Methods and Applications in Fluorescence最新文献

Compensating for photon counting losses in a TCSPC SPAD array enables quantitative time-resolved fluorescence anisotropy imaging. 补偿光子计数损失在TCSPC SPAD阵列使定量时间分辨荧光各向异性成像。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-05-06 DOI: 10.1088/2050-6120/ae6989

Louis Obeid Mogridge, Jakub Nedbal, Istvan Gyongy, Robert Henderson, Klaus Suhling

{"title":"Compensating for photon counting losses in a TCSPC SPAD array enables quantitative time-resolved fluorescence anisotropy imaging.","authors":"Louis Obeid Mogridge, Jakub Nedbal, Istvan Gyongy, Robert Henderson, Klaus Suhling","doi":"10.1088/2050-6120/ae6989","DOIUrl":"https://doi.org/10.1088/2050-6120/ae6989","url":null,"abstract":"We devise and experimentally validate a theoretical model to account for lost photon counts during the exposure time of a time-correlated single photon counting (TCSPC)-based QuantICAM single photon avalanche diode (SPAD) array camera. The work is motivated by the quest for TCSPC-based wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM), implemented by the acquisition of images at orthogonal polarization. For accurate, quantitatively correct TR-FAIM, the two images must be acquired under equivalent conditions and any photons lost during the camera exposures must be precisely quantified. Our model is based on a binomial distribution with a single adjustable parameter. We plot the recorded versus the true photon counts for exposure times of 250 µs and 1000 µs, using photons with random arrival times and from fluorescence decays. Our model describes the experimental data well and the correct number of excitation cycles during the exposure time is extracted from least-squares fits of the binomial model to the experimental data. On the basis of this model, we account for lost photons in TCSPC-based TR-FAIM and show that a compensation for lost photons is essential to obtain quantitatively correct steady state anisotropy and G-factor histograms in TR-FAIM. We also show that, under the conditions used, the rotational correlation time, initial anisotropy r0and hindered rotation parameter r∞histograms are only marginally affected by lost photons. Our work thus paves the way for robust and reliable TCSPC-based TR-FAIM.","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog. 一种本质荧光麦角甾醇类似物的单光子和双光子活细胞成像研究了酵母中的甾醇运输。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-05-04 DOI: 10.1088/2050-6120/ae5e19

Katja Thaysen, Max Lehmann, Vibeke Akkerman, Mohammad Bashawat, Peter Reinholdt, Jenny Leopold, Jürgen Schiller, Holger A Scheidt, Jacob Kongsted, Eric Sperlich, Pablo Wessig, Peter Müller, Daniel Wüstner

{"title":"Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog.","authors":"Katja Thaysen, Max Lehmann, Vibeke Akkerman, Mohammad Bashawat, Peter Reinholdt, Jenny Leopold, Jürgen Schiller, Holger A Scheidt, Jacob Kongsted, Eric Sperlich, Pablo Wessig, Peter Müller, Daniel Wüstner","doi":"10.1088/2050-6120/ae5e19","DOIUrl":"10.1088/2050-6120/ae5e19","url":null,"abstract":"Ergosterol is the main sterol in yeast and an important lipid constituent of the yeast plasma membrane (PM). Methods for analysis of ergosterol trafficking between PM and subcellular compartments often rely on fluorescence microscopy, but existing sterol probes either mimic ergosterol poorly or have inconvenient fluorescence properties. Here, we present a novel intrinsically fluorescent probe that differs from ergosterol only by having a 3'-keto group and two additional conjugated double bonds in the ring system. We show that this analog, named Erg-Tetraene, can order fatty acyl chains of phospholipids and partitions partially into the liquid-ordered phase in model membranes containing cholesterol. The Erg-Tetraene has a red-shifted emission and a much stronger two-photon absorption than the widely used analog dehydroergosterol, allowing for its convenient imaging on commercial microscope systems. Using multi-color confocal and two-photon microscopy, we show that uptake of Erg-Tetraene into yeast depends on the sterol transporters Aus1/Pdr11 and is followed by rapid transport to the vacuole and to lipid droplets. Together, we present a novel analogue of ergosterol with improved fluorescence properties for sterol trafficking studies in yeast and other model organisms.","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147654502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development. 一种新的基于成像的多样品粘度测定方法，用于推进单克隆抗体的开发。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-04-30 DOI: 10.1088/2050-6120/ae5e1a

