Methods and Applications in Fluorescence最新文献

{"title":"Non-invasive assessment of intestinal permeability in healthy volunteers using transcutaneous fluorescence spectroscopy.","authors":"Jonathan Gan, Elena Monfort Sánchez, James Avery, Omar Barbouti, Jonathan Hoare, Hutan Ashrafian, Ara Darzi, Alex J Thompson","doi":"10.1088/2050-6120/ac9513","DOIUrl":"10.1088/2050-6120/ac9513","url":null,"abstract":"<p><p>The permeability of the intestinal barrier is altered in a multitude of gastrointestinal conditions such as Crohn's and coeliac disease. However, the clinical utility of gut permeability is currently limited due to a lack of reliable diagnostic tests. To address this issue, we report a novel technique for rapid, non-invasive measurement of gut permeability based on transcutaneous ('through-the-skin') fluorescence spectroscopy. In this approach, participants drink an oral dose of a fluorescent dye (fluorescein) and a fibre-optic fluorescence spectrometer is attached to the finger to detect permeation of the dye from the gut into the blood stream in a non-invasive manner. To validate this technique, clinical trial measurements were performed in 11 healthy participants. First, after 6 h of fasting, participants ingested 500 mg of fluorescein dissolved in 100 ml of water and fluorescence measurements were recorded at the fingertip over the following 3 h. All participants were invited back for a repeat study, this time ingesting the same solution but with 60 g of sugar added (known to transiently increase intestinal permeability). Results from the two study datasets (without and with sugar respectively) were analysed and compared using a number of analysis procedures. This included both manual and automated calculation of a series of parameters designed for assessment of gut permeability. Calculated values were compared using Student's T-tests, which demonstrated significant differences between the two datasets. Thus, transcutaneous fluorescence spectroscopy shows promise in non-invasively discriminating between two differing states of gut permeability, demonstrating potential for future clinical use.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":"10 4","pages":""},"PeriodicalIF":2.4,"publicationDate":"2022-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10590297","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}

{"title":"Fluorescence measurements: importance of G-factor correction, magic angle, and observation wavelengths.","authors":"Emma Kitchner,&nbsp;Michael Seung,&nbsp;Jose Chavez,&nbsp;Luca Ceresa,&nbsp;Joseph Kimball,&nbsp;Ignacy Gryczynski,&nbsp;Zygmunt Gryczynski","doi":"10.1088/2050-6120/ac92c5","DOIUrl":"https://doi.org/10.1088/2050-6120/ac92c5","url":null,"abstract":"<p><p>Excitation and emission (observation) conditions heavily impact fluorescence measurements. Both observed spectra and intensity decays (fluorescence lifetimes), when incorrectly measured, may lead to incorrect data interpretations. In this report, we discuss the role of observation conditions in steady-state and time-resolved (lifetime) fluorescence measurements. We demonstrate the importance of the correction for uneven transmissions of vertical and horizontal polarizations of emission light through the detection system. The necessity of using so-called total fluorescence intensity or intensity measured under magic angle (MA) conditions has been demonstrated for both steady-state and time-resolved fluorescence measurements. The dependence of lifetime measurements on observation (emission) wavelengths is also discussed. Two fluorophores, rhodamine 6G (R6G) and 4,4 Dimethylamino-cyano stilbene (DCS) in two solvents - ethanol and glycerol have been used in order to cover a broad range of dye polarities and solvent viscosities.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40364646","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}

