Atto 465 Derivative Is a Nuclear Stain with Unique Excitation and Emission Spectra Useful for Multiplex Immunofluorescence Histochemistry

Multiplex immunofluorescence (mIF) is an effective technique for the maximal visualization of multiple target proteins in situ. This powerful tool is mainly limited by the spectral overlap of the currently available synthetic fluorescent dyes. The fluorescence excitation wavelengths ranging between 405 and 488 nm are rarely used in mIF imaging and serve as a logical additional slot for a fluorescent probe. In the present study, we demonstrate that the addition of 2,3,4,5,6-pentafluoroaniline to Atto 465 NHS ester, creating Atto 465-pentafluoroaniline (Atto 465-p), generates a bright nuclear stain in the violet-blue region of the visible spectrum. This allows the 405 nm excitation and emission, classically used for nuclear counterstains, to be used for the detection of another target protein. This increases the flexibility of the mIF panel and, with appropriate staining and microscopy, enables the quantitative analysis of at least six targets in one tissue section. (J Histochem Cytochem XX: XXX–XXX, XXXX)

Effect of Radio-Frequency Treatment on the Changes of Dissolved Organic Matter in Rainwater

Rainwater is a potential source of drinking water, but has various components of dissolved organic matter (DOM). DOM is a reservoir of potential hazards in drinking water. Therefore, a new method is required to purify rainwater as a drinking water source in terms of DOM aspects. A radio-frequency (RF) treatment system is introduced here to purify source water with a small possibility of contamination. RF is generated by applying a frequency of 1.5 MHz through a glass reactor with a diameter of 2 mm which is wrapped by a 2 mm copper wire. The results demonstrate that UV260 value and dissolved organic carbon (DOC) are reduced during RF treatment. DOC was reduced by a lower amount compared to UV260, suggesting the partial transformation of bio-refractory DOM. A fluorescence excitation-emission matrix showed that humic-like substances in rainwater were reduced faster than protein-like ones, indicating that humic-like substances are susceptible to reduction by RF treatment. The results offer information on the use of RF treatment in a rainwater purification process for the production of drinking water.

Fluorescence Excitation–Emission Matrix Spectroscopy and Boosting Regression Tree Model to Detect Dissolved Organic Carbon in Water

In recent years, optical methods have been proven to be a powerful tool for m onitoring dissolved organic carbon (DOC) in natural waters. However, the effectiveness of this method in marine systems with low DOC concentrations remains to be shown. Herein, a new method based on fluorescence excitation–emission matrix spectroscopy for seawater DOC quantification is proposed. Pre-processing method is investigated to achieve a high signal to noise ratio. Peak-picking operation is then performed to obtain feature peaks. In order to combine the information from sparsely located feature peaks, sparse principal component analysis is applied to identifying important variables used in the following regression procedure. Under these conditions the result of regression analysis can be obtained readily in a given data set coupling with boosting regression tree. The method was tested on samples collected from the East China Sea. Compared to the parallel factor analysis–multivariate linear regression method, experimental results show that the proposed method achieved a more consistent regression output and indicate that the boosting regression tree has potential for DOC quantification even at low concentrations.

Effect of the Recycling Process on Drinking Water Treatment: Evaluation Based on Fluorescence EEM Analysis Using the Peak-Picking Technique and Self-Organizing Map

The recycling process is applied in many water treatment plants (WTPs), although this process can lead to adverse effects. The effect of the recycling process on the characteristics of dissolved organic matter was evaluated based on a fluorescence excitation-emission matrix using the peak-picking technique and self-organizing map (SOM). In this study, an evaluation of two WTPs, one with and one without a recycling system, was carried out. Both WTPs show moderate efficiency during the coagulation–flocculation process in removing DOC, fulvic acid-like, humic acid-like, and tryptophan-like substances. The recycling process causes increased values of fulvic acid-like, humic acid-like, and tryptophan-like substances and specific ultraviolet absorbance (SUVA) after the filtration process of about 31.0%, 35.7%, 22.2%, and 6%, respectively. Meanwhile, the WTP without recycling showed a reduction in the level of fulvic acid-like, humic acid-like, and tryptophan-like substances and SUVA by 23.3%, 52.9%, 27.8%, and 21.1%, respectively. Moreover, SOM analysis based on the peak-picking technique can determine differences in sample clusters due to the recycling process.