Karen Leonor Lopez, Francesco Palomba, Marissa Mock, Nithya Srinivasan, Emma Pelegri-O'Day, Michelle A Digman

{"title":"A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development.","authors":"Karen Leonor Lopez, Francesco Palomba, Marissa Mock, Nithya Srinivasan, Emma Pelegri-O'Day, Michelle A Digman","doi":"10.1088/2050-6120/ae5e1a","DOIUrl":"10.1088/2050-6120/ae5e1a","url":null,"abstract":"The increasing demand for monoclonal antibodies (mAbs) as therapeutic agents highlights the ever-growing necessity of optimizing sample-selection procedures; the rate-limiting step of therapy development. One such mAb property test is viscosity, which affects syringeability, concentration dose, and patient experience. Current viscometry methods, such as cone and plate rheometry, although accurate, are limited by low throughput, high sample volume requirements, and lack of automation, driving up the cost and time of mAb development. This paper introduces an innovative imaging-based viscometry application that significantly mitigates these issues. By integrating a wide-field camera with a fluorescence microscope and Python-based single particle tracking (SPT) software, this approach allows for rapid, low-volume (down to 2μl) viscosity measurements of mAb solutions. Using 200 nm yellow-green fluorescent polystyrene beads as tracers, the system ensures accurate macroviscosity assessments of protein solutions and the capability for high-throughput analysis. The platform's precision and sensitivity were validated using bovine serum albumin and viscosity standard solutions, followed by a single-blinded study using Immunoglobulin G1 and G2 (IgG1/IgG2) solutions of varying concentrations (⩽150 mg ml-1) and viscosities (2-31 cP). When compared to the unblinded values, the SPT blinded sample analysis resulted in a linear fit ofR2= 0.97 and an average error of 2.1 cP. Our findings suggest that this novel platform can substantially streamline and enhance the mAb development process, offering a feasible solution to one of the industry's pressing challenges.","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2026-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147654550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A novel carbon dot-based fluorescence/UV dual-mode sensing method for the detection of organophosphorus pesticides. 一种新型碳点荧光/紫外双模传感检测有机磷农药的方法。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-04-29 DOI: 10.1088/2050-6120/ae5443

Bingyu Luo, Qingmiao Pu, Yuru Yang, Ju Wei, Huisan Cai, Song Lu, Xiaodong Su

{"title":"A novel carbon dot-based fluorescence/UV dual-mode sensing method for the detection of organophosphorus pesticides.","authors":"Bingyu Luo, Qingmiao Pu, Yuru Yang, Ju Wei, Huisan Cai, Song Lu, Xiaodong Su","doi":"10.1088/2050-6120/ae5443","DOIUrl":"10.1088/2050-6120/ae5443","url":null,"abstract":"This study presents a fluorescence-colorimetric dual-mode method for the detection of organophosphorus pesticides (OPPs). The method exploits the fact that products of organophosphorus pesticides oxidized by potassium persulfate can both quench the fluorescence of carbon dots and induce color changes. Using Dimethoate (Dim) as the model analyte, the linear detection ranges for the fluorescence and colorimetric modes were 0.05-4.6μg ml-1and 0.05-3.0μg ml-1, respectively, with limits of detection of 0.027μg ml-1and 0.0109μg ml-1. The synergistic dual-mode response significantly enhances the reliability of the measurements. In spiked recovery tests using actual water samples, both detection modes demonstrated high recovery rates, confirming that the method maintains excellent accuracy and precision even in complex matrices. The dual-mode sensing probe developed in this study features a straightforward preparation process and achieves efficient, sensitive, and reliable detection of multiple OPPs, offering a promising analytical tool for monitoring pesticide residues.","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147481082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Quantitative fluorescence imaging of the impact of the nucleocapsid domain deletion on the dynamics of HIV-1 Gag assembly. 核衣壳结构域缺失对HIV-1 Gag组装动力学影响的定量荧光成像。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-04-10 DOI: 10.1088/2050-6120/ae5504

Iryna Lysova, Pascal Didier, Nadia Messaddeq, Jeanne Bouthenet, Yves Mély, Halina Anton