{"title":"Label-free monitoring of proteins in optofluidic hollow-core photonic crystal fibres.","authors":"Jan R Heck,&nbsp;Ermanno Miele,&nbsp;Ralf Mouthaan,&nbsp;Michael H Frosz,&nbsp;Tuomas P J Knowles,&nbsp;Tijmen G Euser","doi":"10.1088/2050-6120/ac9113","DOIUrl":"https://doi.org/10.1088/2050-6120/ac9113","url":null,"abstract":"<p><p>The fluorescent detection of proteins without labels or stains, which affect their behaviour and require additional genetic or chemical preparation, has broad applications to biological research. However, standard approaches require large sample volumes or analyse only a small fraction of the sample. Here we use optofluidic hollow-core photonic crystal fibres to detect and quantify sub-microlitre volumes of unmodified bovine serum albumin (BSA) protein down to 100 nM concentrations. The optofluidic fibre's waveguiding properties are optimised for guidance at the (auto)fluorescence emission wavelength, enabling fluorescence collection from a 10 cm long excitation region, increasing sensitivity. The observed spectra agree with spectra taken from a conventional cuvette-based fluorimeter, corrected for the guidance properties of the fibre. The BSA fluorescence depended linearly on BSA concentration, while only a small hysteresis effect was observed, suggesting limited biofouling of the fibre sensor. Finally, we briefly discuss how this method could be used to study aggregation kinetics. With small sample volumes, the ability to use unlabelled proteins, and continuous flow, the method will be of interest to a broad range of protein-related research.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33456040","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}

{"title":"Deciphering modes of long-range energy transfer in perovskite crystals using confocal excitation and wide-field fluorescence spectral imaging.","authors":"Tejmani Behera,&nbsp;Nithin Pathoor,&nbsp;Rajat Mukherjee,&nbsp;Arindam Chowdhury","doi":"10.1088/2050-6120/ac8f85","DOIUrl":"https://doi.org/10.1088/2050-6120/ac8f85","url":null,"abstract":"<p><p>Excitation energy migration beyond mesoscale is of contemporary interest for both solar photovoltaic and light-emissive devices, especially in context of organometal halide perovskites (OMHPs) which have been shown to have very long (charge carrier) diffusion lengths. While understanding the energy propagation pathways in OMHPs is crucial for further advancement of material design and improvement of opto-electronic features, the simultaneous existence of multiple processes like carrier diffusion, photon recycling, and photon transport makes it often complex to differentiate them. In this study, we unravel the diverse yet dominant excitation energy transfer mode(s) in crystalline MAPbBr₃micron-sized 1D rods and plates by localized (confocal) laser excitation coupled with spectrally-resolved wide-field fluorescence imaging. While rarely used, this technique can efficiently probe excitation migration beyond the diffraction limit and can be realized by simple modification of existing epifluorescence microscopy setups. We find that in rods of length below ∼2 microns, carrier diffusion dominates amongst various energy transfer processes. However, the transient non-radiative defects severely inhibit the extent of carrier migration and also temporarily affect the radiative recombination dynamics of the photo-carriers. For MAPbBr₃plates of several tens of micrometers, we find that the photoluminescence (PL) spectral characteristics remain unaltered at short distances (< ∼3<i>μ</i>m) while at a larger distance, the spectral profile is gradually red-shifted. This implies that carrier diffusion dominates over small distances, while photon recycling,<i>i.e.</i>, repeated re-absorption and re-emission of photons, propagates excitation energy transfer over extended length scales with assistance from wave-guided photon transport. Our findings can potentially be used for future studies on the characterization of energy transport mechanisms in semiconductor solids as well as for organic (molecular) self-assembled microstructures.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40352162","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}

{"title":"Correlation analyses reveal differential diffusion behavior of eisosomal proteins between mother and daughter cells.","authors":"Francisco G Correa Tedesco,&nbsp;Pablo S Aguilar,&nbsp;Laura C Estrada","doi":"10.1088/2050-6120/ac8fe1","DOIUrl":"https://doi.org/10.1088/2050-6120/ac8fe1","url":null,"abstract":"<p><p>Eisosomes are nanoscale plasma membrane domains shaped as furrow-like invaginations. In<i>Saccharomyces cerevisiae</i>these relatively immobile and uniform structures are mainly composed of two cytoplasmic proteins Pil1 and Lsp1. The present work uses fluctuation of fluorescence signals and analytical methods to determine Pil1 and Lsp1 dynamics at different subcellular locations. Using scanning techniques and autocorrelation analysis we determine that the cytoplasmic pools of Pil1 and Lsp1 behave mainly by passive diffusion. Single-point FCS experiments performed at several subcellular locations reveal that Pil1 mobility is faster in daughter cells. Furthermore, pair correlation function analysis indicates a rapid dynamic of Pil1 near the plasma membrane of growing yeast buds, where the membrane is expected to be actively assembling eisosomes.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40353050","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}

