Seven-layer analysis model of an optical waveguide excitation fluorescence microscopy.

In this paper, an optical waveguide evanescent field fluorescence microscopy is studied. Based on Maxwell's equation, a seven-layer theoretical analysis model is developed for the evaluation of an optical waveguide excitation fluorescence microscopy. The optical waveguide excitation fluorescence microscopy structure is systematically and comprehensively analysed at the wavelengths of 488, 532 and 646 nm for fluorescent dyes. The analysis results provide some useful suggestions, which will be beneficial to the research of an optical waveguide evanescent field fluorescence microscopy.

Integrated opto-mechanical cantilever sensor with a rib waveguide.

An integrated opto-mechanical cantilever sensor with a rib waveguide is reported in this paper. The device consists of a rib waveguide cantilever with buried waveguides on silicon. The rib cantilever is introduced to match better with the buried waveguide, further for increasing the interface coupling efficiency. With this configuration, single-mode operating can be achieved in transverse direction without decreasing the width of optical waveguide cantilever. The system sensitivity is 1.1 µm-1 which is increased by about 21%, compared with the conventional structure.

Non-coplanar misalignment optical waveguide cantilever sensor with a monotonic response in a large operation range.

This paper reports a non-coplanar misalignment optical waveguide cantilever sensor realizing a monotonic response with a large operation range. A 1×2 Y-branch optical power splitter cantilever structure was designed, and one of the branches was reduced in thickness at the end, as a non-coplanar structure with respect to another. The misalignment coupling of the two branches due to the thickness of one branch leads to a monotonic response of an optical waveguide cantilever sensor. The simulation results showed a monotonic response with a sensitivity of 6×10-4 n m -1 in a large operation range of -1 to 1 µm.

A density-watershed algorithm (DWA) method for robust, accurate and automatic classification of dual-fluorescence and four-cluster droplet digital PCR data.

Droplet digital PCR (ddPCR) is a single-molecule amplification technology with broad applications in precision medicine and clinical diagnosis. Dual-fluorescence and four-cluster ddPCR (two/four-ddPCR) assay is an effective way to quantify copy numbers. Currently, two/four-ddPCR data are usually classified with manual thresholds. For clinical applications, automatic and accurate methods are required to avoid subjectivity in diagnosis. Although there are some automatic classification algorithms, their accuracy and robustness still need to be improved to meet the needs of clinical diagnosis. Therefore, a new method is in high demand to automatically classify two/four-ddPCR data in an accurate and robust way. Here, a novel density-watershed algorithm (DWA) method was developed for the accurate, automatic and unsupervised classification of two/four-ddPCR data. First, data gridding was applied to a scatter plot of the fluorescence signal intensity to calculate data densities. Based on the data densities, the watershed algorithm was used to divide the gridded scatter plot into isolated regions automatically. Next, an optimal cluster pattern was determined based on these isolated regions, and excess regions were merged. Finally, the two/four-ddPCR data were classified based on the merged regions, and DNA template copy numbers were calculated accordingly. Using the DWA method for the quantification of both wild types and mutants of epidermal growth factor receptor (EGFR) L858R and T790M, the classification results were highly consistent with expectations, and significantly better than commonly-used automatic algorithms for now. The computed template copy numbers scaled proportionally to the relative concentration of input templates (r2 > 0.998) in four orders of magnitude with a good reproducibility, and achieved a limit of detection over 40 times lower than the commonly-used automatic algorithms. Furthermore, the DWA method was validated on 254 clinical DNA samples derived from frozen tissues, formalin-fixed paraffin-embedded tissues and peripheral blood. In most cases, the DWA method derived accurate and valid classification results. This highly effective DWA method may be widely used for automatically classifying two/four-ddPCR data, and it will greatly promote the application of ddPCR in clinical diagnosis.

Flexible Mixed-Potential-Type (MPT) NO2 Sensor Based on An Ultra-Thin Ceramic Film.

