STORM imaging of mitochondrial dynamics using a vicinal-dithiol-proteins-targeted probe.

Stochastic optical reconstruction microscopy (STORM) is a promising method for the visualization of ultra-fine mitochondrial structures. However, this approach is limited to monitoring dynamic intracellular events owing to its low temporal resolution. We developed a new strategy to capture mitochondrial dynamics using a compressed sensing STORM algorithm following raw data pre-treatments by a noise-corrected principal component analysis and K-factor image factorization. Using STORM microscopy with a vicinal-dithiol-proteins targeting probe, visualizing mitochondrial dynamics was attainable with spatial and temporal resolutions of 45 nm and 0.8 s, notably, dynamic mitochondrial tubulation retraction of ~746 nm in 1.2 s was monitored. The labeled conjugate was observed as clusters (radii, ~90 nm) distributed on the outer mitochondrial membranes, not yet reported as far as we know. This strategy is promising for the quantitative analysis of intracellular behaviors below the optical diffraction limit.

Nanoliposomes Co-Encapsulating Photoswitchable Probe and Photosensitizer for Super-Resolution Optical Imaging and Photodynamic Therapy.

Photosensitizers (PSs) are ideal cancer theranostic drugs that can be administered as both fluorescence imaging reagents and photodynamic therapy (PDT) drugs. To improve the tumoritropic behavior of PSs, nanoliposomes are presently being considered as optimal PSs carriers. Although nanoliposomal PSs have been utilized in clinical therapy, PSs localization and photosensitive processing in nanoliposomal PSs are rarely observed on nanoscale. Investigating changes in the fine structure of nanoliposomes under photosensitive processing will further our understanding of the photosensitive effect on nanoliposomal PSs. In this study, nanoliposomes co-encapsulating the PSs benzoporphyrin derivative monoacid A (BPD) and the photoswitchable probe Cy5-927 were prepared to realize PDT and nanoscale super-resolution optical imaging. The fine structures of nanoliposomal BPD and Cy5-927 (LBC) were visualized by a home-built stochastic optical reconstruction microscopy (STORM). Our PDT results showed that the photorelease and PDT efficiency of BPD were not decreased by co-encapsulating with Cy5-927 in LBC. Taken together, LBC can be used as a new optical probe and PDT reagent for investigating changes in nanoliposomes fine structure and micro-interaction in the cellular process of PDT. Therefore, our results deepened our understanding of liposome-based PDT for optimizing cancer treatment. © 2019 International Society for Advancement of Cytometry.

Solo Smart Fluorogenic Probe for Potential Cancer Diagnosis and Tracking in Vivo Tumorous Lymphatic Systems via Distinct Emission Signals.

A versatile twisted-intramolecular-charge-transfer (TICT)-based near-infrared (NIR) fluorescent probe (L) has been judiciously designed and synthesized that could be utilized for potential cancer diagnosis and to track lymph node(s) in mice through distinct emission signals. Essentially, the probe rendered the capability to preferentially recognize the cancer cells over the noncancer cells by polarity-guided lipid droplet specific differential bioimaging (in green emission channel) studies. The probe also exhibited selective turn-on fluorescence response toward HSA/BSA in physiological media (aqueous PBS buffer; pH 7.4) at far-red/NIR regions, because of the 1:1 chelation between the probe and HSA/BSA. Therefore, the fluorescent probe was then maneuvered to track the draining lymphatic system and sentinel lymph node in tumor mice model by fluorescence imaging (NIR/deep-red channel), wherein the accumulated albumin protein in the draining tumor lymphatic system facilitated the in situ formation of the fluorescent albumin-L complex.

Support Vector Machine Classification of Nonmelanoma Skin Lesions Based on Fluorescence Lifetime Imaging Microscopy.

