Im-SCC-FRET: improved single-cell-based calibration of a FRET system.

We recently developed a SCC-FRET (single-cell-based calibration of a FRET system) method to quantify spectral crosstalk correction parameters (ß and δ) and system calibration parameters (G and k) of a Förster resonance energy transfer (FRET) system by imaging a single cell expressing a standard FRET plasmid with known FRET efficiency (E) and donor-acceptor concentration ratio (RC) (Liu et al., Opt. Express30, 29063 (2022)10.1364/OE.459861). Here we improved the SCC-FRET method (named as Im-SCC-FRET) to simultaneously obtain ß, δ, G, k and the acceptor-to-donor extinction coefficient ratio (ε A ε D), which is a key parameter to calculate the acceptor-centric FRET efficiency (EA), of a FRET system when the range of ß and δ values is set as 0-1. In Im-SCC-FRET, the target function is changed from the sum of absolute values to the sum of squares according to the least squares method, and the initial value of ß and δ estimated by the integral but not the maximum value spectral overlap between fluorophore and filter. Compared with SCC-FRET, the experimental results demonstrate that Im-SCC-FRET can obtain more accurate and stable results for ß, δ, G, and k, and add the ratio ε A ε D, which is necessary for the FRET hybrid assay. Im-SCC-FRET reduces the complexity of experiment preparation and opens up a promising avenue for developing an intelligent FRET correction system.

Optical section structured illumination-based Förster resonance energy transfer imaging.

Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were EC32VOSF=0.32±0.02,EC17VOSF=0.38±0.02 , and EC5VOSF=0.45±0.03 , and the measured acceptor-to-donor concentration ratios ( Rc ) were RC32VOSF=1.07±0.03 , RC17VOSF=1.09±0.03 , and RC5VOSF=1.02±0.04 , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.

Automated ExEm-spFRET Microscope.

Excitation­emission-spectral unmixing-based fluorescence resonance energy transfer (ExEm-spFRET) microscopy exhibits excellent robustness in living cells. We here develop an automatic ExEm-spFRET microscope with 3.04 s of time resolution for a quantitative FRET imaging. The user-friendly interface software has been designed to operate in two modes: administrator and user. Automatic background recognition, subtraction, and cell segmentation were integrated into the software, which enables FRET calibration or measurement in a one-click operation manner. In administrator mode, both correction factors and spectral fingerprints are only calibrated periodically for a stable system. In user mode, quantitative ExEm-spFRET imaging is directly implemented for FRET samples. We implemented quantitative ExEm-spFRET imaging for living cells expressing different tandem constructs (C80Y, C40Y, C10Y, and C4Y, respectively) and obtained consistent results for at least 3 months, demonstrating the stability of our microscope. Next, we investigated Bcl-xL-Bad interaction by using ExEm-spFRET imaging and FRET two-hybrid assay and found that the Bcl-xL-Bad complexes exist mainly in Bad-Bcl-xL trimers in healthy cells and Bad-Bcl-xL2 trimers in apoptotic cells. We also performed time-lapse FRET imaging on our system for living cells expressing Yellow Cameleon 3.6 (YC3.6) to monitor ionomycin-induced rapid extracellular Ca2+ influx with a time interval of 5 s for total 250 s.

Measuring calibration factors by imaging a dish of cells expressing different tandem constructs plasmids.

Three-cube Förster resonance energy transfer (FRET) method is the most extensively applied approach for live-cell FRET quantification. Reliable measurements of calibration factors are crucial for quantitative FRET measurement. We here proposed a modified TA-G method (termed as mTA-G) to simultaneously obtain the FRET-sensitized quenching transition factor (G) and extinction coefficients ratio (γ) between donor and acceptor. mTA-G method includes four steps: (1) predetermining the ratio ranges of the sensitized emission of acceptor (FC ) to the donor excitation and donor channel image (IDD [(DA])) for all FRET plasmids; (2) culturing the cells which express every FRET plasmid in one dish respectively; (3) distinguishing and marking the cells expressing different FRET plasmids by detecting their FC /IDD (DA) values; (4) linearly fitting FC /IAA (DA) (acceptor excitation and acceptor channel image) to IDD (DA)/IAA (DA) for different kinds of cells. We implemented mTA-G method by imaging tandem constructs cells with different FRET efficiency cultured in one dish on different days, and obtained consistent G and γ values. mTA-G method not only circumvents switchover of different culture dishes but also keep the constant imaging conditions, exhibiting excellent robustness, and thus will expands the biological applications of quantitative FRET analysis in living cells.

Quantitative Fluorescence Resonance Energy Transfer Analysis on the Direct Interaction of Activation-2b with Histone H3/Switch-3B Protein in Arabidopsis Mesophyll Protoplasts.

Interaction between the alteration/deficiency in activation-2b (ADA2b) and histone H3/switch-3B (SWI3B) proteins was evaluated in arabidopsis mesophyll protoplasts by quantitative fluorescence resonance energy transfer (FRET) analysis. Microscopic image showed that ADA2b, SWI3B and H3 proteins colocalized in nucleus, and quantitative FRET measurements showed 0.31 of FRET efficiency (E) for the protoplasts coexpressing ECFP-ADA2b and EYFP-SWI3B, and 0.285 of E for the protoplasts coexpressing ECFP-H3 and EYFP-ADA2b, demonstrating the direct interaction of ADA2b with SWI3B/H3 protein. Collectively, SWI3B and H3 proteins are the inherent components of the ADA2b complex in which ADA2b directly interacts with SWI3B/H3 protein.

Study of the molecular variation in pre-eclampsia placenta based on micro-Raman spectroscopy.

PURPOSE: Study of the molecular variation in pre-eclampsia placenta based on micro-Raman spectroscopy. METHODS: Five pregnant women with pre-eclampsia from Nanfang hospital were selected as study group whose average age is 28.5 years and 38 ± 2 weeks gestation. The same period of healthy pregnant women, whose average age is 27.6 years and pregnant 39 ± 1 weeks, as control group (n = 5). The normal and pre-eclamptic placental tissues are detected by micro-Raman spectroscopy with the spectrum resolution of 1 cm(-1). RESULTS: We find that the protein structure of α-helix, ß-pleated sheet and ß-turn is overlying in pre-eclamptic placenta, which lead to a disorder of protein structure. The Raman peaks assigned to tryptophan indole ring and phenylalanine in pre-eclamptic placental tissue are more higher than that in normal tissue. CONCLUSIONS: Results suggest that the ordered structures of the main chain in protein molecules are reduced significantly, and the amino acid of side chains is damaged obviously. And a principal component analysis is used to classify the Raman spectra between normal and pre-eclamptic placental tissues. This study presents that Raman spectroscopy has a great potential on the mechanism research and diagnosis of placental lesions.

Nuclear export of PML promotes p53-mediated apoptosis and ferroptosis.

Promyelocytic leukemia protein (PML), a tumor suppressor protein, plays a key role in cell cycle regulation, apoptosis, senescence and cellular metabolism. Here, we report that PML promotes apoptosis and ferroptosis. Our data showed that PML over-expression inhibited cell proliferation and migration. PML over-expression increased apoptotic cells, nuclear condensation and the loss of mitochondrial membrane potential, accompanied by regulation of Bcl-2 family proteins and reactive oxygen species (ROS) level, suggesting that PML enhanced apoptosis. Meanwhile, PML over-expression not only increased lipid ROS accumulation and Malondialdehyde (MDA) content but also downregulated solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression, indicating that PML enhanced ferroptosis. Additionally, knockdown of p53 attenuated the effect of PML on SLC7A11 and GPX4, and inhibited the increase of lipid ROS and ROS by PML over-expression. Moreover, translocation of PML from nucleus to cytoplasm not only promoted apoptosis and ferroptosis, but also inhibited cell proliferation. Taken together, PML promotes apoptosis and ferroptosis, in which the mediation of p53 and the nuclear export of PML play important roles.

Melatonin mediates protective effects on inflammatory response induced by interleukin-1 beta in human mesenchymal stem cells.

Joint diseases like osteoarthritis usually are accompanied with inflammatory processes, in which pro-inflammatory cytokines mediate the generation of intracellular reactive oxygen species (ROS) and compromise survival of subchondral osteoblasts. Melatonin is capable of manipulating bone formation and osteogenic differentiation of mesenchymal stem cells (MSCs). The aim of this work was to investigate the anti-inflammatory effect of melatonin on MSC proliferation and osteogenic differentiation in the absence or presence of interleukin-1 beta (IL-1ß), which was used to induce inflammation. Our data showed that melatonin improved cell viability and reduced ROS generation in MSCs in a dose-dependent manner. When exposed to 10 ng/mL IL-1ß, various concentrations of melatonin resulted in significant reduction of ROS by 34.9% averagely. Luzindole as a melatonin receptor antagonist reversed the anti-oxidant effect of melatonin in MSCs with co-exposure to IL-1ß. Real-time RT-PCR data suggested that melatonin treatment up-regulated the expression of CuZnSOD and MnSOD, while down-regulated the expression of Bax. To investigate the effect of melatonin on osteogenesis, MSCs were cultured in osteogenic differentiation medium supplemented with IL-1ß, melatonin, or luzindole. After exposed to IL-1ß for 21 days, 1 µm melatonin treatment significantly increased the levels of type I collagen, ALP, and osteocalcin, and 100 µm melatonin treatment yielded the highest level of osteopontin. Our study demonstrated that melatonin maintained MSC survival and promoted osteogenic differentiation in inflammatory environment induced by IL-1ß, suggesting melatonin treatment could be a promising method for bone regenerative engineering in future studies.

Dependence of ghost on the incident light angle into dichroic mirror.

The dichroic mirror (DM) is a key component in microscope. We found a ghost in the reflection channel of a dual-channel fluorescence microscope and studied the relationship between the ghost and the incidence angle θ into the DM. The DM emission surface reflection generated ghost if the θ is not 45 ° . We analyzed the distance and intensity relationship between the ghost and the primary image, which is θ -dependent and was demonstrated by imaging live cells and a stage micrometer. The ghost can be eliminated by placing the DM between objective and tube lens, but not between tube lens and detector, ensuring that the incident light into the DM is approximately parallel. Furthermore, the transmitted light of the DM is shifted towards a longer wavelength with increasing θ . Collectively, microscopists must carefully optimize the θ when designing a microscope to avoid the ghost.

[Nonlinear spectral imaging of DNA specimens derived from tumor cells based on second harmonic generation].

Second harmonic generation (SHG) is a second-order nonlinear optical process that has symmetry constraints confining signal to regions lacking a center of symmetry. Using SHG microscopy, a variety of tissue structures have noninvasively been imaged by virtue of intrinsic signal generated by structured proteins such as collagen fibrils in connective tissues or the actomyosin lattice of muscle cells. In biochemistry and structure biology, the high-level structures of DNA and protein macro-molecules are similar in constructing mechanism, although DNAs consist of deoxynucleotides and proteins of amino acid residues. The principal purpose of present work is to detect the SHG signal from different DNA samples by spectral imaging technology based on two-photon excited fluorescence (TPEF) and SHG. These DNA samples include the solution of genomic DNA and extracted nuclei, and cultured living cells. Results show that we can obtain the SHG signal from solution of genomic DNA and extracted nuclei in routine condition, but nothing from cultured cell nuclei. After adding a little of absolute ethanol (less than 5% by volume) in culture medium, the SHG signal is detectable in the interested region of nuclei. The findings suggest that the interaction between ethanol and DNA in living cell gives rise to the shift of molecular conformation, and this shift changes some nonlinear optical properties of DNA molecules.

[Raman spectroscopic study on the influence of ultraviolet-A radiation on collagen I].

Raman spectroscopy was used to study the influence of ultraviolet-A(UV-A) radiation on collagen I. The Raman spectra of collagen I and that after 90 min UV-A radiation were reported. The results proved that irradiation with 90 min UV-A caused the change in the structures of collagen I. Intramolecular hydrogen bonds were broken, and the hydrogen bonding system was changed. The intensity of helix was decreased, while the intensity of the disordered conformation in proteins such as random coil was increased. Otherwise, the UV-A radiation influenced the hydroxylation of proline and the content of hydroxyproline was reduced. The changes caused by UV-A radiation could damage the triple helical structure of collagen I. It would lead to a series of changes, such as the destruction of collagen fibers during the photoaging of skin.

Photo-induced synthesis of molybdenum oxide quantum dots for surface-enhanced Raman scattering and photothermal therapy.

By means of a simple and photo-induced method, four colors of molybdenum oxide quantum dots (MoOx QDs) have been synthesized, using Mo(CO)6 as the structural guiding agent and molybdenum source. The as-prepared MoOx QDs display diverse optical properties due to the different configurations of oxygen vacancies in various nanostructures. Among them, crystalline molybdenum dioxide (MoO2) with a deep blue color shows the most intense localized surface plasmon resonance effect in the near-infrared (NIR) region. The strong NIR absorption endows MoO2 QDs with a high photothermal conversion efficiency of 66.3%, enabling broad prospects as a photo-responsive nanoagent for photothermal therapy of cancer. Moreover, MoO2 QDs can also serve as a novel semiconductor substrate for ultrasensitive surface-enhanced Raman scattering (SERS) analysis of aromatic molecules, amino acids and antibiotics, with SERS performance comparable to that of noble metal-based substrates. The therapeutic applications of MoO2 QDs open up a new avenue for tumor nanomedicine.

