Quantitative characterization of bovine serum albumin thin-films using terahertz spectroscopy and machine learning methods.

The development of new spectral analysis methods in bio thin-film detection has generated intense interest in terahertz (THz) spectroscopy and its application in a wide range of fields. In this paper, it is the first time that machine learning methods are applied to the quantitative characterization of bovine serum albumin (BSA) deposited thin-films detected by terahertz time-domain spectroscopy. The spectra data of BSA thin-films prepared by solutions with concentrations ranging from 0.5 to 35 mg/ml are analyzed using the support vector regression method to learn the underlying model of the frequency against the target concentration. The learned mode successfully predicts the concentrations of the unknown test samples with a coefficient of determination R2 = 0.97932. Furthermore, aiming to identify the relevance of each frequency to the concentration, the maximal information coefficient statistical analysis is used and the three most discriminating frequencies in THz frequency are identified at 1.2, 1.1 and 0.5 THz respectively, which means a good prediction for BSA concentration can be achieved by using the top three relevant frequencies. Moreover, the top discriminating frequencies are in good agreement with the frequencies predicted by a long-wavelength elastic vibration model for BSA protein.

Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation.

Low-power laser irradiation of red light has been recognized as a promising tool across a vast variety of biomedical applications. However, deep understanding of the molecular mechanisms behind laser-induced cellular effects remains a significant challenge. Here, we investigated mechanisms involved in the death process in human hepatic cell line Huh7 at a laser irradiation. We decoupled distinct cell death pathways targeted by laser irradiations of different powers. Our data demonstrate that high dose laser irradiation exhibited the highest levels of total reactive oxygen species production, leading to cyclophilin D-related necrosis via the mitochondrial permeability transition. On the contrary, low dose laser irradiation resulted in the nuclear accumulation of superoxide and apoptosis execution. Our findings offer a novel insight into laser-induced cellular responses, and reveal distinct cell death pathways triggered by laser irradiation. The observed link between mitochondria depolarization and triggering ROS could be a fundamental phenomenon in laser-induced cellular responses.

Optical tweezers system for live stem cell organization at the single-cell level.

Cell manipulation is one of the most impactful applications for optical tweezers, and derived from this promise, we demonstrate a new optical tweezers system for the study of cell adhesion and organization. This method utilizes photonic-crystal-enhanced optical tweezers to manipulate cells with low laser intensities. By doing so, it enables effective cell patterning and culturing within the conditions necessary for successful differentiation and colony formation of human pluripotent stem cells. To this end, the biocompatibility of plasma-treated parylene-C for cell culturing was studied, and a thorough characterization of cellular interactive forces was performed using this system. Furthermore, this study also demonstrates construction of patterned cell arrays at arbitrary positions with micrometer-scale precision.

Non-invasive in vivo quantification of the developing optical properties and graded index of the embryonic eye lens using SPIM.

Graded refractive index lenses are inherent to advanced visual systems in animals. By understanding their formation and local optical properties, significant potential for improved ocular healthcare may be realized. We report a novel technique measuring the developing optical power of the eye lens, in a living animal, by exploiting the orthogonal imaging modality of a selective plane illumination microscope (SPIM). We have quantified the maturation of the lenticular refractive index at three different visible wavelengths using a combined imaging and ray tracing approach. We demonstrate that the method can be used with transgenic and vital dye labeling as well as with both fixed and living animals. Using a key eye lens morphogen and its inhibitor, we have measured their effects both on lens size and on refractive index. Our technique provides insights into the mechanisms involved in the development of this natural graded index micro-lens and its associated optical properties.

On the dysfunctional hemoglobins and cyanosis connection: practical implications for the clinical detection and differentiation of methemoglobinemia and sulfhemoglobinemia.

Methemoglobinemia and sulfhemoglobinemia are potentially life-threatening blood-related disorders characterized by similar symptoms and markedly distinct treatment procedures. In this paper, we investigate the causal relationship between these conditions and the onset of cyanosis, which is typically associated with a purple or bluish skin coloration. More specifically, we perform controlled experiments to elicit cyanotic appearances resulting from different severity levels of these disorders and varying physiological conditions. We note that such experiments cannot be induced in living subjects without posing risks to their health. Accordingly, we have resorted to an in silico experimental approach supported by biophysical data reported in the literature. Besides bringing new insights about cyanotic chromatic variations elicited by methemoglobinemia and sulfhemoglobinemia, our investigation provides the basis for the proposition of a cost-effective protocol for the noninvasive detection and differentiation of these disorders. Our experimental results indicate that its sensitivity range is wider than what is provided by similar protocols employed in these tasks. Moreover, it has lower operational requirements than laboratory tests ordered to enable the diagnosis of these conditions. We believe that these aspects make the proposed protocol particularly suitable for deployment at the point of care of medical settings with limited access to laboratory resources.

Atorvastatin treatment softens human red blood cells: an optical tweezers study.

