the metallurgical process by increasing the carbon content and adding Fe-Nb. The microstructure of the obtained test castings consisted of an austenitic matrix and primary niobium carbides evenly distributed in this matrix. The measured hardness of the samples after heat treatment ranged from 215 to 240 HV and was higher by about 60 units than the hardness of the reference cast GX10CrNi18-9 steel, which had a hardness of about 180 HV. Compared to the reference cast steel, the abrasive wear resistance of the tested cast chromium-nickel steel (measured in Miller test) with contents of 4.4 and 5.4 wt% Nb increased only slightly, i.e., by 5% for the lower niobium content and 11% for the higher niobium content. Compared to ordinary cast GX10CrNi18-9 steel, the addition of 9.2 wt% Nb reduced the abrasive wear by almost 2.5 times. Austenitic steels and cast chromium-nickel steels are used for the manufacture of machine parts and components operating under the conditions of corrosive attack and abrasive wear. The most commonly used grades include GX2CrNi18-9 and X10CrNi18-8 as well as GX2CrNiMo17-12-2 and X6CrNiMoNb17-12-2, all of them being designed for service in numerous branches of the industry. These materials are used not only in the chemical, cellulose, and paper industries or in food processing plants, but also in heavy industry, including mining and materials processing sectors, where they operate as large-size castings often exposed to the effect of abrasion in devices such as heat exchangers, tanks, feeders, screws, and transmission pipelines. Castings made of these materials also operate in pumps and distributors for pumping out suspensions of water and sand, sludge, and also brine. Additionally, when operating as components of the drilling platform equipment, valves, tees, or clamps, they are sometimes exposed to the effect of low temperature combined with impact stresses ,4.The addition of strong carbide-forming elements such as Ti or Nb to these alloys is at the level of tenths of a percent (maximum up to 1%) and is dictated by the technological process. For example, 0.2% Ti acts as a modifier and 0.5% Nb is added to improve technological and functional properties such as weldability or corrosion resistance.The microstructure of the cast steels presented in Unlike steels that are usually austenitic, corrosion-resistant cast steels with the same chemical composition contain small amounts of delta ferrite in their microstructure. Its presence results from the slow cooling of castings and segregation processes occurring during their solidification. In castings made of the austenitic Cr-Ni steel, 5–25% of evenly distributed delta ferrite is often found in the alloy matrix. The amount of this constituent depends on the thickness of the casting wall. The presence of delta ferrite is often desirable owing to its beneficial effect on the weldability of castings and the ability to hinder the spread of microcracks and improve the resistance to intergranular and stress corrosion (it favors the precipitation of carbides inside its own grains and not at the ferrite-austenite interface) . In alloSimilar results of microscopic examinations were obtained by the authors in studies ,7,8,9,10A review of the literature discussing problems of the wear behavior of various materials indicates that current research and development activities mainly focus on the study of dual-phase and multi-phase alloys, including cast austenitic steel whose matrix is reinforced with carbides and nitrides ,12,13,14In , the resThe search for new techniques and technologies of tool production prompted the author to develop a concept that would meet the assumption that tool steels and cast steels may have satisfactory wear resistance, ductility, and crack resistance even at high or low temperatures. These properties are due to the presence of MC and M2C carbides that occur in an austenitic matrix of the alloy. An evident drawback is the presence of “coarse” (thick) MC carbides in castings, significantly deteriorating the cast steel ductility ,20,21.Following the example of tool steels, the essence of the solution proposed by the author consists in the use of a technology that allows the particles of primary carbides to be formed during metallurgical process within the entire volume of steel melt and later in the casting. In earlier research ,22,23,24The idea of introducing carbide-forming elements, successfully applied in cast high-manganese steel, and the satisfactory results of changes in microstructure and abrasion resistance obtained in the Miller test prompted the author to conduct similar research on cast chromium-nickel steel with the addition of titanium , where, The main topic discussed in the present study is the effect of niobium addition to cast Cr-Ni steel on the above-mentioned properties of the