4-Aldrithiol-based photometric assay for detection of methylthioalkylmalate synthase activity.

In Brassica plants, glucosinolates are a diverse class of natural products, of which aliphatic methionine-derived glucosinolates are the most abundant form. Their structural diversity comes from the elongation of some side-chains by up to 9 carbons, which, after the formation of the core glucosinolate structure, can undergo further chemical modifications. Methylthioalkylmalate synthase (MAMS) catalyzes the iterative elongation process for aliphatic methionine-derived glucosinolates. Most biochemical studies on MAMS have been performed using liquid chromatography/mass spectrometry (LC/MS)-based assays or high-performance liquid chromatography (HPLC)-based assays. The LC/MS- and HPLC-based methods are endpoint assays, which cannot be monitored in real time and require a laborious process for data collection. These analytical methods are inefficient for performing multiple enzymatic assays needed to determine steady-state kinetic parameters or for mechanistic evaluation of pH-dependence and kinetic isotope effect studies. Although the function of MAMS has long been defined, there is a gap in knowledge as it pertains to biochemical characterization of this plant enzyme. Part of this may be due to the lack of efficient methods that can be used for this type of research. This chapter describes a continuous photometric assay to track MAMS activity in real time using the 4-aldrithiol reagent for reaction detection.

Simultaneously performing Taylor dispersion analysis with fluorimetry, photometry, and contactless conductometry at the same detection window.

Taylor dispersion analysis (TDA) is a rapid and precise method for determining the hydrodynamic radius (RH) of various substances. We present a versatile TDA system with a flow-through sample injection device, two compact 3-in-1 detectors, and a high-voltage power supply. The 3D-printed detectors combine fluorimetry (FD), photometry (AD@255 nm), and contactless conductometry (C4D) in a single head, enabling simultaneous detection at one capillary window. Using bovine serum albumin (BSA) as a model analyte, we compare TDA with different detection methods. BSA labeled with fluorescein isothiocyanate (FITC) is analyzed in both pulse mode and capillary electrophoresis (CE) TDA. FD and AD detection yield similar RH values, except when FITC binds with small ions in the buffer. In phosphate buffer, C4D underestimates RH values by approximately 18 % due to BSA self-association. In Tris-based buffers, C4D values are 87%-96 % of AD values in pulse mode. With CE-TDA using Tris-CHES buffer, no statistical difference is found across all detections. The system is also applied to CE-TDA of various compounds, particularly charged saccharides. CE-TDA improves the accuracy of TDA results from C4D. We demonstrate the resolution of mixed C4D-TDA signals with assistance from FD and AD signals, successfully resolving gluconate peaks fully covered by another compound. The versatile system with 3-in-1 detection offers a powerful tool for TDA of mixtures and enhances sample throughput.

Lifting the Concentration Limit of Mass Photometry by PEG Nanopatterning.

Mass photometry (MP) is a rapidly growing optical technique for label-free mass measurement of single biomolecules in solution. The underlying measurement principle provides numerous advantages over ensemble-based methods but has been limited to low analyte concentrations due to the need to uniquely and accurately quantify the binding of individual molecules to the measurement surface, which results in diffraction-limited spots. Here, we combine nanoparticle lithography with surface PEGylation to substantially lower surface binding, resulting in a 2 orders of magnitude improvement in the upper concentration limit associated with mass photometry. We demonstrate the facile tunability of degree of passivation, enabling measurements at increased analyte concentrations. These advances provide access to protein-protein interactions in the high nanomolar to low micromolar range, substantially expanding the application space of mass photometry.

A chemogenetic approach for dopamine imaging with tunable sensitivity.

Genetically-encoded dopamine (DA) sensors enable high-resolution imaging of DA release, but their ability to detect a wide range of extracellular DA levels, especially tonic versus phasic DA release, is limited by their intrinsic affinity. Here we show that a human-selective dopamine receptor positive allosteric modulator (PAM) can be used to boost sensor affinity on-demand. The PAM enhances DA detection sensitivity across experimental preparations (in vitro, ex vivo and in vivo) via one-photon or two-photon imaging. In vivo photometry-based detection of optogenetically-evoked DA release revealed that DETQ administration produces a stable 31 minutes window of potentiation without effects on animal behavior. The use of the PAM revealed region-specific and metabolic state-dependent differences in tonic DA levels and enhanced single-trial detection of behavior-evoked phasic DA release in cortex and striatum. Our chemogenetic strategy can potently and flexibly tune DA imaging sensitivity and reveal multi-modal (tonic/phasic) DA signaling across preparations and imaging approaches.

Protocol for fiber photometry recording from deep brain regions in head-fixed mice.