Optical tweezers are proven indispensable single-cell micro-manipulation and mechanical phenotyping tools. In this study, we have used optical tweezers for measuring the viscoelastic properties of human red blood cells (RBCs). Comparison of the viscoelastic features of the healthy fresh and atorvastatin treated cells revealed that the drug softens the cells. Using a simple modeling approach, we proposed a molecular model that explains the drug-induced softening of the RBC membrane. Our results suggest that direct interactions between the drug and cytoskeletal components underlie the drug-induced softening of the cells.

Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells.

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells.

Label-free sensing of the binding state of MUC1 peptide and anti-MUC1 aptamer solution in fluidic chip by terahertz spectroscopy.

The aptamer and target molecule binding reaction has been widely applied for construction of aptasensors, most of which are labeled methods. In contrast, terahertz technology proves to be a label-free sensing tool for biomedical applications. We utilize terahertz absorption spectroscopy and molecular dynamics simulation to investigate the variation of binding-induced collective vibration of hydrogen bond network in a mixed solution of MUC1 peptide and anti-MUC1 aptamer. The results show that binding-induced alterations of hydrogen bond numbers could be sensitively reflected by the variation of terahertz absorption coefficients of the mixed solution in a customized fluidic chip. The minimal detectable concentration is determined as 1 pmol/µL, which is approximately equal to the optimal immobilized concentration of aptasensors.

Optical surface plasmon resonance sensor modified by mutant glucose/galactose-binding protein for affinity detection of glucose molecules.

Transdermal extraction of interstitial fluid (ISF) offers an attractive method for minimally invasive blood glucose monitoring. However, only a minute volume of ISF could be transdermally extracted, which is required to be diluted to form a manipulable volume of fluid for easy collection, transportation, and glucose detection. Therefore, a high-resolution glucose detection method is required for detecting glucose concentration in diluted ISF. In this paper, an optical surface plasmon resonance (SPR) sensor modified by the glucose/galactose-binding (GGB) protein which has good affinity to glucose molecules was presented for specific and sensitive glucose detection. The GGB protein was mutated at different sites for thiol coupling with the SPR surface and adjusting the affinity between glucose molecules and GGB protein. And the immobilization process of the GGB protein onto the surface of SPR sensor was optimized. Then, the stability of the SPR sensor modified with GGB protein was tested immediately and two weeks after immobilization. The coefficient of variation for glucose concentration measurement was less than 4.5%. By further mutation of the GGB protein at the A213S and L238S sites, the measurement range of the SPR sensor was adjusted to 0.1-100 mg/dL, which matches the glucose concentration range of 5-10 times diluted ISF (3-100 mg/dL). These results suggest that the SPR biosensor immobilized with GGB protein has the potential for continuous glucose monitoring by integrating into the microfluidic ISF extraction chip.

Speckle variance OCT for depth resolved assessment of the viability of bovine embryos.

The morphology of embryos produced by in vitro fertilization (IVF) is commonly used to estimate their viability. However, imaging by standard microscopy is subjective and unable to assess the embryo on a cellular scale after compaction. Optical coherence tomography is an imaging technique that can produce a depth-resolved profile of a sample and can be coupled with speckle variance (SV) to detect motion on a micron scale. In this study, day 7 post-IVF bovine embryos were observed either short-term (10 minutes) or long-term (over 18 hours) and analyzed by swept source OCT and SV to resolve their depth profile and characterize micron-scale movements potentially associated with viability. The percentage of en face images showing movement at any given time was calculated as a method to detect the vital status of the embryo. This method could be used to measure the levels of damage sustained by an embryo, for example after cryopreservation, in a rapid and non-invasive way.

Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings.

This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests - ELISA and BIAcore - the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria.

Terahertz identification and quantification of neurotransmitter and neurotrophy mixture.

Terahertz spectroscopy has been widely used for investigating the fingerprint spectrum of different substances. For cancerous tissues, the greatest difficulty is the absorption peaks of various substances contained in tissues overlap with each other, which are hard to identify and quantitative analyze. As a result, it is very hard to measure the presence of cancer cell and then to diagnose accurately. In this paper, we select three typical neurotransmitters (γ-aminobutyric acid, L-glutamic acid, dopamine hydrochloride) and two typical metabolites (inositol and creatine) in neurons to measure their terahertz spectra with different mixture ratios. By choosing characteristic absorption peaks, removing baseline and using the least square method, we can identify the components and proportions of each mixture, where the goodness of fit to practical situation is up to 94%. These results provide important evidences for identifying nerve substances and obtaining exact quantitative analysis.

Characterization of femtosecond-laser pulse induced cell membrane nanosurgical attachment.

This article provides insight into the mechanism of femtosecond laser nanosurgical attachment of cells. We have demonstrated that during the attachment of two retinoblastoma cells using sub-10 femtosecond laser pulses, with 800 nm central wavelength, the phospholipid molecules of both cells hemifuse and form one shared phospholipid bilayer, at the attachment location. In order to verify the hypothesis that hemifusion takes place, transmission electron microscope images of the cell membranes of retinoblastoma cells were taken. It is shown that at the attachment interface, the two cell membranes coalesce and form one single membrane shared by both cells. Thus, further evidence is provided to support the hypothesis that laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane, which resulted in cross-linking of the phospholipid molecules in each membrane. This process of hemifusion occurs throughout the entire penetration depth of the femtosecond laser pulse train. Thus, the attachment between the cells takes place across a large surface area, which affirms our findings of strong physical attachment between the cells. The femtosecond laser pulse hemifusion technique can potentially provide a platform for precise molecular manipulation of cellular membranes. Manipulation of the cellular membrane is an important procedure that could aid in studying diseases such as cancer; where the expression level of plasma proteins on the cell membrane is altered.