Early diagnosis of malignant skin lesions is critical for prompt treatment and a clinical prognosis of skin cancers. However, it is difficult to precisely evaluate the development stage of nonmelanoma skin cancers because they are derived from the same tissues as a result of the uncontrolled growth of abnormal squamous keratinocytes in the epidermis layer of the skin. In the present study, we developed a linear-kernel support vector machine (LSVM) model to distinguish basal cell carcinoma (BCC) from actinic keratosis (AK) and Bowen's disease (BD). The input parameters of the LSVM model consist of appropriate lifetime components and entropy values, which were extracted from two-photon fluorescence lifetime imaging of hematoxylin and eosin (H&E)-stained biopsy sections. Different features used as inputs for SVM training were compared and evaluated. In constructing the SVM models, features obtained from the lifetime (τ2) of the second component were found to be significantly more predictive than the average fluorescence lifetime (τm) in terms of diagnostic accuracy, sensitivity, and specificity. The above findings were confirmed on the basis of the receiver operating characteristic (ROC) curves of diagnostic models. Shannon entropy was added to the SVM models as an independent feature to further improve the diagnostic accuracy. Therefore, fluorescence lifetime analysis and entropy calculations can provide highly informative features for the accurate detection of skin neoplasm disorders. In summary, fluorescence lifetime imaging microscopy (FLIM) combined with the SVM classification exhibited great potential for developing an effective computer-aided diagnostic criterion and accurate cancer detection in dermatology.

Third-Party Allogeneic Mesenchymal Stromal Cells Prevent Rejection in a Pre-sensitized High-Risk Model of Corneal Transplantation.

High-risk cornea transplant recipients represent a patient population with significant un-met medical need for more effective therapies to prevent immunological graft rejection due to heightened anti-donor immune response. In this study, a rat model of pre-existing anti-donor immunity was developed in which corneal allografts were rejected earlier than in non-pre-sensitized recipients. In this model, third-party (non-donor, non-recipient strain) allogeneic mesenchymal stromal cells (allo-MSC) were administered intravenously 7 and 1 days prior to transplantation. Rejection-free graft survival to 30 days post-transplant improved from 0 to 63.6% in MSC-treated compared to vehicle-treated control animals (p = < 0.0001). Pre-sensitized animals that received third-party allo-MSC prior to transplantation had significantly higher proportions of CD45+CD11b+ B220+ monocytes in the lungs 24 h after the second MSC injection and significantly higher proportions of CD4+ FoxP3+ regulatory T cells in the graft-draining lymph nodes at the average day of rejection of control animals. In in vitro experiments, third-party allo-MSC polarized primary lung-derived CD11b/c+ myeloid cells to a more anti-inflammatory phenotype, as determined by cytokine profile and conferred them with the capacity to suppress T cell activation via prostaglandin E2 and TGFß1. In experiments designed to further validate the clinical potential of the protocol, thawed cryopreserved, third-party allo-MSC were shown to be similarly potent at prolonging rejection-free corneal allograft survival as their freshly-cultured counterparts in the pre-sensitized high-risk model. Furthermore, thawed cryopreserved third-party allo-MSC could be co-administered with mycophenolate mofetil without adversely affecting their immunomodulatory function. In conclusion, a clinically-relevant protocol consisting of two intravenous infusions of third-party allo-MSC during the week prior to transplantation, exerts a potent anti-rejection effect in a pre-sensitized rat model of high-risk corneal allo-transplantation. This immune regulatory effect is likely to be mediated in the immediate post-transplant period through the promotion, by allo-MSC, of alternatively-activated macrophages in the lung and, later, by enhanced regulatory T-cell numbers.

Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that has achieved significant results in breaking the resolution limit and relevant applications. In principle, STED super resolution is obtained by stimulated emission partially inhibiting the spontaneous emission in the periphery of a diffraction-limited area. However, very high depletion laser power is generally necessary for the enhancement of imaging resolution, which is harmful to live biological specimens due to its high phototoxicity and photo-bleaching effects. Therefore, further improving the STED resolution at a lower depletion power level has recently attracted increasing interest from researchers in various fields. In this work, a phasor plot approach combined with fluorescence lifetime imaging microscopy (FLIM) is used to resolve the abovementioned problem based on a long- and short-lifetime criterion. Firstly, the time-resolved data obtained by STED-FLIM is converted to the frequency domain via a phasor approach. Next, partial data is extracted according to the information on the phase and amplitude for resolution improvement. Then, fluorescent microspheres (100 nm in diameter) are observed under different depletion powers, resulting in a series of improved resolution through phasor plots. Finally, this method is applied to image human Nup153 in fixed HeLa cells, providing a 86 nm higher resolution than that in traditional STED imaging at a depletion power of 20 mW.

OCT imaging detection of brain blood vessels in mouse, based on semiconducting polymer nanoparticles.

