The 9th Chinese–Russian Workshop on Biophotonics and Biomedical Optics was held online on 28– 30 September 2020. The bilateral workshop brought together both Russian and Chinese scientists, engineers, and clinical researchers from a variety of disciplines engaged in applying optical science, photonics, and imaging technologies to problems in biology and medicine. During the workshop, 2 plenary lectures, 35 invited presentations, 5 oral presentations, and 8 internet reports were presented. This special issue selects some papers from the attendees and includes both research and review articles.The papers from this special issue will provide the readers an update on the latest developments in biophotonics and biomedical optics. This issue includes two reviews, focusing on the application of optical nanoprobes in bacterial infection by Ding et al. and the application of tissue optical clearing in diabetes-induced pathological changes by Zhu et al. In addition, thirteen original research articles are presented, covering the topics of optical imaging, tissue optical clearing, optical interactions with tissue and cells, optical techniques for clinical application.Specifically, many advanced optical imaging techniques have been developed in recent years and applied to various biomedical applications. In this issue, Wang et al. described a two-photon nonlinear SIM (2P-SIM) technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern, which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process. Kazachkina et al. performed a pilot study of the dynamics of tumor oxygenation determination using phosphorescence lifetime imaging of mesotetra (sulfopheny1) tetrabenzoporphyrin Pd (II) (TBP), and observed a low oxygen content with increased phosphorescence lifetime of TBP in the tumor. Mylnikov et al. used the fluorescence imaging methods to reveal the indicated forms of tumor cell death under the combined effect of flavonoidcontaining extract of Gratiola officinalis and cytostatic, and found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect. In addition, for the label-free imaging, Dyachenko et al. used laser speckle contrast imaging to monitor acute pancreatitis at ischemia-reperfusion of pancreas in rats. Zou et al. applied the OCT to investigate the influence of different sized nanoparticles and thermal coagulation-induced changes in the optical properties of normal, benign, and cancerous human breast tissues. Liu et al. presented an adaptive Watershed algorithm to automatically extract foveal avascular zone (FAZ) from retinal optical coherence tomography angiography (OCTA) images. Liang et al. revealed the underlying mechanisms of artifacts for thermoacoustic imaging (TAI) by investigating the specific absorption rate (SAR) distribution inside tissue phantoms, and showed its high dependence with the geometries of the imaging targets and the polarizing features of the microwave.Tissue optical clearing technique has become a powerful tool in deep tissue detection. The underlying mechanisms of optical clearing will help better understanding and application of the clearing protocols. In this issue, Genin et al. performed complex study of glycerol effects on rat skin tissue from different aspects involving the optical, weight and geometrical properties, and discussed the possible mechanism under the action of glycerol solutions. Jaafar et al. presented an investigation of optical clearing agents’ in°uence on probing depth using porcine skin with confocal Raman microspectroscopy (CRM). In addition, Kozintseva et al. studied the time dependence of optical clearing by monitoring the luminescence intensity of the upconverting particles (UCNPs), and demonstrated the possibility to use the UCNPs for studying the dynamics of optical clearing of biological tissues under local compression.The optical interactions with tissue and cells are hotspots in biomedical photonics. In this issue, Gyulkhandanyan et al. determined and tested the most effective meso-substituted cationic pyridylporphyrins and metalloporphyrins with high photoactivity against Gram negative and Gram positive microorganisms. Kapkov et al. also used the laser tweezers for quantitative measurement of interaction forces between red blood cells (RBSs) and endothelial cells (ECs) in stationary conditions. They showed that the interaction force raises along with increasing concentration of fibrinogen and dextran in all considered cases of ECs interaction with RBCs.The photonics plays important roles in not only fundamental researches but also clinical diagnosis. In this issue, Hou et al. applied the Muller matrix microscope to distinguish microstructural features between high-grade cervical squamous intraepithelial lesion (HSIL) and cervical squamous cell carcinoma (CSCC), and presented results from 37 clinical patients with analysis regions of cervical squamous epithelium. This work provides an efficient method for digital pathological diagnosis and points out a new way for automatic screening of pathological sections.This issue provides a broad and frontier view about the recent developments in biophotonics and biomedical optics, hence we strongly recommend this issue.It should be noted that this special issue is just a part of the Chinese–Russian workshop on Biophotonics and Biomedical Optics 2020. Some of the above papers will be published in the first issue of next year. Finally, we thank all the contributing authors for making this issue possible.