{"title":"<i>i-scope</i>: a compact automated fluorescence microscope for cell counting applications in low resource settings.","authors":"Arti Tyagi,&nbsp;Neha Khaware,&nbsp;Bramha Tripathi,&nbsp;Tushar Jeet,&nbsp;Prabhu Balasubramanian,&nbsp;Ravikrishnan Elangovan","doi":"10.1088/2050-6120/ac8f84","DOIUrl":"https://doi.org/10.1088/2050-6120/ac8f84","url":null,"abstract":"<p><p>Fluorescence microscopy has widespread applications across biological sciences. It has been routinely used for cell counting, which provides a preliminary diagnostic test for many infectious diseases. Conventional fluorescence microscopes are bulky, expensive, time-intensive and laborious. They often require trained operators to acquire and analyze data. We report a compact automated digital fluorescence microscopy system,<i>i-scope</i>, for cell counting applications. The<i>i-scope</i>employs a total internal reflection fluorescence (TIRF) mode of sample illumination, along with a brightfield mode. It has a magnification of 30X, an optical resolution of ∼0.2<i>μ</i>m/pixel and offers sample scanning over 20 mm × 20 mm. A custom-written program enables automated image acquisition and analysis, thereby enhancing ease of operation. It has a compact form-factor and has been developed into a standalone system with a processing unit, screen, and other accessories to offer a portable and economic point-of-care diagnostic solution in low-resource settings. We analysed the performance of the i<i>-scope</i>for milk somatic cell enumeration and benchmarked it against that of a conventional fluorescence microscope.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40352160","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}

{"title":"A high-sensitivity rapid acquisition spectrometer for lanthanide(III) luminescence.","authors":"Patrick R Nawrocki,&nbsp;Villads R M Nielsen,&nbsp;Thomas Just Sørensen","doi":"10.1088/2050-6120/ac8d4d","DOIUrl":"https://doi.org/10.1088/2050-6120/ac8d4d","url":null,"abstract":"<p><p>Detecting luminescence beyond 750-800 nm becomes problematic as most conventional detectors are less sensitive in this range, and as simple corrections stops being accurate. Lanthanide luminescence occurs in narrow bands across the spectrum from 350-2000 nm. The most emissive lanthanide(III) ions have bands from 450 nm to 850 nm, some with additional bands in the NIR. Investigating NIR bands are hard, but the difficulties already start at 700 nm. In general, the photon flux from lanthanide(III) emitters is not great, and the bands beyond 700 nm are very weak, we therefore decided to build a spectrometer based on cameras for microscopy with single-photon detection capabilities. This was found to allieviate all limitations and to allow for fast and efficient recording of luminescence spectra in the range from 450 to 950 nm. The spectrometer characteristics were investigated and the performance was benchmarked against two commercial spectrometers. We conclude that this spectrometer is ideal for investigating lanthanide luminescence, and all other emitters with emission in the target range.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40446158","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}