obtained alloys.2O3 crucible. The charge was 18-10 steel scrap and high-purity pig iron of known chemical composition used as a carburizer. After melting the charge, to obtain the required chemical composition, alloy additives such as the Fe-Mn65 and Fe-Si65 ferroalloys, metallic chromium, and electrolytic nickel were successively introduced. After melting the alloy additives, stirring the melt, and heating to 1600 °C, the metal was deoxidized with aluminum added in the amount of 1 g/1 kg of steel [Test melts were carried out in a Balzers VSG-02 laboratory induction vacuum furnace  using a 1 kg capacity Alof steel . In the Chemical analysis of the tested alloys was carried out under industrial conditions using a Spectro Maxx LMF04 spectrometer  and was next completed with laboratory examinations using a Spectro Midex energy-dispersive X-ray fluorescence spectrometer . Chemical analysis of the composition of test samples showed that the content of the main elements, i.e., Mn, Cr, and Ni, in the melted cast steel was comparable to the content of these elements in the reference cast GX10CrNiMo18-9 steel. High silicon content (from 0.6 to 0.9%) was the result of double deoxidation with Fe-Ca-Si and aluminum, where the content of the latter one at the level of 0.03–0.04% indicates correct deoxidation of the steel melt. The content of niobium in the three tested specimens was 4.4%, 5.4%, and 9.2% and increased with the increasing carbon content, which was 0.3%, 0.5%, and 0.8%, respectively. The Nb/C ratio was, in each case, above 10, which guarantees complete bonding of carbon into carbides and the absence of cementite in the microstructure of the tested alloys. The specimens cut out for testing were subjected to solution treatment, i.e., water cooling from the temperature of 1050 °C and holding for 30 min.Hardness was measured with a Vickers hardness tester  under a standard load of 30 kg, applied to both as-cast samples and samples after solution treatment.The microstructure of the tested alloys was examined under a Neophot 32 light microscope  equipped with a camera for digital image recording. Chemical analysis of the composition of carbides present in the tested alloys and of the alloy matrix was performed with a JEOL JSM 7100 F secondary electron (SE) field emission scanning electron microscope (SEM) made in Tokyo, Japan, and with a scanning electron microscope equipped with an EDS detector supplied by Oxford Instruments in Abingdon, UK.Phases present in the tested samples were identified with a Kristalloflex 4H X-ray diffractometer from Siemens, Munich, Germany, using the characteristic Cu radiation (Kα = 0.154 nm) with a step size of 0.052 theta/1 s.The abrasive wear response was determined in the Miller test conforming to ASTM G75, which is used to compare the abrasive wear behavior of various construction materials. The applied method of wear testing allows the author to compare the currently obtained results with the results of their own experiments conducted previously, and with the results obtained by other research teams ,29,30,31The test samples with dimensions of 25.4 × 12.7 mm and a thickness of 9 mm were placed in the holders of the device under a constant load of 22.2 N and were next subjected to abrasion in a mixture of water and silicon carbide in a 1:1 ratio. The counter-sample was the rubber lining of the trough bottom where the abrasion process took place. Silicon carbide with grain number 220 according to the FEPA standard and a grain size of 53–73 µm was used. Two 16 h abrasion tests were performed in 4 cycles for each sample, calculating next the mean. Every four hours, the samples were weighed with an accuracy of 0.001 g. Based on the obtained values of weight losses, abrasive wear curves were plotted for the tested samples. The values of wear obtained for the samples of the tested alloys were compared with the values of wear obtained for the reference sample cast from the GX10CrNi18-9 steel containing 0.1% C, 18.4% Cr, 8.3% Ni, and 0.4% Mo, subjected to standard heat treatment, i.e., solution treatment in water from the temperature of 1050 °C, and characterized by the hardness of 180 HV.The surfaces of the samples after abrasive wear tests were macroscopically compared with the surface of the reference sample cast from the GX10CrNi18-9 steel.Detailed analyses of the wear mechanism operating in the tested cast steel and of the surface condition of samples after the abrasion test require additional profilometric tests, which is a standard method used for the assessment of surface conditions after various technological and tribological operations. Examinations of the surface condition after wear test will be the subject of further studies carried out by the author.In this study, various issues related to the microstructure characterization, hardness, and