Biodegradable Black Phosphorus-based Nanomaterials in Biomedicine: Theranostic Applications.

Ascribe to the unique two-dimensional planar nanostructure with exceptional physical and chemical properties, black phosphorous (BP) as the emerging inorganic twodimensional nanomaterial with high biocompatibility and degradability has been becoming one of the most promising materials of great potentials in biomedicine. The exfoliated BP sheets possess ultra-high surface area available for valid bio-conjugation and molecular loading for chemotherapy. Utilizing the intrinsic near-infrared optical absorbance, BPbased photothermal therapy in vivo, photodynamic therapy and biomedical imaging has been realized, achieving unprecedented anti-tumor therapeutic efficacy in animal experiments. Additionally, the BP nanosheets can strongly react with oxygen and water, and finally degrade to non-toxic phosphate and phosphonate in the aqueous solution. This manuscript aimed to summarize the preliminary progresses on theranostic application of BP and its derivatives black phosphorus quantum dots (BPQDs), and discussed the prospects and the state-of-art unsolved critical issues of using BP-based material for theranostic applications.

Effect of Red Light-Emitting Diodes Irradiation on Hemoglobin for Potential Hypertension Treatment Based on Confocal Micro-Raman Spectroscopy.

Red light-emitting diodes (LED) were used to irradiate the isolated hypertension hemoglobin (Hb) and Raman spectra difference was recorded using confocal micro-Raman spectroscopy. Differences were observed between the controlled and irradiated Hb by comparing the spectra records. The Raman spectrum at the 1399 cm-1 band decreased following prolonged LED irradiation. The intensity of the 1639 cm-1 band decreased dramatically in the first five minutes and then gradually increased in a time-dependent manner. This observation indicated that LED irradiation increased the ability of oxygen binding in Hb. The appearance of the heme aggregation band at 1399 cm-1, in addition to the oxygen marker band at 1639 cm-1, indicated that, in our study, 30 min of irradiation with 15.0 mW was suitable for inhibiting heme aggregation and enhancing the oxygen-carrying capacity of Hb. Principal component analysis showed a one-to-one relationship between irradiated Hb at different time points and the corresponding Raman spectra. Our approach could be used to analyze the hemoglobin from patients with confocal micro-Raman spectroscopy and is helpful for developing new nondrug hypertension therapy.

Facile synthesis of black phosphorus-Au nanocomposites for enhanced photothermal cancer therapy and surface-enhanced Raman scattering analysis.

Black phosphorus (BP), a new type of two-dimensional nanomaterial, has attracted crucial attention in recent years owing to its excellent properties and great potential in various chemical, physical, and biological fields. In this study, BP nanosheets loaded with Au nanoparticles (BP-Au NSs) are obtained by a one-step facile synthetic method. The Au nanostructures can not only enhance the photothermal efficiency of the nanocomposites, but also endow BP-Au NSs with the potential to act as effective surface-enhanced Raman scattering (SERS) substrates for Raman biodetection. Cancer photothermal therapy (PTT) has been carried out in vitro and in vivo using BP-Au NSs as nanoagents. Under irradiation by an 808 nm laser, BP-Au NSs are capable of producing sufficient hyperthermia to destroy cancer cells, and the transplanted tumors in most of the tumor-bearing mice disappeared; BP-Au NSs are more effective than bare BP nanosheets. The PTT effect can also be monitored by a Raman technique that benefits from the high SERS activity of the BP-Au NSs. The molecular fingerprint features of breast tumors before and after PTT treatment were clearly identified using SERS analysis. The theranostic applications of BP-Au NSs exhibit promising potential in biomedicine.

Synthesis of Au NP@MoS2 Quantum Dots Core@Shell Nanocomposites for SERS Bio-Analysis and Label-Free Bio-Imaging.

