Channel Fluorescence Research Articles

Double-layer focal plane microscopy for high throughput DNA sequencing.

Throughput is one of the most important properties in DNA sequencing. We propose a novel double-layer focal plane microscopy that doubles the DNA sequencing throughput. Each fluorescence channel is divided into two tube lens channels by energy splitting, and the camera is adjusted to take images corresponding to different defocus positions of the objective, thus doubling the information capacity of the microscopy. The microscopy is applied to gene chip, which has high spatial frequency and good uniformity, so the simultaneous imaging of the two tubes has little influence on each other due to the spatial averaging effect. Experimental results show that the image signal to noise ratio (SNR) is reduced by 1%, while the sequencing throughput is doubled.

Hyperfine structure investigations of Mn I

In this work the hyperfine structure (hfs) of 30 spectral lines of the manganese atom was investigated with the method of laser induced fluorescence in a hollow cathode discharge with spectral selection of the fluorescence channels. As a result, the hyperfine structure constants A and B for altogether 12 odd-parity and 16 even-parity levels were determined. For 3 odd-parity and 15 even-parity levels the A and B values are reported for the first time, for another 8 levels new constants B were evaluated, and for the remaining 8 levels the accuracy of the hitherto available literature data was improved; in some cases, where discrepancies between the earlier experimental results, obtained by other authors, and the theoretical predictions were observed, the new data constituted an unequivocal verification, which data are correct. The available database of the hyperfine structure constants for the manganese atom was thus further extended. Semi-empirical parametrization of the hyperfine structure for the even-parity level system of the atomic manganese were also performed in the present work; the consistency between the experimental and semi-empirically predicted hfs constants was found generally satisfactory, and in many cases even very good.

Rapid screening of aptamers for fluorescent targets by integrated digital PCR and flow cytometry.

In this paper, we report the development of a new strategy termed integrated digital PCR-fluorescence activated sorting based SELEX (IFS-SELEX) that enables rapid screening of aptamers against fluorescent targets. Initially, this strategy employs an integrated digital PCR system to amplify each sequence of a preliminarily enriched library, which is obtained by a traditional SELEX method, on the surface of polystyrene beads. Then, the as-prepared beads are incubated with the fluorescent target and then subjected to fluorescence-activated sorting. Since only those sequences with high binding affinity for the target are collected and sequenced, unnecessary analysis of ineligible sequences is avoided by this method, and the aptamer selection process is thereby greatly streamlined. As a proof-of-concept, we applied this strategy for the screening of aptamers against two fluorescent targets, i.e., ciprofloxacin (CFX) and thioflavin T (ThT), and successfully obtained corresponding sequences with low dissociation constants. The binding affinities of aptamers for ThT were well associated with the sorting regions defined in the fluorescence channel of the flow cytometry process. The experimental results demonstrated that the as-designed IFS-SELEX method can serve as a universal platform for rapid, facile, and efficient aptamer selection against various fluorescent targets.

Imparting down/up-conversion dual channels fluorescence to luminescence metal-organic frameworks by carbon dots-induced for fluorescence sensing.

The rapid and simple synthesis of highly efficient multi-emission luminescent metal-organic frameworks (LMOFs) with down/up-conversion fluorescence properties becomes critical in potential optical sensing. Here, we present a luminescent dual-ligand co-assisted strategy to one-pot prepare LMOFs for multiplex fluorescence sensing using carbon dots (CDs) and 2-methylimidazole (2-MIM) as dual-ligand to induce Zn2+ on zeolitic imidazolate framework-8 surface (CDs/MIM-Zn). The CDs/MIM-Zn exhibited efficient fluorescence-enhanced dual channels triple-emission with Stokes and anti-Stokes type excitation profiles. With the assistance of Stokes and anti-Stokes type optical property of CDs and the intrinsic porosity of MOFs structure, a rapid and sensitive dual-channel detection excited at 370nm and 790nm was performed to fluorescence turn off-on detect metal ions and reduced glutathione in real blood samples with the satisfactory recoveries of 98.3-106.4%. The dual channels triple-emission strategy ensured multiplex detection of more analytes in parallel.

