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Quantitative phase imaging has gained popularity in bioimaging because it can avoid the need for cell staining, which, in some cases, is difficult or impossible. However, as a result, quantitative phase imaging does not provide the labelling of various specific intracellular structures. Here we show a novel computational segmentation method based on statistical inference that makes it possible for quantitative phase imaging techniques to identify the cell nucleus. We demonstrate the approach with refractive index tomograms of stain-free cells reconstructed using tomographic phase microscopy in the flow cytometry mode. In particular, by means of numerical simulations and two cancer cell lines, we demonstrate that the nucleus can be accurately distinguished within the stain-free tomograms. We show that our experimental results are consistent with confocal fluorescence microscopy data and microfluidic cyto-fluorimeter outputs. This is a remarkable step towards directly extracting specific three-dimensional intracellular structures from the phase contrast data in a typical flow cytometry configuration.The accurate identification of the three-dimensional quantitative shape of a cell nucleus is now possible without fluorescent staining by applying computational segmentation to refractive index tomograms recorded in the flow cytometry mode.

Main conclusionETR/AN ratios should be in the range 7.5–10.5 for non-stressed C3 plants. Ratios extremely out of this range can be reflecting both uncontrolled plant status and technical mistakes during measurements. We urge users to explicitly refer to this ratio in future studies as a proof for internal data quality control.For the last few decades, the use of infra-red gas-exchange analysers (IRGAs) coupled with chlorophyll fluorometers that allow for measurements of net CO2 assimilation rate and estimates of electron transport rate over the same leaf area has been popularized. The evaluation of data from both instruments in an integrative manner can result in additional valuable information, such as the estimation of the light respiration, mesophyll conductance and the partitioning of the flux of electrons into carboxylation, oxygenation and alternative processes, among others. In this review, an additional and more ‘straight’ use of the combination of chlorophyll fluorescence and gas exchange-derived parameters is presented, namely using the direct ratio between two fully independently estimated parameters, electron transport rate (ETR)—determined by the fluorometer—and net CO2 assimilation rate (AN)—determined by the IRGA, i.e., the ETR/AN ratio, as a tool for fast detection of incongruencies in the data and potential technical problems associated with them, while checking for the study plant’s status. To illustrate this application, a compilation of 75 studies that reported both parameters for a total of 178 species under varying physiological status is presented. Values of ETR/AN between 7.5 and 10.5 were most frequently found for non-stressed C3 plants. C4 species showed an average ETR/AN ratio of 4.7. The observed ratios were larger for species with high leaf mass per area and for plants subjected to stressful factors like drought or nutritional deficit. Knowing the expected ETR/AN ratio projects this ratio as a routinary and rapid check point for guaranteeing both the correct performance of equipment and the optimal/stress status of studied plants. All known errors associated with the under- or overestimation of ETR or AN are summarized in a checklist that aims to be routinely used by any IRGA/fluorometer user to strength the validity of their data.

Analysis of plant behavior under diverse environmental conditions would be impossible without the methods for adequate assessment of the processes occurring in plants. The photosynthetic apparatus and its reaction to stress factors provide a reliable source of information on plant condition. One of the most informative methods based on monitoring the plant biophysical characteristics consists in detection and analysis of chlorophyll a fluorescence. Fluorescence is mainly emitted by chlorophyll a from the antenna complexes of photosystem II (PSII). However, fluorescence depends not only on the processes in the pigment matrix or PSII reaction centers but also on the redox reactions at the PSII donor and acceptor sides and even in the entire electron transport chain. Presently, a large variety of fluorometers from various manufacturers are available. Although application of such fluorometers does not require specialized training, the correct interpretation of the results would need sufficient knowledge for converting the instrumental data into the information on the condition of analyzed plants. This review is intended for a wide range of specialists employing fluorescence techniques for monitoring the physiological plant condition. It describes in a comprehensible way the theoretical basis of light emission by chlorophyll molecules, the origin of variable fluorescence, as well as relations between the fluorescence parameters, the redox state of electron carriers, and the light reactions of photosynthesis. Approaches to processing and analyzing the fluorescence induction curves are considered in detail on the basis of energy flux theory in the photosynthetic apparatus developed by Prof. Reto J. Strasser and known as a “JIP-test.” The physical meaning and relation of each calculated parameter to certain photosynthetic characteristics are presented, and examples of using these parameters for the assessment of plant physiological condition are outlined.

Reefs in the Northern Straits of Malacca are exposed to low-light conditions mainly due to sedimentation. Corals can be found in the urban reefs of Pulau Kendi (PK) and Pulau Songsong (PS) despite being exposed to low-light stress. Rapid Light Curve (RLC) measurements were performed in situ using a Diving Pulse Amplitude Modulated (PAM) fluorometer to investigate the photoacclimation of hard corals in the turbid waters of the non-protected reefs in PK and PS. The photosynthetic responses of corals suggested two distinct patterns of photoacclimation which are preferential dynamic non-photochemical quenching and preferential photochemical quenching. When the light response curves were plotted against E / E k , all coral species from PK and Pavona danai from PS were light saturated ( E / E k  > 1) indicating the activation of the NPQ mechanism. However, Goniastrea aspera and Cyphastrea chalcidicum exhibited a different trend of photoacclimation in which the light did not reach saturation (light is limited ( E / E k  < 1) indicating the preferential photochemical quenching as photoacclimation strategy. The results indicated that the photoacclimation mechanism may vary between species and corals can acclimate to changes in the environment. However, the extent of the acclimation may depend on other physiological factors such as Symbiodiniaceae type which needs to be investigated in the future.