A novel flexible mixed-potential-type (MPT) sensor was designed and fabricated for NO2 detection from 0 to 500 ppm at 200 °C. An ultra-thin Y2O3-doped ZrO2 (YSZ) ceramic film 20 µm thick was sandwiched between a heating electrode and reference/sensing electrodes. The heating electrode was fabricated by a conventional lift-off process, while the porous reference and the sensing electrodes were fabricated by a two-step patterning method using shadow masks. The sensor's sensitivity is achieved as 58.4 mV/decade at the working temperature of 200 °C, as well as a detection limit of 26.7 ppm and small response time of less than 10 s at 200 ppm. Additionally, the flexible MPT sensor demonstrates superior mechanical stability after bending over 50 times due to the mechanical stability of the YSZ ceramic film. This simply structured, but highly reliable flexible MPT NO2 sensor may lead to wide application in the automobile industry for vehicle emission systems to reduce NO2 emissions and improve fuel efficiency.

Precise cell patterning using cytophobic self-assembled monolayer deposited on top of semi-transparent gold.

This paper reports a simple and effective method for cell patterning by using a self-assembled monolayer (SAM)-treated glass surface which is surrounded by semi-transparent gold coated with another type of SAM. Specifically, a hydrophobic SAM, derived from 1-hexadecanethiol (HDT), was coated on the gold surface to prevent cell growth, and a hydrophilic SAM, derived from 3-trimethoxysilyl propyl-diethylenetriamine (DETA), was coated on the exposed glass surface to promote cell growth. The capabilities of this technique are as follows: 1) single-cell resolution, 2) easy alignment of the cell patterns to the structures already existing on the substrate, 3) visualization and verification of the predefined cytophobic/cytophilic pattern prior to cell growth, and 4) convenient monitoring cell growth at the same location for an extended long term period of time. Whereas a number of earlier techniques have demonstrated the single cell resolution, or visualization and verification of the cytophobic/cytophilic patterns prior to cell growth, we believe that our technique is unique in possessing all of these beneficial qualities at the same time. The distinguishing characteristic of our technique is, however, that the use of semi-transparent Cr/Au film allows for convenient brightfield pattern visualization and offers an advantage over previously developed methods which require fluorescent imaging. We have successfully demonstrated the patterning of four different kinds of cells using this technique: immortalized mouse hypothalamic neurons (GT1-7), mouse osteoblast cells (MC3T3), mouse fibroblast cells (NIH3T3) and primary rat hippocampal neurons. This study was performed with a specific ultimate application-the creation of a multi electrode array (MEA) with predefined localization of cell bodies on top of the electrodes, as well as predefined patterns for cell extensions to grow in between the electrodes. With that goal in mind, we have also determined critical parameters for patterning of each of these cell types, such as the minimum size of a cell-adherent island for exclusively anchoring one cell or two cells, as well as the width of the cytophilic pathway between two islands that enables cell extensions to grow, while preventing the anchoring of the cell bodies. Additionally, we have provided statistical analysis of the occupancy for various sizes and shape of cell-anchoring islands. As demonstrated here, we have developed a novel and reliable cell patterning technique, which can be utilized in various applications, such as biosensors or tissue engineering.

A "quasi" confocal droplet reader based on laser-induced fluorescence (LIF) cytometry for highly-sensitive and contamination-free detection.

Highly-sensitive and contamination-free droplet digital PCR (ddPCR) is an enabling technology and widely needed for accurate quantification of nucleic acid in clinical applications. In this paper, a novel droplet reader was developed by combining a "quasi" confocal laser-induced fluorescence (LIF) cytometry with a delicate microfluidic chip design. The droplets with a size of 90 µm was illuminated at an out-of-focus position by two aligned laser beams to generate maximum fluorescent signal. Additionally, the lateral offset position of the microfluidic chip should be precisely tuned so that the bandwidth of the FAM and VIC channels were configured at the matching sizes. Then, PMT gain voltages and pneumatic pressures were optimized for better droplet detection efficiencies. An aerosol adsorption experiment was performed to demonstrate that there was no aerosol contamination, and detected copy numbers of both mutants and wild types scaled linearly with the expected input copy numbers (r2>0.998) with a LoB of 0.0 copies and LoD of 3.0 copies. The results demonstrated that this droplet reader with the delicate chip is a convenient, highly-sensitive and contamination-free to detect fluorescence signals inside droplets after ddPCR, which is highly promising for broad applications of ddPCR in clinical diagnosis.

Spectral characterization of the voltage-sensitive dye di-4-ANEPPDHQ applied to probing live primary and immortalized neurons.