The characterization of long-range transported North American biomass burning plumes: what can a multi-wavelength Mie–Raman-polarization-fluorescence lidar provide?

Abstract. This article presents a study of long-range transported biomass burning aerosols (BBA) originated from the North American wildfires in September 2020. The BBA plumes presented in this study were in the troposphere and underwent 1–2 weeks of aging before arriving at the lidar station ATOLL (ATmospheric Observatory of LiLle) in northern France. A novel lidar-derived dataset, 2α+3β+3δ+ϕ (α: extinction coefficient; β: backscatter coefficient; δ: particle linear depolarization ratio, i.e., PLDR; ϕ: fluorescence capacity), is provided for the characterization of BBA. The fluorescence capacity is an intensive aerosol parameter describing the ability of aerosols in producing fluorescence when exposed to UV excitation. In our BBA observations, obvious variations in aerosol intensive parameters, reflecting the variability of BBA properties, were detected. The PLDRs varied from less than 0.03 at all wavelengths to 0.15–0.22 and 0.12–0.16, respectively, at 355 and 532 nm. The extinction related Angström exponent was within the range of −0.3 to 1.0 and the fluorescence capacity was 1.0 × 10−4–4.0 × 10−4. Lidar ratio as low as 24 ± 4 sr (50 ± 8 sr) was observed in the BBA plumes at 355 (532) nm on 17–18 September, which was lower than most previously observed aged BBAs. These variations are likely correlated with the combustion process, the lifting of BBA plumes and the conditions (temperature, humidities, etc.) in the aging process. In addition, our results indicate BBA could act as ice nucleating particles in tropospheric conditions. The lidar fluorescence channel proves to be an important added value in aerosol characterization and aerosol–cloud interactions studies, due to its high sensitivity. With the increase in wildfire occurrence and intensity, BBAs become a more and more important atmospheric component. In this context, we show the potential of our novel lidar-derived dataset for aged BBA particles' characterization and for the understanding of their role in cloud processes.

A 3-(2′-nitro vinyl)-4-phenylselenyl coumarin as a fluorescent probe for distinguishing detection of Cys/Hcy and GSH

Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play important roles in regulating the redox balance of life process and are closely associated with human health and diseases. In order to better understand their roles in physiological process and the early diagnosis of related diseases, it is crucial for the selective detection of biothiols in biological systems. In this paper, we developed a 4-phenylselenium coumarin-nitroolefin conjugate as the fluorescent probe for the detection of biothiols. By introducing the 4-phenyselenium as the active site, probe CouSeNO2 is capable of detecting Cys/Hcy and GSH in dual fluorescence channels. For the biothiols, the first-step sensing reaction is proved to be nucleophilic substitution of 4-phenylselenium by the thiol group. Furthermore, through two-channel fluorescence imaging, probe CouSeNO2 has been successfully applied to sense the exogenous and endogenous biothiols in living cells.

Ensemble of nucleic acid absolute quantitation modules for copy number variation detection and RNA profiling

Current gold standard for absolute quantitation of a specific DNA sequence is droplet digital PCR (ddPCR), which has been applied to copy number variation (CNV) detection. However, the number of quantitation modules in ddPCR is limited by fluorescence channels, which thus limits the CNV sensitivity due to sampling error following Poisson distribution. Here we develop a PCR-based molecular barcoding NGS approach, quantitative amplicon sequencing (QASeq), for accurate absolute quantitation scalable to over 200 quantitation modules. By attaching barcodes to individual target molecules with high efficiency, 2-plex QASeq exhibits higher and more consistent conversion yield than ddPCR in absolute molecule count quantitation. Multiplexed QASeq improves CNV sensitivity allowing confident distinguishment of 2.05 ploidy from normal 2.00 ploidy. We apply multiplexed QASeq to serial longitudinal plasma cfDNA samples from patients with metastatic ERBB2+ (HER2+ ) breast cancer seeking association with tumor progression. We further show an RNA QASeq panel for targeted expression profiling.

A coumarin-based fluorescence chemosensor for the determination of Al3+ and ClO− with different fluorescence emission channels

A coumarin-based chemosensor FCOC (1 N'-(furan-2-carbonyl)-2-oxo-2H-chromene-3-carbohydrazide) was synthesized, and its sensing behaviors towards different analytes were investigated. The chemosensor FCOC showed discriminative detection pathways to aluminum (III) and hypochlorite anion with distinct fluorescence channels (blue and yellow channels). While FCOC displayed no fluorescence, after the addition of Al3+ ions into the FCOC solution an intense yellow fluorescence was observed. Also, the chemosensor FCOC showed blue fluorescence toward ClO−. Limit of detection (LOD) values were found to be 14.2 nM for Al3+ and 0.238 µM for ClO−, respectively. Response mechanisms of FCOC to Al3+ and ClO− were proven using different spectroscopic techniques and theoretical calculation. Overall, this study demonstrated that the single chemosensor has the capability of monitoring both Al3+ and ClO− through two different sensing modes.

A Series of Photosensitizes for Fe3+ in Aqueous Solution and Cells With Colorimetric and Fluorescent Channels: Synthesis and Performance.

Ferrum (Fe) is a widely existing metal element and nearly the most important trace element in living species, including human beings. The design of chemosensors for Fe ions faces issues related to the d-d transition of Fe(II) and Fe(III) ions, which makes them efficient electron trappers and energy quenchers. Most fluorescent dyes cannot afford such d-d quenching, showing emission turn off effect towards both Fe(II) and Fe(III) ions with poor selectivity. As a consequence, the development for Fe with emission turn on effect and good selectivity shall be continued and updated. In this work, three rhodamine-derived chemosensors modified by different lengths of alkyl chains having electron-donating N and O atoms were synthesized and explored for the selective optical sensing of Fe(III). These chemosensors showed colorimetric and fluorescent emission turn on sensing for Fe(III), showing two sensing channels. These chemosensors showed good selectivity, which was assigned to the sieving effect of alkyl chains with electron-donating N and O atoms. The N atom was found to be more effective in associating with Fe(III), compared to the O atom. Their fluorescent cell imaging experiment was carried out to confirm the possibility of being used for cell imaging.

Monitoring the different changing behaviors of •OH and cysteine in two ferroptosis pathways by a dual-functional fluorescence probe

Ferroptosis is a unique iron-dependent modality of regulated cell death, which attracted growing research interests in the past few years because of its potential pathological roles and therapeutic functions in many critical diseases. Hydroxyl radical (•OH) and cysteine (Cys) play not only crucial but also highly related roles in the development and redox regulation in ferroptosis. Herein, by virtue of a special sequential response strategy, we developed a new dual-functional fluorescence probe Coum-HCy for •OH and Cys. Coum-HCy is capable of selective, sensitive and especially sequential detection of •OH and Cys in two separated fluorescence channels (λem = 650 nm for •OH and λem = 453 nm for Cys). With Coum-HCy, we have monitored the different changing behaviors of •OH and Cys in two ferroptosis pathways initiated by erastin or RSL3 respectively. The results show that •OH is significantly generated during whether erastin or RSL3 initiating ferroptosis. However, due to the distinct mechanism of erastin and RSL3, the intracellular Cys level obviously decrease when cells treated with erastin but keep unaffected by RSL3. The good analytical properties of Coum-HCy may enable it to be widely used in the studies of more ferroptosis-relative physiological or pathological processes, which would not only simplify the detection procedures but also provide more accurate information to indicate the possible mutual correlation between •OH and Cys during ferroptosis.

Using the Autofluorescence Finder on the Sony ID7000TM Spectral Cell Analyzer to Identify and Unmix Multiple Highly Autofluorescent Murine Lung Populations.

Autofluorescence (AF) is a feature of all cell types, though some have more than others. In tissues with complex heterogeneous cellularity, AF is frequently a source of high background, masking faint fluorescent signals and reducing the available dynamic range of detectors for detecting fluorescence signals from markers of interest in a flow cytometry panel. Pulmonary flow cytometry presents unique challenges because lung cells are heterogeneous and contain varying amounts of high AF. The goal of this study was to demonstrate how a novel AF Finder tool on the Sony ID7000™ Spectral Cell Analyzer can be used to identify and screen multiple AF subsets in complex highly AF tissues like murine lungs. In lung single cell suspensions, the AF Finder tool identified four distinct AF spectra from six highly AF subsets. The subtraction of these distinct AF spectra resulted in a resolution increase by several log decades in several fluorescent channels. The major immune and lung tissue resident cells in a murine model of asthma were easily identified in a multi-color panel using AF subtraction. The findings demonstrate the practicality of the AF Finder tool, particularly when analyzing samples with multiple AF populations of varying intensities, in order to reduce fluorescence background and increase signal resolution in spectral flow cytometry.

