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Clean surface assured for more sensitive inspection with fluorescent penetrant dye. Simple procedure devised to ensure visible (red) penetrant dye used to identify flaws in welded surface completely removed from surface. Consists in applying reversible penetrant developer to surface to be inspected.
Transfer of dye shows quality of contact between two mating parts. Mating parts checked for fit by spreading fluorescent dye on one, making brief light contact with other, and looking (under UV light) for transferred dye. Dye offers greater visibility under ultraviolet illumination, allowing better indication of how precisely parts match and what areas interfere.
A new method for generating photogrammetric targets by projecting an array of laser beams onto a membrane doped with fluorescent laser dye has recently been developed. In this paper we review this new fluorescence based technique, then proceed to show how it can be used for dynamic measurements, and how a short pulsed (10 ns) laser allows the measurement of vibration modes at frequencies several times the sampling frequency. In addition, we present experimental results showing the determination of fundamental and harmonic vibration modes of a drum style dye-doped polymer membrane tautly mounted on a 12-inch circular hoop and excited with 30 Hz and 62 Hz sinusoidal acoustic waves. The projected laser dot pattern was generated by passing the beam from a pulsed Nd:YAG laser though a diffractive optical element, and the resulting fluorescence was imaged with three digital video cameras, all of which were synchronized with a pulse and delay generator. Although the video cameras are capable of 240 Hz frame rates, the laser s output was limited to 30 Hz and below. Consequently, aliasing techniques were used to allow the measurement of vibration modes up to 186 Hz with a Nyquist limit of less than 15 Hz.
Fluorescent dye-doped polystyrene latex microspheres (PSLs) are being developed for velocimetry and scalar measurements in variable property flows. Two organic dyes, Rhodamine B (RhB) and dichlorofluorescence (DCF), are examined to assess laser-induced fluorescence (LIF) properties for flow imaging applications and single-shot temperature measurements. A major interest in the current research is the application of safe dyes, thus DCF is of particular interest, while RhB is used as a benchmark. Success is demonstrated for single-point laser Doppler velocimetry (LDV) and also imaging fluorescence, excited via a continuous wave 2 W laser beam, for exposures down to 10 ms. In contrast, when exciting with a pulsed Nd:YAG laser at 200 mJ/pulse, no fluorescence was detected, even when integrating tens of pulses. We show that this is due to saturation of the LIF signal at relatively low excitation intensities, 4-5 orders of magnitude lower than the pulsed laser intensity. A two-band LIF technique is applied in a heated jet, indicating that the technique effectively removes interfering inputs such as particle diameter variation. Temperature measurement uncertainties are estimated based upon the variance measured for the two-band LIF intensity ratio and the achievable dye temperature sensitivity, indicating that particles developed to date may provide about +/-12.5 C precision, while future improvements in dye temperature sensitivity and signal quality may enable single-shot temperature measurements with sub-degree precision.
No abstract available
Calcium has been implicated as a key component in the signal transduction process of root gravitropism. We measured cytoplasmic free calcium in protoplasts isolated from the elongation zone and cap of primary roots of light-grown, vertically oriented seedlings of Zea mays L. Protoplasts were loaded with the penta-potassium salts of fura-2 and indo-1 by incubation in acidic solutions of these calcium indicators. Loading increased with decreasing pH but the pH dependence was stronger for indo-1 than for fura-2. In the case of fura-2, loading was enhanced only at the lowest pH (4.5) tested. Dyes loaded in this manner were distributed predominantly in the cytoplasm as indicated by fluorescence patterns. As an alternative method of loading, protoplasts were incubated with the acetoxymethylesters of fura-2 and indo-1. Protoplasts loaded by this method exhibited fluorescence both in the cytoplasm and in association with various organelles. Cytoplasmic calcium levels measured using spectrofluorometry, were found to be 160 +/- 40 nM and 257 +/- 27 nM, respectively, in populations of protoplasts from the root cap and elongation zone. Cytoplasmic free calcium did not increase upon addition of calcium to the incubation medium, indicating that the passive permeability to calcium was low.
Light-induced blinking, an inherent feature of many forms of super-resolution microscopy, has been linked to transient reduction of the fluorescent cyanine dye used as an imaging agent. There is, however, only scant literature information related to one-electron reduced cyanine dyes, especially in an aqueous environment. Here, we examine a small series of cyanine dyes, possessing disparate π-conjugation lengths, under selective reducing or oxidizing conditions. The experiment allows recording of both differential absorption spectra and decay kinetics of the resultant one-electron reduced or oxidized transient species in water. Relative to the ground state, absorption transitions for the various radicals are weak and somewhat broadened but do allow correlation with the π-conjugation length. In all cases, absorption maxima lie to the blue of the main ground-state transition. Under anaerobic conditions, the transient species decay on the microsecond to millisecond time scale, with the mean lifetime depending on molecular structure, radiation dose, and dye concentration. The experimental absorption spectra recorded for the one-electron reduced radicals and the presumed dimer cation radical compare well to spectra obtained from time-dependent density functional theory calculations. Furthermore, the results allow conclusions to be drawn regarding the plausibility of the reduced species being responsible for light-induced blinking in direct stochastic optical reconstruction microscopy.
Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.
Abstract The advent of chatbots raises the possibility of a paradigm shift across society including the most technical of fields with regard to access to information, generation of knowledge, and dissemination of education and training. Photochemistry is a scientific endeavor with roots in chemistry and physics and branches that encompass diverse disciplines ranging from astronomy to zoology. Here, five chatbots have each been challenged with 13 photochemically relevant queries. The chatbots included ChatGPT 3.5, ChatGPT 4.0, Copilot, Gemini Advanced, and Meta AI. The queries encompassed fundamental concepts (e.g., “Why is the fluorescence spectrum typically the mirror image of the absorption spectrum?”), practical matters (e.g., “What is the inner filter effect and how to avoid it?”), philosophical matters (“Please create the most important photochemistry questions.”), and specific molecular features (e.g., “Why are azo dyes non‐fluorescent?”). The chatbots were moderately effective in answering queries concerning fundamental concepts in photochemistry but were glaringly deficient in specialized queries for dyes and fluorophores. In some instances, a correct response was embedded in verbose scientific nonsense whereas in others the entire response, while grammatically correct, was utterly meaningless. The unreliable accuracy makes present chatbots poorly suited for unaided educational purposes and highlights the importance of domain experts.
This minireview focuses on the application of fluorogenic compounds in the detection of bacteria with particular emphasis on the assessment of physiological activity using epifluorescence microscopy. Microbiological applications of several related methods will also be reviewed.
Laser-induced fluorescence target generation in dye-doped polymer films has recently been introduced as a promising alternative to more traditional photogrammetric targeting techniques for surface profiling of highly transparent or reflective membrane structures. We investigate the photophysics of these dye-doped polymers to help determine their long-term durability and suitability for laser-induced fluorescence photogrammetric targeting. These investigations included experimental analysis of the fluorescence emission pattern, spectral content, temporal lifetime, linearity, and half-life. Results are presented that reveal an emission pattern wider than normal Lambertian diffuse surface scatter, a fluorescence time constant of 6.6 ns, a pump saturation level of approximately 20 micro J/mm(exp 2), and a useful lifetime of more than 300,000 measurements. Furthermore, two demonstrations of photogrammetric measurements by laser-induced fluorescence targeting are presented, showing agreement between photogrammetric and physically measured dimensions within the measurement scatter of 100 micron.
This report introduces fluorescence strobo-stereoscopy (FSS) to suppress strong specular reflection and enable the full field of view (FFOV) 3D surface imaging while the part is rotating. Specular reflection off the target surface significantly degrades the image quality and becomes critical for highly reflective surface measurements. In FSS, the fluorescent dye-doped fluid applied on the machined surface is excited upon incident ultra-violet light and becomes a new light source by Stokes' Law. Thus, specular reflection off of smooth surface can be suppressed by separating the fluorescent light from the excitation light. The developed FSS comprises a pair of imaging cameras, spatial filters, and an excitation light source. As a result, FSS effectively rejected the specular reflection and improved the FFOV 3D surface image quality of the machined part by enhancing contrast in the rotating target surface. Such enhancements in 3D imaging allowed to identify manufacturing tolerance of the part and to detect the surface features. The axial and lateral accuracy errors of FSS were 2.3% and 1.4% with the target size of 4.07 mm and 0.215 mm, respectively. A whole view reconstruction of the cylindrical target sample was performed, and the corresponding cylindricity and diameter deviation were assessed. The fluid media effect and the target surface quality effect were discussed.
The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.
The external surfaces of covalent organic frameworks (COFs) are presumably composed of free terminal functional groups from their constituent monomers. These terminal groups have been utilized to immobilize molecules onto COF surfaces for a variety of studies and potential applications. Despite the utility of these latent functional groups for COF functionalization and composite formation, the surface chemistry of the COFs has not been quantitatively investigated and is not well understood. In this work, fluorescent organic dyes bearing reactive amine and aldehyde functional groups are used to probe the surface chemistry of representative two-dimensional (2D) imine-based COFs. Furthermore, systematic dye binding and displacement experiments provide new insights into the characteristics of COF surfaces and the reactivity of different molecules with the surface groups on the COF crystallites.
SwissSPAD3 is the latest of a family of widefield time-gated SPAD imagers developed for fluorescence lifetime imaging (FLI) applications. Its distinctive features are (i) the ability to define shorter gates than its predecessors (width W < 1 ns), (ii) support for laser repetition rates up to at least 80 MHz and (iii) a dual-gate architecture providing an effective duty cycle of 100%. We present widefield macroscopic FLI measurements of short lifetime NIR dyes, analyzed using the phasor approach. Furthermore, the results are compared with those previously obtained with SwissSPAD2 and to theoretical predictions.
This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Here, each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.
By directly measuring the donor fluorescence lifetime as a function of acceptor concentration in the laser dye mixture Rhodamine 6G-Cresyl violet, we found that the Stern-Volmer relation is obeyed, from which the rate of excitation transfer is determined. The experimental results indicate that the dominant mechanism responsible for the efficient excitation transfer is that of resonance transfer due to long range dipole-dipole interaction.
Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.