{"title":"The innards of the cell: studies of water dipolar relaxation using the ACDAN fluorescent probe.","authors":"Santiago Otaiza-González,&nbsp;Manuel Cabadas,&nbsp;German Robert,&nbsp;Roberto P Stock,&nbsp;Leonel Malacrida,&nbsp;Ramiro Lascano,&nbsp;Luis A Bagatolli","doi":"10.1088/2050-6120/ac8d4c","DOIUrl":"https://doi.org/10.1088/2050-6120/ac8d4c","url":null,"abstract":"<p><p>This article reviews the use of the 6-acetyl-2-(dimethylamino)naphthalene (ACDAN) fluorophore to study dipolar relaxation in cells, tissues, and biomimetic systems. As the most hydrophilic member of the 6-acyl-2-(dimethylamino)naphthalene series, ACDAN markedly partitions to aqueous environments. In contrast to 6-lauroyl-2-(dimethylamino)naphthalene (LAURDAN), the hydrophobic and best-known member of the series used to explore relaxation phenomena in biological (or biomimetic) membranes, ACDAN allows mapping of spatial and temporal water dipolar relaxation in cytosolic and intra-organelle environments of the cell. This is also true for the 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) derivative which, unlike LAURDAN, partitions to both hydrophobic and aqueous environments. We will (i) summarize the mechanism which underlies the solvatochromic properties of the DAN probes, (ii) expound on the importance of water relaxation to understand the intracellular environment, (iii) discuss technical aspects of the use of ACDAN in eukaryotic cells and some specialized structures, including liquid condensates arising from processes leading to liquid immiscibility and, (iv) present some novel studies in plant cells and tissues which demonstrate the kinds of information that can be uncovered using this approach to study dipolar relaxation in living systems.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40446147","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}

{"title":"Spectroscopic investigation on the interaction of direct yellow-27 with protein (BSA).","authors":"Babita Bisht,&nbsp;Pinki Dey,&nbsp;Avinash Kumar Singh,&nbsp;Sanjay Pant,&nbsp;Mohan Singh Mehata","doi":"10.1088/2050-6120/ac8a8b","DOIUrl":"https://doi.org/10.1088/2050-6120/ac8a8b","url":null,"abstract":"<p><p>Direct yellow 27 (DY-27) interaction with bovine serum albumin (BSA) was investigated using multi-spectroscopic techniques to understand the toxicity mechanism. Fluorescence quenching of BSA by DY-27 was observed as a result of the formation of a BSA-DY27 complex with a binding constant of 1.19 × 10⁵M^-1and followed a static quenching mechanism with a quenching constant K_svof 7.25 × 10⁴M^-1. The far UV circular dichroism spectra revealed the conformational changes in the secondary structure of BSA in the presence of DY-27. The calculated average lifetime of BSA is 6.04 ns and is nearly constant (5.99 ns) in the presence of dye and supports the proposed quenching mechanism. The change in free energy (ΔG) was calculated to be -28.96 kJ mol^-1and confirmed the spontaneity of the binding process. Further, docking studies have been conducted to gain more insights into the interactions between DY-27 and serum albumin.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40618699","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}

{"title":"Development of resistance to 5-fluorouracil affects membrane viscosity and lipid composition of cancer cells.","authors":"Liubov Shimolina,&nbsp;Alexander Gulin,&nbsp;Aleksandra Khlynova,&nbsp;Nadezhda Ignatova,&nbsp;Irina Druzhkova,&nbsp;Margarita Gubina,&nbsp;Elena Zagaynova,&nbsp;Marina K Kuimova,&nbsp;Marina Shirmanova","doi":"10.1088/2050-6120/ac89cd","DOIUrl":"https://doi.org/10.1088/2050-6120/ac89cd","url":null,"abstract":"<p><p>The investigations reported here were designed to determine whether the bulk plasma membrane is involved in mechanisms of acquired resistance of colorectal cancer cells to 5-fluorouracil (5-FU). Fluorescence lifetime imaging microscopy (FLIM) of live cultured cells stained with viscosity-sensitive probe BODIPY 2 was exploited to non-invasively assess viscosity in the course of treatment and adaptation to the drug. In parallel, lipid composition of membranes was examined with the time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results showed that a single treatment with 5-FU induced only temporal changes of viscosity in 5-FU sensitive cells immediately after adding the drug. Acquisition of chemoresistance was accompanied by persistent increase of viscosity, which was preserved upon treatment without any changes. Lipidomic analysis revealed that the resistant cells had a lower level of monounsaturated fatty acids and increased sphingomyelin or decreased phosphatidylcholine in their membranes, which partly explain increase of the viscosity. Thus, we propose that a high membrane viscosity mediates the acquisition of resistance to 5-FU.</p>","PeriodicalId":18596,"journal":{"name":"Methods and Applications in Fluorescence","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40614244","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}