Production of Labile Protein-Like Dissolved Organic Carbon Associated With Anaerobic Methane Oxidization in the Haima Cold Seeps, South China Sea

Cold seeps where methane-rich fluids escape from the seafloor generally support enormous biomass of chemosynthetic organisms and associated fauna. In addition to transporting a great amount of methane toward the seafloor, cold seeps also contribute to the aged, dissolved organic carbon (DOC) pool in the deep ocean. Here, two sediment cores from the “Haima cold seeps,” northern South China Sea and a nearby reference core were analyzed for pore-water sulfate and DOC concentrations, δ13C of DOC, and optical properties of dissolved organic matter (DOM). High DOC concentrations (0.9–3.7 mM) accompanied by extremely low δ13C values (−43.9 to −76.2‰) suggest the conversion of methane into sedimentary DOC pool in the seep sediments. Parallel factor analysis (PARAFAC) of the fluorescence excitation-emission matrices shows higher fluorescent intensities of labile protein-like components (C2 and C4) and lower fluorescent intensities of refractory humic-like components (C1 and C3) in the seep cores compared to the reference core. The intensity of C2 is positively correlated with DOC concentrations and δ13C-DOC in the seep sediments, suggesting that the labile protein-like DOM was produced by the anaerobic oxidation of methane (AOM). Moreover, low humification index (HIX) and high biological index (BIX) values also indicate intensified production of relatively labile DOM with lower degradation degree in the seep cores compared to the reference core. Hence, we highlight that methane-derived DOC may serve as important carbon and energy sources for heterotrophic microbial communities due to its relatively labile nature.

A review on fluorescence spectroscopic analysis of water and wastewater

In recent years, the application of fluorescence spectroscopy has been widely recognized in water environment studies. The sensitiveness, simplicity, and efficiency of fluorescence spectroscopy are proved to be a promising tool for effective monitoring of water and wastewater. The fluorescence excitation-emission matrix (EEMs) and synchronous fluorescence spectra have been widely used analysis techniques of fluorescence measurement. The presence of organic matter in water and wastewater defines the degree and type of pollution in water. The application of fluorescence spectroscopy to characterize dissolved organic matter (DOM) has made the water quality assessment simple and easy. With the recent advances in this technology, components of DOM are identified by employing parallel factor analysis (PARAFAC), a mathematical trilinear data modeling with EEMs. The majority of wastewater studies indicated that the fluorescence peak of EX/EM at 275nm/340nm is referred to tryptophan region (Peak T1). However, some researchers identified another fluorescence peak in the region of EX/EM at 225-237nm/340-381nm, which described the tryptophan region and labeled it as Peak T2. Generally, peak T is a protein-like component in the water sample, where T1 and T2 signals were derived from the <0.20µm fraction of pollution. Therefore, a more advanced approach, such as an online fluorescence spectrofluorometer, can be used for the online monitoring of water. The results of various waters studied by fluorescence spectroscopy indicate that changes in peak T intensity could be used for real-time wastewater quality assessment and process control of wastewater treatment works. Finally, due to its effective use in water quality assessment, the fluorescence technique is proved to be a surrogate online monitoring tool and early warning equipment.

Handheld Multispectral Fluorescence Imaging System to Detect and Disinfect Surface Contamination

Contamination inspection is an ongoing concern for food distributors, restaurant owners, caterers, and others who handle food. Food contamination must be prevented, and zero tolerance legal requirements and damage to the reputation of institutions or restaurants can be very costly. This paper introduces a new handheld fluorescence-based imaging system that can rapidly detect, disinfect, and document invisible organic residues and biofilms which may host pathogens. The contamination, sanitization inspection, and disinfection (CSI-D) system uses light at two fluorescence excitation wavelengths, ultraviolet C (UVC) at 275 nm and violet at 405 nm, for the detection of organic residues, including saliva and respiratory droplets. The 275 nm light is also utilized to disinfect pathogens commonly found within the contaminated residues. Efficacy testing of the neutralizing effects of the ultraviolet light was conducted for Aspergillus fumigatus, Streptococcus pneumoniae, and the influenza A virus (a fungus, a bacterium, and a virus, respectively, each commonly found in saliva and respiratory droplets). After the exposure to UVC light from the CSI-D, all three pathogens experienced deactivation (> 99.99%) in under ten seconds. Up to five-log reductions have also been shown within 10 s of UVC irradiation from the CSI-D system.

Multiple Compact Camera Fluorescence Detector for Real-Time PCR Devices

The polymerase chain reaction is an important technique in biological research because it tests for diseases with a small amount of DNA. However, this process is time consuming and can lead to sample contamination. Recently, real-time PCR techniques have emerged which make it possible to monitor the amplification process for each cycle in real time. Existing camera-based systems that measure fluorescence after DNA amplification simultaneously process fluorescence excitation and emission for dozens of tubes. Therefore, there is a limit to the size, cost, and assembly of the optical element. In recent years, imaging devices for high-performance, open platforms have benefitted from significant innovations. In this paper, we propose a fluorescence detector for real-time PCR devices using an open platform camera. This system can reduce the cost, and can be miniaturized. To simplify the optical system, four low-cost, compact cameras were used. In addition, the field of view of the entire tube was minimized by dividing it into quadrants. An effective image processing method was used to compensate for the reduction in the signal-to-noise ratio. Using a reference fluorescence material, it was confirmed that the proposed system enables stable fluorescence detection according to the amount of DNA.