Thermal therapy with magnetic nanoparticles for cell destruction.

In this article we suggest a new concept for cell destruction based upon manipulating magnetic nanoparticles (MNPs) by applying external, low frequency alternating magnetic field (AMF) that oscillates the particles, together with focused laser illumination. Assessment of temperature profiles in a head and neck squamous cell carcinoma sample showed that cells with MNPs, treated with AMF (3 Hz, 300 mW) and laser irradiation (30 mW), reached 42°C after 4.5 min, as opposed to cells treated with laser but without AMF. Moreover, a theoretical model was developed to assess the overall theoretical temperature rise, which was shown to be 50% lower than the experimental temperature. Furthermore, we found that the combination of laser irradiation and AMF decreased the number of live cells by ~50%. Thus, the concentrated assembly of laser heating with AMF-induced MNP oscillations leads to more rapid and efficient cell death. These results suggest that the manipulated MNP technique can serve as a superior agent for PTT, with improved cell death capabilities.

Label-free fast 3D coherent imaging reveals pancreatic islet micro-vascularization and dynamic blood flow.

In diabetes, pancreatic ß-cells play a key role. These cells are clustered within structures called islets of Langerhans inside the pancreas and produce insulin, which is directly secreted into the blood stream. The dense vascularization of islets of Langerhans is critical for maintaining a proper regulation of blood glucose homeostasis and is known to be affected from the early stage of diabetes. The deep localization of these islets inside the pancreas in the abdominal cavity renders their in vivo visualization a challenging task. A fast label-free imaging method with high spatial resolution is required to study the vascular network of islets of Langerhans. Based on these requirements, we developed a label-free and three-dimensional imaging method for observing islets of Langerhans using extended-focus Fourier domain Optical Coherence Microscopy (xfOCM). In addition to structural imaging, this system provides three-dimensional vascular network imaging and dynamic blood flow information within islets of Langerhans. We propose our method to deepen the understanding of the interconnection between diabetes and the evolution of the islet vascular network.

Mitochondrial swelling and restorable fragmentation stimulated by femtosecond laser.

Mitochondria play a key role in all cellular physiology, processes, and behaviors. It is very difficult to precisely stimulate single mitochondria noninvasively in traditional biomedical research. In this study, we report that femtosecond laser can stimulate fragmentation or swelling of single mitochondria in human mesenchymal stem cells rather than physical disruption or ablation. In experiments, fragmented mitochondria can recover normal very soon but swelling ones cannot. At the same time, laser-induced generation of mitochondrial reactive oxygen species and opening of mitochondria permeability transition pores are involved in mitochondrial responses to photostimulation. Furthermore, the localized translocation of proapoptotic molecules are found in those stimulated mitochondria. Those results suggest femtosecond-laser photostimulation as a noninvasive and precise method for mitochondrial manipulation and related research.

Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration.

We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines.

Rapid and noninvasive technique to assess the metabolomics profile of bovine embryos produced in vitro by Raman spectroscopy.

Morphological assessments are used to select embryos with the highest implantation potential, however it is still very limited. The development of new technologies, such as Raman spectroscopy have improved quantitative and qualitative analysis, and consequently led to a better characterization of embryos and improvements on the prediction of their potential. Therefore, we propose a method based on the conventional in vitro culture system of bovine embryos, and the subsequent analysis of the culture media drops by Raman spectroscopy. Our results obtained by PCA analysis clearly showed a separation of the spectral profiles from culture media drops with and without embryos.

Erratum: Single-molecule force measurement via optical tweezers reveals different kinetic features of two BRaf mutants responsible for cardio-facial-cutaneous (CFC) syndrome: errata.

We correct the omission of the construct and protein purification method in our recent paper [Biomed. Opt. Express 4(12), 2835-2845 (2013)].[This corrects the article on p. 2835 in vol. 4, PMID: 24409384.].

Three-wavelength light control of freely moving Drosophila Melanogaster for less perturbation and efficient social-behavioral studies.

We developed a real-time automated laser-tracking system combined with continuous wave 1064-nm infrared or 473-nm blue lasers to provide punishment for studying memory in Drosophila Melanogaster. Combining optogenetic tools with laser properties, such as 473-nm and 593-nm lasers that activate light sensitive proteins in artificial transgenic flies, we can manipulate the specific neuron of an assigned fly among multiple flies to investigate neuron circuit relationships in social interactions. In restraining condition assay or optogenetic experiments, a ventral irradiated system would be more efficient due to higher ventral cuticle transmissions and neuron ganglia locations. Therefore, ventral irradiated systems cause less perturbation during behavior studies.