In this work, we report a facile method using MoS2 quantum dots (QDs) as reducers to directly react with HAuCl4 for the synthesis of Au nanoparticle@MoS2 quantum dots (Au NP@MoS2 QDs) core@shell nanocomposites with an ultrathin shell of ca. 1 nm. The prepared Au NP@MoS2 QDs reveal high surface enhanced Raman scattering (SERS) performance regarding sensitivity as well as the satisfactory SERS reproducibility and stability. The limit of detection of the hybrids for crystal violet can reach 0.5 nM with a reasonable linear response range from 0.5 µM to 0.5 nM (R² ≈ 0.974). Furthermore, the near-infrared SERS detection based on Au NP@MoS2 QDs in living cells is achieved with distinct Raman signals which are clearly assigned to the various cellular components. Meanwhile, the distinguishable SERS images are acquired from the 4T1 cells with the incubation of Au NP@MoS2 QDs. Consequently, the straightforward strategy of using Au NP@MoS2 QDs exhibits great potential as a superior SERS substrate for chemical and biological detection as well as bio-imaging.

Multifunctional Nanoplatform Based on Black Phosphorus Quantum Dots for Bioimaging and Photodynamic/Photothermal Synergistic Cancer Therapy.

A multifunctional nanoplatform based on black phosphorus quantum dots (BPQDs) was developed for cancer bioimaging and combined photothermal therapy (PTT) and photodynamic therapy (PDT). BPQDs were functionalized with PEG chains to achieve improved biocompatibility and physiological stability. The as-prepared nanoparticles exhibite prominent near-infrared (NIR) photothermal and red-light-triggered photodynamic properties. The combined therapeutic application of PEGylated BPQDs were then performed in vitro and in vivo. The results demonstrate that the combined phototherapy significantly promote the therapeutic efficacy of cancer treatment in comparison with PTT or PDT alone. BPQDs could also serve as the loading platform for fluorescent molecules, allowing reliable imaging of cancer cells. In addition, the low cytotoxicity and negligible side effects to main organs were observed in toxicity experiments. The theranostic characteristics of PEGylated BPQDs provide an uplifting potential for the future clinical applications.

Simulation study of second-harmonic microscopic imaging signals through tissue-like turbid media.

We establish, for the first time, a simulation model for dealing with the second-harmonic signals under a microscope through a tissue-like turbid medium, based on the Monte Carlo method. With this model, the angle-resolved distribution and the signal level eta of second-harmonic light through a slab of the turbid medium are demonstrated and the effects of the thickness (d) of the turbid medium, the numerical aperture (NA) of the objective as well as the size (rho) of the scatterers forming the turbid medium are explored. Simulation results reveal that the use of a small objective NA results in a narrow angle distribution but strong second-harmonic signals. A turbid medium consisting of large scattering particles has a strong influence on the angle distribution and the signal level eta, which results in a low penetration limit for second-harmonic signals made up of ballistic photons. It is approximately 30 microm in our situation.

Study of molecule variations in human xanthelasma skin based on confocal micro-raman spectroscopy.

Confocal micro-Raman spectroscopy was used to distinguish human xanthelasma skin (HXS) from the human normal skin (HNS). Results showed that intensive Raman peaks at 1,269, 1,336, 1,448, and 1,656 cm(-1) increased obviously. Both 1,269 and 1,656 cm(-1) peaks showed that the proteins in HXS were mostly in the anti-parallel ß sheet conformation. While the intensities of bands at 1,032, 1,087, 1,300, and 1,448 cm(-1) belonged to lipids were enhanced in HXS spectrum compared to those in HNS spectrum. There were main intercellular lipids alkyl chains with minor proteins contribution at 1,087 cm(-1) and phenylalanine at 1,032 cm(-1) . To quantitative analysis of the difference, the ratio of I852/I829 was calculated, which was 1:1.04 ± 0.04 and 1:1.11 ± 0.02 for HNS and HXS (p < 0.01), respectively. The data indicated that some tyrosine residues, which form a hydrogen bond with H2 O prior to aggregation, were captured by strong hydrogen-bond acceptors in the aggregate. The decreased ratio of I852/I829 indicated more hydrophobic in HXS than HNS. Principal component analysis showed a one-to-one relationship between human xanthelasma skin and the corresponding Raman spectra. It could be given useful help for the diagnostication of xanthelasma.

Second harmonic microscopic imaging and spectroscopic characterization in prostate pathological tissue.

Second harmonic microscopic imaging and spectroscopy technology has become a powerful tool for biomedical studies, especially in cancer research. In this paper, second harmonic generation in benign prostatic hyperplasia (BPH) and prostate cancer (PC) tissues in mouse model (C57BL6) have been reported. Excitated samples with different wavelength near-infrared laser from 780 to 850 nm we found that second harmonic signals from BPH nuclei stronger than that from PC, and a wavelength sensitivity was also observed in this experiment. Providing useful help for prostate malignancy diagnosis and identifying tissue components on clinic.