Optical Coherence Tomography (OCT) is a valuable technology that has been used to obtain microstructure images of tissue, and has several advantages, though its applications are limited in high-scattering tissues. Therefore, semiconducting polymer nanoparticles (SPNs) that possess strong absorption characteristics are applied to decrease light scattering in tissues and used as exogenous contrast agents for enhancing the contrast of OCT imaging detection. In this paper, we prepared two kinds of SPNs, termed PIDT-TBZ SPNs and PBDT-TBZ SPNs, as the contrast agents for OCT detection to enhance the signal. Firstly, we proved that they were good contrast agents for OCT imaging in agar-TiO2. After that, the contrast effects of these two SPNs were quantitatively analyzed, and then cerebral blood vessels were monitored by a home-made SD-OCT system. Finally, we created OCT images in vitro and in vivo with these two probes and performed quantitative analysis using the images. The results indicated that these SPNs created a clear contrast enhancement of small vessels in the OCT imaging process, which provides a basis for the application of SPNs as contrast agents for bioimaging studies.

Individual traveller health priorities and the pre-travel health consultation.

The purpose of this study was to examine the principal travel health priorities of travellers. The most frequently selected travel health concerns were accessing medical care abroad, dying abroad, insect bites, malaria, personal safety and travel security threats. The travel health risks of least concern were culture shock, fear of flying, jet lag and sexually transmitted infections. This study is the first to develop a hierarchy of self-declared travel health risk priorities among travellers.

Controllable Virus-Directed Synthesis of Nanostructured Hybrids Induced by Organic/Inorganic Interactions.

The bioinspired synthesis of hierarchical hybrid nanomaterials using biological objects as a template attracts growing interest for the design of new technologically relevant nanostructured materials. To ensure control over the shape and properties of the fabricated hybrid structures, understanding of the growth mechanism is required. In this work, tobacco mosaic virus (TMV) is used as a template to direct the synthesis of zinc sulfide (ZnS) at ambient conditions and different pH from additive-free aqueous solution. TMV/ZnS hybrid nanowires or thin films are obtained with controllable thickness of the inorganic layer. The deposition mechanism is studied by monitoring the optical properties, band gap (Eg ) and particle size, respectively, of ZnS particles mineralized on the TMV template and ZnS reference nanoparticles. A heterogeneous nucleation of the inorganic phase on the template surface is proposed. Band gap measurements reveal that the average size of the ZnS nanoparticles grown on the virus surface is smaller compared to solution-grown nanoparticles. Moreover, a blue shift of the ZnS photoluminescence peak indicates a dominance of different crystal lattice defects in both systems. The present method for the selective template-directed mineralization opens new possibilities in the synthesis of well-organized functional hybrid materials.

Quantitative measurement of optical parameters of cell lines 5-8F and 6-10B using polarization sensitive optical coherence tomography.

The aim was to test whether the typical NPC cell lines of 5-8F (high tumorigenesis and metastasis) and 6-10B (low tumorigenesis and metastasis) could be differentiated by polarization sensitive optical coherence tomography (PS-OCT). We imaged the two types of low cellular differentiated NPC cell lines 5-8F and 6-10B pellets using PS-OCT; then extracted the optical parameters of attenuate coefficient and anisotropy from the A-scan lines based on the multiple scattering model; and compared their phase retardation. The fitting scattering coefficients were µs=10.91±0.45 and µs=11.33±0.27 cm(-1) for 5-8F and 6-10B pellets (p<0.05), respectively; and the anisotropy factors were g=0.900±0.013 and g=0.885±0.008 for 5-8F and 6-10B pellets (p<0.01), respectively. While the phase retardation of 6-10B was a little faster than 5-8F. These results indicated that PS-OCT could differentiate the two cell lines, and had the potential ability for typing the tissue of NPC.

Quantitative discrimination of NPC cell lines using optical coherence tomography.

We tried to explore the intrinsic differences in the optical properties of the four representative NPC cell lines on the models of radiobiology and metastasis by OCT. The scattering coefficients and anisotropies were extracted by fitting the average a-scan attenuation curves based on the multiple scatter effect. The values of scattering coefficients and anisotropy factors were 5.21 ± 0.11, 5.30 ± 0.09, 5.92 ± 0.21, 6.97 ± 0.22, and 0.892 ± 0.009, 0.886 ± 0.006, 0.884 ± 0.009, 0.86 ± 0.01 for CNE1, CNE2, 5-8F and 6-10B pellets (p < 0.05, P = 0.07 for CNE1 and CNE2), respectively. The results showed that the radiobiology and metastasis cell's model could be distinguished obviously; which implied that the corresponding types of NPC tissue might be potentially differentiated by OCT.