{"title":"Quantitative fluorescence imaging of the impact of the nucleocapsid domain deletion on the dynamics of HIV-1 Gag assembly.","authors":"Iryna Lysova, Pascal Didier, Nadia Messaddeq, Jeanne Bouthenet, Yves Mély, Halina Anton","doi":"10.1088/2050-6120/ae5504","DOIUrl":"10.1088/2050-6120/ae5504","url":null,"abstract":"The human immunodeficiency virus 1 (HIV-1) group specific antigen (Gag) polyprotein, the main structural protein of HIV-1 is sufficient to mimic the late stages of the viral replication cycle. When expressed in cells, Gag binds to RNAs and assembles at the plasma membrane to form virus-like particles (VLPs) with a morphology similar to that of HIV-1 virions. The nucleocapsid (NC) domain of Gag plays a critical role in RNA binding and selective encapsidation of the viral genome. This work focuses on the impact of NC deletion on Gag assembly, VLP formation, and intracellular trafficking. By combining several fluorescence-based quantitative microscopy techniques, we show that in the absence of the NC domain, Gag cytoplasmic oligomerization and formation of initial ribonucleoprotein complexes are impacted, resulting in a significant delay in the kinetics of VLP formation. Interestingly, VLPs formed from the Gag-ΔNC mutant display greater diversity in size and shape. Single particle tracking experiments revealed that VLPs formed at the plasma membrane are immobile, while intracellular VLPs are mostly mobile. For the latter, the motions and diffusion coefficients of VLPs formed by the Gag-ΔNC mutant were highly similar to those of VLPs formed with the wild-type Gag, indicating that the NC domain is not implicated in the intracellular trafficking. These findings illustrate how fluorescence-based microscopy techniques can provide quantitative insights into the role of the NC domain in Gag assembly.","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2026-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147486613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease. 尼罗红光谱评价肾小球肾病疾病特异性脂质改变。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-04-09 DOI: 10.1088/2050-6120/ae56af

Asha Swamy, Wulin Teo, Jason T Bau, Adrienne Kline, Aysa Imanzadeh, Kevin Chapman, Graciela Andonegui, Hallgrimur Benediktsson, Daniel A Muruve, Peter K Stys, Justin Chun

{"title":"Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease.","authors":"Asha Swamy, Wulin Teo, Jason T Bau, Adrienne Kline, Aysa Imanzadeh, Kevin Chapman, Graciela Andonegui, Hallgrimur Benediktsson, Daniel A Muruve, Peter K Stys, Justin Chun","doi":"10.1088/2050-6120/ae56af","DOIUrl":"10.1088/2050-6120/ae56af","url":null,"abstract":"Lipid accumulation has been implicated in the progression of chronic kidney disease, yet its role in glomerulonephritis (GN) remains poorly characterized. Here we use Nile Red (NR), a solvatochromic fluorophore that stains for lipid droplets (LDs) to examine LD distribution and lipid chemistry in glomerular diseases. Seventy-two kidney biopsy samples, including histologically diagnosed GN subtypes and control nephrectomy tissues, were stained with NR. LDs were quantified using a semi-automated MATLAB-based image analysis pipeline and spectral emission profiles were generated to assess the emission ratio, reflecting differences in lipid composition. We found that GN subtypes exhibit distinct patterns of lipid accumulation and lipid polarity as evaluated by using a NR emission ratio. Our study demonstrates the utility of NR fluorescence spectroscopy in detecting disease-specific lipid alterations in GN. The findings suggest that distinct lipid signatures may serve as pathological indicators, offering potential new diagnostic and mechanistic insights into glomerular disease.","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2026-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147513281","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A review on application of laser induced fluorescence spectroscopy in exploring bioaerosol characteristics. 激光诱导荧光光谱技术在生物气溶胶特性研究中的应用综述。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-04-01 DOI: 10.1088/2050-6120/ae5505

Pragya Parmita Konwar, Manoj Saikia, Simanta Hazarika

引用次数: 0

Smartphones with multispectral imaging for medical testing. 用于医学测试的多光谱成像智能手机。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-03-23 DOI: 10.1088/2050-6120/ae501c