To exclude the influence of motion on in vivo calcium imaging, animals usually need to be fixed. However, the whole-body restraint can cause stress in animals, affecting experimental results. In addition, some brain regions are prone to bleeding during surgery, which lowers the success rate of calcium imaging. Here, we present a protocol for calcium imaging using heparin-treated fiber in head-fixed mice. We describe steps for stereotaxic surgery, including virus injection and optic fiber implantation, fiber photometry, and data analysis. For complete details on the use and execution of this protocol, please refer to Du et al.1.

Identifying behavioral links to neural dynamics of multifiber photometry recordings in a mouse social behavior network.

Objective.Distributed hypothalamic-midbrain neural circuits help orchestrate complex behavioral responses during social interactions. Given rapid advances in optical imaging, it is a fundamental question how population-averaged neural activity measured by multi-fiber photometry (MFP) for calcium fluorescence signals correlates with social behaviors is a fundamental question. This paper aims to investigate the correspondence between MFP data and social behaviors.Approach:We propose a state-space analysis framework to characterize mouse MFP data based on dynamic latent variable models, which include a continuous-state linear dynamical system and a discrete-state hidden semi-Markov model. We validate these models on extensive MFP recordings during aggressive and mating behaviors in male-male and male-female interactions, respectively.Main results:Our results show that these models are capable of capturing both temporal behavioral structure and associated neural states, and produce interpretable latent states. Our approach is also validated in computer simulations in the presence of known ground truth.Significance:Overall, these analysis approaches provide a state-space framework to examine neural dynamics underlying social behaviors and reveals mechanistic insights into the relevant networks.

Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions.

Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa-5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20-100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.

Monitoring norepinephrine release in vivo using next-generation GRABNE sensors.

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter. The first-generation NE sensor makes in vivo, real-time, cell-type-specific and region-specific NE detection possible, but its low NE sensitivity limits its utility. Here, we developed the second-generation GPCR-activation-based NE sensors (GRABNE2m and GRABNE2h) with a superior response and high sensitivity and selectivity to NE both in vitro and in vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell-type-specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRABNE sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo, providing new insights into the physiological and pathophysiological roles of NE.

Protocol for in vivo dual-color fiber photometry in the mouse thalamus.

In vivo calcium imaging of neural activity is an indispensable approach for understanding the mechanisms and functions of neural system. Development of advanced imaging tools and various genetically encoded calcium indicators allows us to simultaneously record the activity of different neural populations. Here, we present a protocol for acquiring neural activity of two discrete neural populations in mice using dual-color fiber photometry. We describe steps for injecting viral constructs and implanting the fiber optic through stereotaxic surgery, calcium signal acquisition, and data analysis. We also describe the incorporation of electroencephalogram and electromyography recordings with dual-color fiber photometry analysis. For complete details on the use and execution of this protocol, please refer to Shin et al.1.

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

LASER FLARE PHOTOMETRY IN PRIMARY RHEGMATOGENOUS RETINAL DETACHMENT: An Evaluation of 2,487 Cases.

PURPOSE: Exploratory analysis associated with the prospective, multicenter, randomized PRIVENT trial. To characterize the associations between laser flare photometry and anatomical and epidemiological features of rhegmatogenous retinal detachment (RRD). METHODS: The authors measured laser flare values of all 3,048 prescreened patients excluding those with comorbidities. A mixed regression analysis evaluated the strength of the influencing factors like age, sex, lens status, and presence and extent of RRD on laser flare. RESULTS: Rhegmatogenous retinal detachment was more frequent in men (65.8%) than in women (34.2%, P < 0.001) and in right (52%) than in left eyes (48%, P = 0.045). Phakic RRD affected less quadrants and was less likely to be associated with macula-off status than pseudophakic RRD (48.4% vs. 58.0% macula off, 23% vs. 31% ≥3 quadrants, P < 0.001). Laser flare of affected eyes was significantly higher compared with fellow eyes (12.6 ± 15.2 vs. 8.3 ± 7.4 pc/ms, P < 0.001). The factors age, sex, lens status, presence of RRD, and the number of quadrants affected were independent influencing factors on laser flare. R 2 was 0.145 for phakic and 0.094 for pseudophakic eyes. CONCLUSION: The results indicate that there may be more factors affecting laser flare than previously assumed. This might limit flare as predictive value for PVR and retinal redetachment.

Lights, fiber, action! A primer on in vivo fiber photometry.