wear resistance of cast austenitic Cr-Ni steels with primary niobium carbides produced in the metallurgical process are discussed.Examinations using light microscopy showed that the as-cast microstructure of the tested alloys with the niobium content of 4.4–9.2% consisted of an austenitic matrix and plate-like shaped primary niobium carbides evenly distributed in this matrix. The presence of cementite was not traced in the examined microsections. The tested samples were subjected to a heat treatment (solution treatment in water), and then their hardness was measured. The heat treatment parameters and the measured hardness values are given in Compared with as-cast samples, the solution treatment from the temperature of 1050 °C followed by cooling in water caused an increase in the obtained values of hardness, which further increased with the increasing content of carbon and niobium. In samples with the lowest content of carbon and niobium, the hardness after solution treatment increased by about 5 HV units, while for the niobium content of 5.4%, the increase in hardness amounted to about 15 HV units. The highest hardness was obtained in samples with the niobium content of 9.2%. Its value exceeded by 30 HV units the hardness obtained in as-cast state. The small scatter of the measured hardness values proves the homogeneity of the tested alloys and even distribution of carbide precipitates in the alloy matrix. This is also the reason why the average hardness values are not given in The abrasion resistance was measured in a Miller test on samples after solution treatment from the temperature of 1050 °C, as this type of heat treatment is recommended as a standard procedure for cast Cr-Ni steel, and on the sample with the highest niobium content, i.e., 9.2%, in the as-cast state. From the obtained partial weight losses of samples, the total weight loss was calculated for each test cycle, and using these data, a graph was plotted to show the cumulative weight loss of samples as a function of abrasion time. From the obtained results, it follows that the wear resistance of the tested cast chromium-nickel steel increased with the increasing content of niobium addition, but for the contents of 4.4 and 5.4%, this increase was insignificant. The value of wear decreased from 0.314 g/16 h for the reference cast steel to 0.296 g/16 h for the content of 5.4% Nb and to 0.279 g/16 h for the content of 4.4% Nb. Compared to the reference cast GX10CrNiMo18-9 steel, the increase in niobium content to 9.2% reduced the wear almost twice (even in as-cast state) and then its value amounted to 0.173 g/16 h. The lowest wear of 0.132 g/16 h was obtained for samples of this alloy after solution treatment in water.Primary niobium carbides produced in molten steel were evenly distributed in the austenitic matrix, but in the interdendritic spaces, their tendency to form small clusters was observed.No precipitation of cementite was observed in the tested samples after heat treatment.The measured as-cast hardness of the tested samples amounted to about 210 HV and was independent of both carbon content and niobium addition.After heat treatment, the hardness of the tested samples increased with the increase in niobium content, ranging from 215 to 240 HV.The formation of primary niobium carbides in the matrix of chromium-nickel cast steel increased the abrasion resistance by even 2.5 times.Carbides evenly distributed in the austenitic chromium-nickel matrix changed the wear behavior of the tested samples. Owing to their presence, the surfaces of the produced samples were free from any furrows and deep scratches.The results of the studies discussed in this article lead to the conclusion that the introduction of strongly carbide-forming elements, such as niobium, into the molten chromium-nickel steel produced primary carbides in the alloy matrix, the presence of which increased, sometimes even many times, the wear resistance of the steel. When properly satisfied, this condition allows the tested alloy to be used for new material applications, mainly the tools and components exposed to wear. As shown by the results of microstructural examinations, in the majority of cases, the obtained niobium carbides were of a faceted type. They were also characterized by a slight tendency to form clusters in the interdendritic spaces. This distribution of carbides in the steel matrix may increase the risk of crack formation in the resultant castings and reduce their impact strength. Therefore, the next stage of the research on particle-reinforced cast steel should be devoted to the development of a method of the steel melting and modification that will allow the changing of the size, shape, and distribution of primary carbides in the matrix. The promising results obtained so far allow the following conclusions to be drawn: