Structured illumination microscopy (SIM) is one of the mainstream real-time super imaging techniques due to its fast temporal resolution, fluorescent probe compatibility, and low phototoxicity. However, in real-time imaging, parameter estimation and image reconstruction take a long time, making it impossible to observe image details in real time. To improve the imaging speed of the SIM system without losing spatial resolution, we introduced the Particle Swarm Optimization (PSO) method to estimate the illumination light parameters. By constructing a function with the same form as the cosine light and using the normalized cross-correlation (NCC) as the objective function, we initialize the parameter range and apply the PSO algorithm to perform parameter fitting within the specified range, comparing with the original image. Experimental results show that we can increase the reconstruction speed by approximately 2.5 times without affecting the reconstruction quality. The PSO algorithm achieves a good balance between temporal and spatial resolution in imaging.
The cytoskeleton plays an important role for maintaining cell morphology and movement. Extracting the fine feature of cytoskeleton would be helpful to investigate the role of cytoskeleton in biological and pathological process. This paper proposes a feature analysis method based on machine vision to extract the fine feature of cytoskeleton. The nasopharyngeal epithelia cells (NP69), human high metastatic nasopharyngeal carcinoma cells (5-8F), and human high differentiation nasopharyngeal carcinoma cells (CNE1) were imaged with a total internal reflection fluorescence microscope (TIRFM). To extract the fine feature of cytoskeleton from these TIRFM images, three machine vison methods including the conventional threshold method, the adaptive threshold-connected-domain method (ATCD), and the maximum interclass variance method (OTSU) were used to segment the cytoskeleton. The ATCD method has great advantage over the conventional threshold and OTSU. The extracted length of the nasopharyngeal carcinoma cells (5-8F and CNE1) cytoskeleton is shorter than that of nasopharyngeal epithelia cell (NP69). These results indicate that ATCD machine vison method can be used to extract the fine feature of cytoskeleton with high resolution TIRFM images.
Adenosine aggravates pain via the interactions of adenosine A2A receptor (A2AR) and adenosine A1 receptor (A1R). And heteromerization of A2AR and A1R can put adenosine into operation. Simultaneously, a growing body of information indicates Ca2+ participate in pain management. Our team proved the interaction between A1R and A2AR by Iem-spFRET method. In the present study, we estimated the influence of the dimerization on the concentration of intracellular Ca2+. These dates demonstrate that the interaction of pain-related receptors (A2AR and A1R) has a significant effect on intracellular Ca2+ signaling.
Serum proteins contain many biomarkers of diseases, such as cancer. It would be important to purify the serum proteins for disease diagnosis. In this paper, cellulose acetate (CA) membrane was employed to purify serum proteins from human serum while removing other serum constituents and exogenous substances. The purified serum proteins were mixed with silver nanoparticles for SERS measurement. A total of 42 SERS spectra were recorded from purified serum proteins obtained from 20 liver cancer patients and 22 healthy volunteers. Subtle but discernible spectral changes of the two groups could be observed in the SERS spectra. Principal components analysis (PCA) and linear discriminate analysis (LDA) algorithm were introduced to analyze the difference between the two groups. Additionally, this method is nondestructive, fast and easy to operate, which is greatly important for clinical serum sample detection. These results indicated that SERS signal of serum proteins purified with CA membrane has a good prospect in liver cancer screening.
Mast cells (MCs) degranulation have an extremely momentous role in the progresses of immunoreaction, anaphylaxis as well as the variation of the tumor microenvironment (TME). The emergence of the substances due to MCs degranulation will arouse multiple changes of optical characteristics, such as energy transfer, fluorescence and spectra, etc. In this study, we implement the simultaneous spectral unmixing of excitation and emission by adjusting the cube filters and optical path to solely trigger the donor excitation and obtain the acceptor fluorescence emission. In addition, we add another channel to collect the real-time spectra with a portable and mobile spectroscopy equipment. Here, we developed graphene oxide (GO) and reduced GO (rGO)-based fluorescence resonance energy transfer (FRET) biosensors for MCs degranulation to verify the performance of the dual-channel system on an Inverted Fluorescence Microscope. MCs undergo degranulation can rapidly release tryptase, one proteases of the highest concentration in cytozoic pre-formed mediator. The acceptor fluorescence emission and spectra are detected simultaneously in real-time by tryptase-sensitized FRET biosensor on the dual-channel system. Moreover, the dual-channel can be switched by rapid adjusting optical channel during excitation at any moment. Results showed that the MCs degranulation could be monitoring in real-time on the dual-channel optical system. In particular, the minimal changes of the initial degranulation also could be measured with high response rate. Consequently, this dual-channel system may serve as a potential tool for the investigation of protein-protein interaction, single molecule dynamics and the working mechanism of membrane proteins using FRET-related techniques.