Accurate detection and delineation boundary of renal cell carcinoma based on dual-targeted magnetic-fluorescent carbon dots

Accurate tumor localization and tumor boundary delineation are significant to improve the effectiveness of cancer therapeutics. In the present study, a fluorescence/magnetic resonance (MR) nanoprobe AS1411-CLT1-Gd-CDs with dual targeting function was developed for precise detection and delineation boundary of renal cell carcinoma (RCC). The dual-targeted AS1411-CLT1-Gd-CDs nanoprobe was initially established by co-conjugating peptide CLT1 and aptamer AS1411 to magnetic-fluorescent gadolinium(III)-doped carbon dots (Gd-CDs). The proposed AS1411-CLT1-Gd-CDs with wide-range multi-color fluorescence emission could target-specific image 786-O RCC cells through three distinct fluorescence channels and achieved the quantitativedetermination of 786-O cells with a low detection limit of 552 cells mL−1. Besides, the dual-targeted AS1411-CLT1-Gd-CDs nanoprobe could accurately delineate tumor boundary and generate greater and prolonged plateau contrast enhancement than the non-targeted and single-targeted Gd-CDs. Furthermore, the small-sized nanoprobe could be easily excreted from the body by the renal excretory system after imaging without any obvious long-term toxicity. These studies demonstrated that the proposed dual-targeted nanoprobe appeared as a very promising fluorescence/MR imaging agent for accurate identification and localization of renal cancer tumors, which could be beneficial to the early diagnosis and treatment of RCC.

PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel.

The probe technology described in this paper facilitates detection and discrimination of multiple targets in a single fluorescent channel during PCR. This provides a strategy for doubling the number of targets that can be analysed simultaneously on existing PCR instruments. These probes are referred to as PlexProbes and produce fluorescence that can be switched ‘on’ or ‘off’ in the presence of target by manipulating the temperature. During PCR, fluorescence can be measured at multiple temperatures allowing discrimination of specific targets at defined temperatures. In a single fluorescent channel, a model duplex assay allowed either real-time or endpoint detection of Chlamydia trachomatis (CT) at 52°C and end-point detection of Neisseria gonorrhoeae (GC) at 74°C. Using this model system, as few as 40 copies of each specific target could be detected as single infection or co-infection, regardless of the presence or absence of the other target. A PlexProbe prototype assay for sexually transmitted infections (PP-STI) which simultaneously enables detection and differentiation of six targets using only three fluorescent channels was then constructed and evaluated. The PP-STI assay detects GC (2 gene targets), CT, Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) and an internal control (IC). To evaluate assay performance, a panel of archived clinical samples (n = 337) were analysed using PP-STI and results compared to those obtained with a commercially available diagnostic assay. The overall agreement between results obtained with the PP-STI assay and the reference test was greater than 99.5%. PlexProbes offer a method of detecting more targets from a single diagnostic test, empowering physicians to make evidence-based treatment decisions while conserving time, labour, sample volume and reagent costs.

Label-free imaging of intracellular organelle dynamics using flat-fielding quantitative phase contrast microscopy (FF-QPCM)

Panoramic and long-term observation of nanosized organelle dynamics and interactions with high spatiotemporal resolution still hold great challenge for current imaging platforms. In this study, we propose a live-organelle imaging platform, where a flat-fielding quantitative phase contrast microscope (FF-QPCM) visualizes all the membrane-bound subcellular organelles, and an intermittent fluorescence channel assists in specific organelle identification. FF-QPCM features a high spatiotemporal resolution of 245 nm and 250 Hz and strong immunity against external disturbance. Thus, we could investigate several important dynamic processes of intracellular organelles from direct perspectives, including chromosome duplication in mitosis, mitochondrial fusion and fission, filaments, and vesicles' morphologies in apoptosis. Of note, we have captured, for the first time, a new type of mitochondrial fission (entitled mitochondrial disintegration), the generation and fusion process of vesicle-like organelles, as well as the mitochondrial vacuolization during necrosis. All these results bring us new insights into spatiotemporal dynamics and interactions among organelles, and hence aid us in understanding the real behaviors and functional implications of the organelles in cellular activities.