Spectral properties of a recently developed voltage-sensitive dye, di-4-ANEPPDHQ, were characterized as the dye was dissolved in the solvent dimethyl sulfoxide as the stock solution, in Hank's buffered salt solution as the staining solution, and bound to the plasma membrane of primary rat hippocampal neurons and immortalized mouse hypothalamic neurons (GT1-7) in vitro. Their dependence on the local chemical and electrical environment of dye molecules was determined. The excitation and emission peaks are 479 nm and 570 nm for the stained primary neurons, and 476 nm and 585 nm for the stained immortalized neurons. The excitation and emission bands of the stained GT1-7 neurons, defined as 50% peak intensity, are 429-516 nm and 544-648 nm, respectively.

A Cosine Similarity Algorithm Method for Fast and Accurate Monitoring of Dynamic Droplet Generation Processes.

Droplet microfluidics has attracted significant interests in functional microcapsule synthesis, pharmaceuticals, fine chemicals, cosmetics and biomedical research. The low variability of performing chemical reactions inside droplets could benefit from improved homogeneity and reproducibility. Therefore, accurate and convenient methods are needed to monitor dynamic droplet generation processes. Here, a novel Cosine Similarity Algorithm (CSA) method was developed to monitor the droplet generation frequency accurately and rapidly. With a microscopic droplet generation video clip captured with a high-speed camera, droplet generation frequency can be computed accurately by calculating the cosine similarities between the frames in the video clip. Four kinds of dynamic droplet generation processes were investigated including (1) a stable condition in a single microfluidic channel, (2) a stable condition in multiple microfluidic channels, (3) a single microfluidic channel with artificial disturbances, and (4) microgel fabrication with or without artificial disturbances. For a video clip with 5,000 frames and a spatial resolution of 512 × 62 pixels, droplet generation frequency up to 4,707.9 Hz can be calculated in less than 1.70 s with an absolute relative calculation error less than 0.08%. Artificial disturbances in droplet generation processes can be precisely determined using the CSA method. This highly effective CSA method could be a powerful tool for further promoting the research of droplet microfluidics.

Cell patterning using molecular vapor deposition of self-assembled monolayers and lift-off technique.

This paper reports a precise, live cell-patterning method by means of patterning a silicon or glass substrate with alternating cytophilic and cytophobic self-assembled monolayers (SAMs) deposited via molecular vapor deposition. Specifically, a stack of hydrophobic heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane SAMs and a silicon oxide adhesion layer were patterned on the substrate surface, and a hydrophilic SAM derived from 3-trimethoxysilyl propyldiethylenetriamine was coated on the remaining non-treated areas on the substrate surface to promote cell growth. The primary characteristics of the reported method include: (i) single-cell resolution; (ii) easy alignment of the patterns with the pre-existing patterns on the substrate; (iii) easy formation of nanoscale patterns (depending on the exposure equipment); (iv) long shelf life of the substrate pattern prior to cell culturing; (v) compatibility with conventional, inverted, optical microscopes for simple visualization of patterns formed on a glass wafer; and (vi) the ability to support patterned cell (osteoblast) networks for at least 2 weeks. Here, we describe the deposition technique and the characterization of the deposited layers, as well as the application of this method in the fabrication of multielectrode arrays supporting patterned neuronal networks.

Developing rapid detection of mycobacteria using microwaves.

In this paper, we describe the development of a culture-based biochip device for rapid detection of mycobacteria in environmental samples. Individual biochips rely upon the unique paraffinophilic nature of mycobacteria to rapidly and selectively adhere to the surface of the device. We used prototype biochips to experimentally demonstrate the concept of rapid and selective detection of mycobacteria by testing pure cultures and using epifluorescence microscopy to visualize microorganisms on the surface. As an alternative, rapid approach for identifying the biomass on the biochip surface, we used microwaves in the 10 to 26 GHz frequency range. The results of this study indicate that different microorganisms are responsible for specific shifts in resonance frequencies of a microwave cavity. By combing the semi-selective paraffin surface of the biochip with the microorganism-specific response to the microwaves, we have developed an improved analytical system with the potential to rapidly identify and enumerate mycobacteria in environmental samples in as little as 2 h.