Bacterial infection is an acute infection caused by pathogens or conditional pathogens, which leads to severe disease and even death. It has become a significant reason for diseases and deaths worldwide. Therefore, rapid and precise detection, diagnosis, and treatment in the early stage are the key to deal with bacterial infections. Over recent years, along with the advances in biomaterials and nanotechnology, numerous nanomaterial-based multifunctional probes have been extensively explored in the biomedical field. Because of their excellent optical properties, inorganic optical nanoprobes are used to rapidly detect bacterial infection in the early stage and show excellent antibacterial properties, which has a great application prospect in antibacterial therapy expected to reduce the risk of bacterial infection. In this mini-review, we generally overviewed and summarized recent progress on inorganic nanoparticle-based optical imaging techniques as a platform to construct functional theranostics for the efficient treatment of bacterial infections. The opportunities and challenges in the application of fluorescent optical nanoprobes are prospected.

Oxygenation of tissues plays an important role in the development and progression of tumor to treatment effects. Method of metalloporphyrines phosphorescence quenching by oxygen is one of the ways to measure dynamics of the oxygen concentration in the tissues by phosphorescence lifetime imaging of meso-tetra(sulfopheny1)tetrabenzoporphyrin Pd (II) (TBP) using the timecorrelated single photon counting (TCSPC) method. It has been shown that phosphorescence lifetime of the sensor in S37 tumor in vivo varied in the range of 130 to 290 μs after both topical and intravenous administration of TBP. It indicates that oxygen level in tumors was lower compared to normal tissues where TBP phosphorescence has not been detected. Phosphorescence lifetimes of TBP increased in the solid tumor and in the muscle after photodynamic therapy of solid tumor that demonstrates oxygen consumption during treatment and possibly stopping the blood flow and hence the oxygen supply to the tissues.

Structured illumination microscopy (SIM) is an essential super-resolution microscopy technique that enhances resolution. Several images are required to reconstruct a super-resolution image. However, linear SIM resolution enhancement can only increase the spatial resolution of microscopy by a factor of two at most because the frequency of the structured illumination pattern is limited by the cutoff frequency of the excitation point spread function. The frequency of the pattern generated by the nonlinear response in samples is not limited; therefore, nonlinear SIM (NL-SIM), in theory, has no inherent limit to the resolution. In the present study, we describe a two-photon nonlinear SIM (2P-SIM) technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern, which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process. The theoretical models of super-resolution imaging were established through our simulation, which describes the working mechanism of the multi-frequency structure of the nonsinusoidal function to improve the resolution. The simulation results predict that a 5-fold improvement in resolution in the 2P-SIM is possible.

Confocal Raman microspectroscopy (CRM) with 633- and 785-nm excitation wavelengths combined with optical clearing (OC) technique was used for ex-vivo study of porcine skin in the Raman fingerprint region. The optical clearing has been performed on the skin samples by applying a mixture of glycerol and distilled water and a mixture of glycerol, distilled water and chemical penetration enhancer dimethyl sulfoxide (DMSO) during 30 min and 60 min of treatment. It was shown that the combined use of the optical clearing technique and CRM at 633nm allowed one to preserve the high probing depth, signal-to-noise ratio and spectral resolution simultaneously. Comparing the effect of different optical clearing agents on porcine skin showed that an optical clearing agent containing chemical penetration enhancer provides higher optical clearing efficiency. Also, an increase in treatment time allows to improve the optical clearing efficiency of both optical clearing agents. As a result of optical clearing, the detection of the amide-III spectral region indicating well-distinguishable structural differences between the type-I and type-IV collagens has been improved.