Fiber photometry is a key technique for characterizing brain-behavior relationships in vivo. Initially, it was primarily used to report calcium dynamics as a proxy for neural activity via genetically encoded indicators. This generated new insights into brain functions including movement, memory, and motivation at the level of defined circuits and cell types. Recently, the opportunity for discovery with fiber photometry has exploded with the development of an extensive range of fluorescent sensors for biomolecules including neuromodulators and peptides that were previously inaccessible in vivo. This critical advance, combined with the new availability of affordable "plug-and-play" recording systems, has made monitoring molecules with high spatiotemporal precision during behavior highly accessible. However, while opening exciting new avenues for research, the rapid expansion in fiber photometry applications has occurred without coordination or consensus on best practices. Here, we provide a comprehensive guide to help end-users execute, analyze, and suitably interpret fiber photometry studies.

Integrating operant behavior and fiber photometry with the open-source python library Pyfiber.

Despite the popularity of fiber photometry (FP), its integration with operant behavior paradigms is progressing slowly. This can be attributed to the complex protocols in operant behavior - resulting in a combination of diverse non-predictable behavioral responses and scheduled events, thereby complicating data analysis. To overcome this, we developed Pyfiber, an open-source python library which facilitates the merge of FP with operant behavior by relating changes in fluorescent signals within a neuronal population to behavioral responses and events. Pyfiber helps to 1. Extract events and responses that occur in operant behavior, 2. Extract and process the FP signals, 3. Select events of interest and align them to the corresponding FP signals, 4. Apply appropriate signal normalization and analysis according to the type of events, 5. Run analysis on multiple individuals and sessions, 6. Collect results in an easily readable format. Pyfiber is suitable for use with many different fluorescent sensors and operant behavior protocols. It was developed using Doric lenses FP systems and Imetronic behavioral systems, but it possesses the capability to process data from alternative systems. This work sets a solid foundation for analyzing the relationship between different dimensions of complex behavioral paradigms with fluorescent signals from brain regions of interest.

Evaluation of photometric stereo and elastomeric sensor imaging for the non-destructive 3D analysis of questioned documents - A pilot study.

Photometric Stereo and Elastomeric Sensor Imaging were assessed for measuring the 3-dimensional (3D) morphology of questioned document samples. Photometric stereo is shown to be a powerful non-contact technique for revealing micron level detail of the samples examined. Elastomeric Sensor Imaging is shown to complement photometric stereo by yielding equivalent results. Additionally, this technique allows quantification of the morphological depth information. The techniques were applied to 2 different types of questioned document sample. Firstly, written signatures were examined. Both techniques were able to reveal characteristic features that could be used to infer stroke direction and ink line application sequence. Secondly toner/ink intersections were examined. Both techniques allowed visualisation of 3D features which were used to infer the sequence of application.

Dynamic Analysis of Aerosol Release from a Pressurized Metered Dose Inhaler Combined with a Valved Holding Chamber Using Simplified Laser Photometry.

Background: A pressurized metered dose inhaler combined with a valved holding chamber (pMDI+VHC) is used to prevent upper airway complications and improve the efficiency of inhaled drug delivery; however, the aerodynamic behavior of the released particles has not been well investigated. This study aimed at clarifying the particle release profiles of a VHC using simplified laser photometry. Methods: An inhalation simulator comprised a computer-controlled pump and a valve system that withdrew aerosol from a pMDI+VHC using a jump-up flow profile. A red laser illuminated the particles leaving VHC and evaluated the intensity of the light reflected by the released particles. Results: The data suggested that the output (OPT) from the laser reflection system represented particle concentration rather than particle mass, and the latter was calculated as OPT × instantaneous withdrawn flow (WF). Summation of OPT hyperbolically decreased with flow increment, whereas summation of OPT × instantaneous flow was not influenced by WF strength. Particle release trajectories consisted of three phases, namely increment with a parabolic curve, flat, and decrement with exponential decay phases. The flat phase appeared exclusively at low-flow withdrawal. These particle release profiles suggest the importance of early phase inhalation. The hyperbolic relationship between WF and particle release time revealed the minimal required withdrawal time at an individual withdrawal strength. Conclusions: The particle release mass was calculated as laser photometric output × instantaneous flow. Simulation of the released particles suggested the importance of early phase inhalation and predicted the minimally required withdrawal time from a pMDI+VHC.

A paired emitter-detector diode-based photometer for the determination of sodium hypochlorite adulteration in milk.

This paper reports on developing a low cost but efficient paired emitter-detector diode (PEDD)-based photometer. The photometer consists of a white light-emitting diode (LED) as the emitter diode, an RGB LED as the detector diode, and a multimeter for recoding the signal. The developed PEDD-based photometer was utilized for the determination of liquid bleach adulteration in cow milk samples. N,N-Diethyl-p-phenylenediamine sulfate aqueous solution of pH 6 was used as a probe to monitor the presence of residual active chlorine in milk. The results showed that the developed method could be used to determine sodium hypochlorite in the concentration range of 0.5 to 20.0 ppm Cl2 with 0.14 and 0.46 ppm Cl2 limit of detection and limit of quantification, respectively. The intraday and interday precisions of the method at two concentration levels of 5.5 and 13.7 ppm Cl2 were 1.04% and 0.52%, and 1.81% and 1.02%, respectively. The recoveries of 114.2% and 106.9% were obtained for 5.5 and 13.7 ppm Cl2 concentrations levels, respectively. Real sample analyzes results showed that "maybe" liquid bleach adulteration in milk is the case for local distributors of raw milk.