Joseph R Lakowicz, Kundan Sivashanmugan, E Albert Reece

引用次数: 0

FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets. FLIM质量度量可视化是验证大面积非同质FLIM数据集一致性的一种手段。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-03-18 DOI: 10.1088/2050-6120/ae4e7b

Helen M Wilson, Jenu V Chacko, David J Odde, Paolo P Provenzano, Kevin W Eliceiri

{"title":"FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets.","authors":"Helen M Wilson, Jenu V Chacko, David J Odde, Paolo P Provenzano, Kevin W Eliceiri","doi":"10.1088/2050-6120/ae4e7b","DOIUrl":"10.1088/2050-6120/ae4e7b","url":null,"abstract":"Robust and interpretable analysis of fluorescence lifetime imaging microscopy (FLIM) data requires careful assessment of data across biological samples. Due to limitations in sample availability, difference in protein expression, photobleaching, or acquisition time, FLIM datasets are often susceptible to signal variability. This is only exacerbated with large field-of-view FLIM data, such as examining metabolic fluxes across whole tissue slices due to morphology changes. We adapt the FLIM F-value (or figure-of-merit) within our analysis as a statistical metric to capture the confidence in lifetime by comparing variance across fitted parameters, analogous to typical image SNR. In this study, we apply pixelwise and regional analysis of F-values across large-area FLIM datasets to identify image regions with similar confidence levels. Visualization of F-value distribution enables detection of acquisition outliers or poor-quality regions within a large mosaic collection, which can be flagged for reacquisition or removal. This approach enhances the statistical power of downstream biological interpretation by ensuring that only data with quantifiable and stable lifetime information are retained. To our knowledge, this is the first application of F-value mapping as a dataset-wide quality control measure in FLIM.","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2026-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147369885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Normalization is essential for accurate intraoperative perfusion assessment using near-infrared fluorescence imaging. 归一化对于近红外荧光成像准确评估术中灌注至关重要。

IF 2.4 3区化学

Methods and Applications in Fluorescence Pub Date : 2026-03-16 DOI: 10.1088/2050-6120/ae4e7c

Roderick C Peul, Szymon M Kielbasa, Ferran Soebrata, Stefan Koning, Mo W Kruiswijk, Tom H Dijkhuis, Louelle E van der Aa, Indy Planting, Pim van den Hoven, Richelle J M Hoveling, Jaap F Hamming, Alexander L Vahrmeijer, Joost R van der Vorst

{"title":"Normalization is essential for accurate intraoperative perfusion assessment using near-infrared fluorescence imaging.","authors":"Roderick C Peul, Szymon M Kielbasa, Ferran Soebrata, Stefan Koning, Mo W Kruiswijk, Tom H Dijkhuis, Louelle E van der Aa, Indy Planting, Pim van den Hoven, Richelle J M Hoveling, Jaap F Hamming, Alexander L Vahrmeijer, Joost R van der Vorst","doi":"10.1088/2050-6120/ae4e7c","DOIUrl":"10.1088/2050-6120/ae4e7c","url":null,"abstract":"Significance. Near-infrared fluorescence (NIRF) imaging is increasingly used for perfusion assessment in clinical care and research settings. While subjective interpretation demonstrates clinical benefits, quantitative analysis is crucial for broader adoption and classification of perfusion patterns. However, strict standardization often conflicts with fast-paced clinical workflow, obstructing broad implementation.Aim and approach. This study hypothesized that normalization of fluorescence measurements could effectively correct for measurement variability, reducing the need for strict standardization in quantitative NIRF perfusion imaging. To evaluate this, a model capable of consistently simulating fluorescence perfusion patterns was employed. Experiments were conducted in an operating room using four NIRF camera systems under varying measurement conditions.Results. Normalization to maximum signal intensity provided consistent perfusion parameter values across differences in camera type, angle, rotation and settings (Δ≤1%). In cases of severe malperfusion where peak-intensity was not reached within the measurement period, normalization did not adequately correct for measurement variability (Δ increased).Conclusion. While standardization remains valuable beyond parameter accuracy, appropriate normalization substantially reduces dependence on strict measurement protocols. These findings support broader clinical adoption of quantitative NIRF imaging, extending its utility beyond specialized tertiary centers and facilitating widespread integration into routine surgical care.","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147369857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0