Background Photosynthetic efficiency might be a key factor determining plant resistance to abiotic stresses. Plants can sense when growing conditions are not favorable and trigger an internal response at an early stage before showing external symptoms. When a high amount of salt enters the plant cell, the membrane system and function of thylakoids in chloroplasts could be destroyed and affect photosynthetic performance if the salt concentration is not regulated to optimal values. Oryza species have salt-tolerant and salt-sensitive genotypes; however, very few studies have investigated the genetic architecture responsible for photosynthetic efficiency under salinity stress in cultivated rice. Results We used an imaging-based chlorophyll fluorometer to monitor eight rice varieties that showed different salt tolerance levels for four consecutive days under control and salt conditions. An analysis of the changes in chlorophyll fluorescence parameters clearly showed the maximum quantum efficiency of PSII in sensitive varieties was significantly reduced after NaCl treatment when compared to tolerant varieties. A panel of 232 diverse rice accessions was then analyzed for chlorophyll fluorescence under salt conditions, the results showed that chlorophyll fluorescence parameters such as F 0 and NPQ were higher in Japonica subspecies, ΦPSII of Indica varieties was higher than that in other subgroups, which suggested that the variation in photosynthetic efficiency was extensively regulated under salt treatment in diverse cultivated rice. Two significant regions on chromosome 5 were identified to associate with the fraction of open PSII centers (qL) and the minimum chlorophyll fluorescence (F 0 ). These regions harbored genes related to senescence, chloroplast biogenesis and response to salt stress are of interest for future functional characterization to determine their roles in regulating photosynthesis. Conclusions Rice plant is very sensitive to salinity stress, especially at young seedling stage. Our work identified the distribution pattern of chlorophyll fluorescence parameters in seedlings leaf and their correlations with salt tolerance level in a diverse gene pool. We also revealed the complexity of the genetic architecture regulating rice seedling photosynthetic performance under salinity stress, the germplasm analyzed in this study and the associated genetic information could be utilized in rice breeding program.

The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10 −6  ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10 −5  ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing. Key points • The platform realizes one-pot reaction of RPA and Cas12a. • Sensitivity is 100 times more than qPCR. • Three output modes are suitable to be used to quantitative test or POC testing.

Micelles are of increasing importance as versatile carriers for hydrophobic substances and nanoprobes for a wide range of pharmaceutical, diagnostic, medical, and therapeutic applications. A key parameter indicating the formation and stability of micelles is the critical micelle concentration (CMC). In this respect, we determined the CMC of common anionic, cationic, and non-ionic surfactants fluorometrically using different fluorescent probes and fluorescence parameters for signal detection and compared the results with conductometric and surface tension measurements. Based upon these results, requirements, advantages, and pitfalls of each method are discussed. Our study underlines the versatility of fluorometric methods that do not impose specific requirements on surfactants and are especially suited for the quantification of very low CMC values. Conductivity and surface tension measurements yield smaller uncertainties particularly for high CMC values, yet are more time- and substance consuming and not suitable for every surfactant.

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS–11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A 260/280 . In case DNA was pure (A 260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A 260/280 values > 2.0. The A 260/280 and A 260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S . pneumoniae , and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

Dye tracing using fluorescent dyes is an essential tool for investigating flow path distribution, conduit networks, and connectivity in complex hydrogeologic systems, such as karst and fractured aquifers. The breakthrough curve, derived from the dye concentration measured at a downstream location with near‐continuous time resolution, provides quantitative information on water travel time distribution. Accurate measurement of dye concentration, however, is often challenged by the presence of natural organic materials (NOMs), including humic acids, fulvic acids, and chlorophylls, which contribute significant background fluorescence and interfere with dye signals. This study introduces a robust, automated spectral deconvolution framework aimed at precisely quantifying multiple fluorescent dye concentrations in hydrologic dye tracing. The framework employs an automated multi‐peak fitting algorithm, equipped with fluorescence peak information for both common NOMs and dye compounds, to effectively separate dye‐specific fluorescence from NOM background signals. The methodology is capable of processing large data sets generated from the high‐frequency water sampling required for breakthrough curve estimation. Three dye tracing field campaigns were conducted to compare the proposed approach with common practice of using in situ fluorometers. The results from these campaigns demonstrate significant improvements in both accuracy and efficiency when using the automated approach. A Python‐based code, along with the water tracing data from the three campaigns, is provided as open source in the public domain, inviting the community to utilize the proposed dye tracing platform for accurate and efficient dye tracing in natural water systems.

A systematic study of the luminescence properties of monodisperse β-NaYF 4 : 20% Yb 3+ , 2% Er 3+ upconversion nanoparticles (UCNPs) with sizes ranging from 12–43 nm is presented utilizing steady-state and time-resolved fluorometry. Special emphasis was dedicated to the absolute quantification of size- and environment-induced quenching of upconversion luminescence (UCL) by high-energy O–H and C–H vibrations from solvent and ligand molecules at different excitation power densities ( P ). In this context, the still-debated population pathways of the 4 F 9/2 energy level of Er 3+ were examined. Our results highlight the potential of particle size and P value for color tuning based on the pronounced near-infrared emission of 12 nm UCNPs, which outweighs the red Er 3+ emission under “strongly quenched” conditions and accounts for over 50% of total UCL in water. Because current rate equation models do not include such emissions, the suitability of these models for accurately simulating all (de)population pathways of small UCNPs must be critically assessed. Furthermore, we postulate population pathways for the 4 F 9/2 energy level of Er 3+ , which correlate with the size-, environment-, and P-dependent quenching states of the higher Er 3+ energy levels.