Objective of the study: We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavonoid-containing extract of Gratiola officinalis and cytostatic (cyclophosphamide). Materials and methods: The dyes were propidium iodide and acridine orange, which were used in the "alive and dead" test. This test helped us to identify the total number of dead cells in the forms of necrosis and apoptosis and the number of cells in which apoptosis had started, it was characterized by the appearance of apoptotic bodies or nucleus pyknosis. Results: We found the most pronounced cytotoxic activity at the ratio of extract of Gratiola officinalis and cyclophosphamide concentrations of 1:1. The number of living cells decreased when exposed to the ratio of extract and cytostatic concentrations of 2:1. When the ratio of concentration of the extract relative to the cytostatic increased to 3:1, the cytostatic activity of the extract began to appear, the total number of tumor cells decreased. The number of cells with nucleus pyknosis and the number of cells with apoptosis signs significantly increased at a 3:1 ratio of extract and cytostatic concentrations, which confirms the presence of pro-apoptotic activity of the studied combination. This trend indicates the dependence of a certain form of cell death (apoptosis, necrosis) on the ratio of extract and cytostatic doses, and it also demonstrates the cytostatic and cytotoxic effects of this combination. Conclusion: Fluorescence methods of investigation in the "alive and dead" test allowed us to visualize the forms of cell death of human kidney carcinoma A498 by combined exposure to the flavonoid-containing extract of Gratiola officinalis and cytostatic (cyclophosphamide) 24 h after exposure. We found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect.

Red blood cells (RBCs) are able to interact and communicate with endothelial cells (ECs). Under some pathological or even normal conditions, the adhesion of RBCs to the endothelium can be observed. Presently, the mechanisms and many aspects of the interaction between RBCs and ECs are not fully understood. In this work, we considered the interaction of single RBCs with single ECs in vitro aiming to quantitatively determine the force of this interaction using laser tweezers. Measurements were performed under different concentrations of proaggregant macromolecules and in the presence or absence of tumor necrosis factor (TNF-α) activating the ECs. We have shown that the strength of interaction depends on the concentration of fibrinogen or dextran proaggregant macromolecules in the environment. A nonlinear increase in the force of cells interaction (from 0.4 pN to 21 pN) was observed along with an increase in the fibrinogen concentration (from 3 mg/mL to 9 mg/mL) in blood plasma, as well as with the addition of dextran macromolecules (from 10 mg/mL to 60 mg/mL). Dextran with a higher molecular mass (500 kDa) enhances the adhesion of RBCs to ECs greater compared to the dextran with a lower molecular mass (70 kDa). With the preliminary activation of ECs with TNF-α, the force of interaction increases. Also, the adhesion of echinocytes to EC compared to discocytes is significantly higher. These results may help to better understand the process of interaction between RBCs and ECs.

Complex study of glycerol effects on the skin tissue was performed. The change in optical, weight and geometrical parameters of the rat skin under the action of the glycerol solutions was studied ex vivo. Possible mechanisms of the skin optical clearing under the action of glycerol solutions of different concentrations were discussed. The results can be helpful for refinement of models developed to evaluate the effective diffusion coefficients of glycerol in tissues.

The current work is focused on the study of optical clearing of skeletal muscles under local compression. The experiments were performed on in vitro bovine skeletal muscle. The time dependence of optical clearing was studied by monitoring the luminescence intensity of NaYF4: Er,Yb upconverting particles located under tissue layers. This study shows the possibility to use upconverting nanoparticles (UCNPs) both for studying the dynamics of the optical clearing of biological tissue under compression and to detect moments of cell wall damage under excessive pressure. The advantage of using UCNPs is the presence of several bands in their luminescence spectra, located both at close wavelengths and far apart.

Microwave-induced thermoacoustic imaging (MI-TAI) remains one of the focus of attention among biomedical imaging modalities over the last decade. However, the transmission and distribution of microwave inside bio-tissues are complicated, thus result in severe artifacts. In this study, to reveal the underlying mechanisms of artifacts, we deeply investigate the distribution of specific absorption rate (SAR) inside tissue-mimicking phantoms with varied morphological features using both mathematical simulations and corresponding experiments. Our simulated results, which are confirmed by the associated experimental results, show that the SAR distribution highly depends on the geometries of the imaging targets and the polarizing features of the microwave. In addition, we propose the potential mechanisms including Mie-scattering, Fabry- Perot-feature, small curvature effect to interpret the diffraction effect in different scenarios, which may provide basic guidance to predict and distinguish the artifacts for TAI in both fundamental and clinical studies.