Relevance of Laser Flare Photometry in Retinal Pathologies. / Relevanz der Laser-Flare-Photometrie bei retinalen Pathologien.

Laser flare photometry provides a non-invasive and objective measurement of the Tyndall effect in the anterior chamber. The laser flare value (measured in photon number per millisecond [pc/ms]) thus quantifies the extent of disruption to the blood-aqueous barrier and can be used in clinical applications to monitor uveitis therapy or to measure the postoperative degree of inflammation. Standardised performance must be observed during measurement. Publications of the last 35 years on laser flare photometry deal not only with the measurement procedure but also with its use in clinical practice for different ophthalmological pathologies. Likewise, various influencing factors have already been investigated and described that must be considered when measuring and evaluating laser flare values. The focus of this article is the relevance of laser flare photometry in retinal pathologies. In recently published studies, the level of objective tyndallometry in primary rhegmatogenous retinal detachment is shown to depend on lens status, symptom duration, and extent of retinal detachment. The greater is the area of the retina affected, the greater the disruption of the blood-aqueous barrier appears to be. Elevated laser flare values have also been considered as a predictor for the development of proliferative vitreoretinopathy (PVR). However, based on current knowledge, this assumption must be put into perspective. According to current data, objective tyndallometry can be used to monitor the progression of intraocular inflammation and to quantify the blood-aqueous barrier, and the values correlate with the extent and anatomical features, as well as the symptom duration in retinal detachment. Many influencing factors have already been identified. But further evaluation is desirable and needed. It is still unclear whether laser flare values can be used in the future as a predictor for sequelae such as PVR development.

The Value of Laser Flare Photometry as a Monitoring Tool for Uveitis. / Der Stellenwert von Laser-Flare-Messungen zur Verlaufsbeurteilung von Uveitiden.

Laser flare (LF) photometry (P) is used to quantify the protein concentration in the aqueous humor, and therefore assess the blood-aqueous humor barrier. LFP is more reliable than the clinical assessment of the Tyndall effect, and is thus especially useful in the follow-up of uveitis patients. In active uveitis, LFP correlates well with the anterior chamber cell grading. Various studies have shown that high LF values are associated with an increased risk of uveitic complications, such as macular edema, glaucoma, and posterior synechiae. LFP can also be used to assess the response to anti-inflammatory treatments as well as the optimal timing and selection of the surgical technique for intraocular surgeries.

Recent Advances in Single Cell Analysis by Electrochemiluminescence.

Understanding biological mechanisms operating in cells is one of the major goals of biology. Since heterogeneity is the fundamental property of cellular systems, single cell measurements can provide more accurate information about the composition, dynamics, and regulatory circuits of cells than population-averaged assays. Electrochemiluminescence (ECL), the light emission triggered by electrochemical reactions, is an emerging approach for single cell analysis. Numerous analytes, ranging from small biomolecules such as glucose and cholesterol, proteins and nucleic acids to subcellular structures, have been determined in single cells by ECL, which yields new insights into cellular functions. This review aims to provide an overview of research progress on ECL principles and systems for single cell analysis in recent years. The ECL reaction mechanisms are briefly introduced, and then the advances and representative works in ECL single cell analysis are summarized. Finally, outlooks and challenges in this field are addressed.

Spectrally Resolved Fiber Photometry for In Vivo Multi-Color Fluorescence Measurements.

This article describes how to assemble and operate a spectrometer-based fiber photometry system for in vivo simultaneous measurements of multiple fluorescent biosensors in freely moving mice. The first section of the article describes the step-by-step procedure to assemble a basic single-spectrometer fiber photometry system and how to expand it into a dual-spectrometer system that allows for simultaneous recordings from two sites. The second part describes the steps for a typical fiber probe implantation surgery. The last section describes how to acquire and analyze the time-lapsed spectral data. This article is intended for teaching labs how to build their own fiber photometry systems (with a video tutorial) from commercially available parts and perform in vivo recordings in behaving mice. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Assembling a dual-laser, single-spectrometer fiber photometry system Support Protocol: Dual-spectrometer fiber photometry assembly Basic Protocol 2: Optical fiber probe implantation Basic Protocol 3: Data acquisition and analysis.