Independent emission-spectral unmixing fluorescence resonance energy transfer, Iem-spFRET, is a novel and powerful tool for measuring FRET efficiency in real time. In this paper, we simulate the measurement error of the Iem-spFRET by introducing random noise in sample data, donor fingerprint, and acceptor fingerprint. The random noise intensity is set from 0.0005 to 0.0025, corresponding to 5% - 25% of the maximum donor fingerprint intensity. The simulated results show the effect of random noise on apparent FRET efficiency (EfD) is less than on receptor-to-donor concentration ratio (Rc). Random noise with 10% maximum donor fingerprint intensity only leads to 0.33% variation of when the noise is added to both sample and fingerprints. These results indicate that Iem-spFRET is a robust method and could be applied on cases with weak FRET signal.
We report a novel approach to selectively close single blood vessels within tissue using multiphoton absorption-based photothermolysis. The treatment process is monitored by in vivo reflectance confocal microscopy in real-time. Closure of single targeted vessels of varying sizes ranging from capillaries to venules was demonstrated. We also demonstrated that deeply situated vessels could be closed precisely while preserving adjacent overlying superficial vessels. Partial vessel occlusion could also be achieved, and it is accompanied by intravascular blood cell speed increasing. This approach provides a novel precision medicine method for non-invasive precise microsurgery treatment of vascular diseases on per vessel/per lesion basis.
Cancer cells secrete copious amounts of secretory granules, exosomes, proteases. Recently, studies reviewed that mast cells (MCs) play crucial roles in the growth, spread and metastasis of tumor. MCs are one of the earliest cell types to infiltrate developing tumors. MCs undergo degranulation in response to various stimuli and rapidly release diverse bioactive mediators, such as histamine, tryptase, serotonin, tumor necrosis factor α (TNFα), which will tremendously affect the tumor microenvironment (TME). However, the mechanisms between the secretion of MCs degranulation and tumor remain unclear. Therefore, we developed a nanobiosensor based on fluorescence resonance energy transfer (FRET) for the determination of P815 mast cells and HeLa cells by secretagogues. With the pep-FITC as an energy donor and reduced grapheme oxide (rGO) as an energy acceptor, the two parts assemble an efficient FRET biosensor through electrostatic and stacking interaction (- interaction). Sensitized secretory cells can produce tryptase which would hydrolyze the specific cleavage site of the peptide, leading to ruin FRET system and then yield intensive fluorescence (FL) recovery of quenched FITC. Results showed that P815 cells are more sensitive and intense secretory than HeLa cells owing to more amount secretory mediators of P815 can change the microenvironment and further exacerbate the degree of degranulation in return. Our findings suggest that FRET biosensor have the ability to detect the extracellular dynamics of the cancer cells microenvironment. In addition, targeting mast cells may serve as a novel therapeutic scheme for cancer treatment and that inhibiting mast cell function may lead to tumor regression.
Degranulation in mast cell is usually characterized by the release of tryptase. We developed a fluorescence resonance energy transfer (FRET) probe based on graphene oxide (GO) to detect tryptase released from mast cells. The GO based FRET probe is composed of GO and a self-assembled complex of tryptase-specific recognition peptide chains labeled with isothiocyanate fluorescein. The fluorescence intensity around the mast cells increased when the mast cells were stimulated with C48/80, a kind of reagent promoting degranulation. The fluorescence distribution is inhomogenous. The fluorescence intensity was dependent on the concentration of C48/80 and the stimulation time. These results demonstrate that GO-base FRET probe could be used to study degranulation in mast cells.
In this study, two FRET-based probes are constructed to research oligomerization of Epstein-Barr virus Oncoprotein LMP1 in live cells. The images of wide-field fluorescence microscopy display that the majority of two LMP1-associated probes co-localized in internal perinuclear membranes. Furthermore, the fluorescence spectra of single cell co-expressed two probes indicated that the ratio of two emission peaks is around one, and the fluorescence spectra changed insignificantly during an hour observation. These findings indicated that LMP1/LMP1 interacted stably in live cells.
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