Quantification of mitophagy using mKeima-mito in cultured human primary retinal pigment epithelial cells

The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live-cell time-lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.

A robust dual-channel fluorescence-enhanced probe for duplex imaging of endogenous HClO/ClO- in living cells and in zebrafish

A unique fluorescent probe, MCB, for HClO/ClO- was rationally constructed by integrating methylene blue, 7-hydroxycoumarin, and two HClO-specific reacting moieties into a single molecular structure. Distinct from other published cases, MCB displayed remarkable dual-channel fluorescence enhancement, peaked at 464 and 700 nm respectively, which simultaneously provided two crosstalk-free fluorescence channels, blue and NIR channels, for mutual corroboration, and hence improving the reliability of the detection results. More importantly, MCB exhibited excellent sensing properties for HClO/ClO-, such as fast response (within 90 s), high sensitivity (LOD as low as 3.9 nM based on blue fluorescence channel), and sufficient selectivity. Significantly, MCB was successfully utilized for dual-channel imaging of HClO/ClO-in living cells and zebrafish, thereby holding the potential for research that focused on elucidating the biological roles of HClO/ClO-.

Imaging Flow Cytometry as a Quick and Effective Identification Technique of Pollen Grains from Betulaceae, Oleaceae, Urticaceae and Asteraceae.

Despite the continuous and intensive development of laboratory techniques, a light microscope is still the most common tool used in pollen grains differentiation. However, microscopy is time-consuming and needs well-educated and experienced researchers. Other currently used techniques can be categorised as images and non-images analysis, but each has certain limitations. We propose a new approach to differentiate pollen grains using the Imaging Flow Cytometry (IFC) technique. It allows for high-throughput fluorescence data recording, which, in contrast to the standard FC, also enables real-time control of the results thanks to the possibility of digital image recording of cells flowing through the measuring capillary. The developed method allows us to determine the characteristics of the pollen grains population based on the obtained fluorescence data, using various combinations of parameters available in the IDEAS software, which can be analysed on different fluorescence channels. On this basis, we distinguished pollen grains both between and within different genera belonging to the Betulaceae, Oleaceae, Urticaceae and Asteraceae families. Thereby, we prove that the proposed methodology is sufficient for accurate, fast, and cost-effective identification and potentially can be used in the routine analysis of allergenic pollen grains.

Visualizing the Interplay of Lipid Droplets and Protein Aggregates During Aging via a Dual-Functional Fluorescent Probe.

Fluorescence imaging the interplay between lipid droplets (LDs) and protein aggregates (PAs) is extremely valuable for elucidating molecular mechanisms of aging. Here, we describe the first dual-functional fluorescent probe, LW-1, for simultaneously imaging LDs and PAs in distinct fluorescence channels to dissect interplaying roles between LDs and PAs during aging. Notably, based on an intriguing mechanism of hydrogen bonds regulating single bond rotation, LW-1 selectively detected LDs in a red channel. Meanwhile, based on another mechanism of the hydrogen bond regulating intramolecular charge transfer efficiency, probe LW-1 further detected PAs in an NIR channel. Practical applications showed that LW-1 was capable of concurrently detecting LDs and PAs in living cells. Moreover, simultaneously imaging LDs and PAs in intestine tissues of mice at different aging degrees was conducted. The results denoted that the PAs level in the intestine tissue increased dramatically with aging, accompanying the buildup of LDs. Significantly, the interplay between LDs and PAs during aging was observed. These evidences demonstrated that the PAs level was closely related with aging processes in intestine tissues, while LDs were formed correspondingly to interact with PAs, suggesting that excessive PAs can be loaded into LDs and then be removed by lipophagy.

An ESIPT fluorescent probe based on Staudinger reaction for detection of triphenylphosphine and tetrakis(triphenyl-phosphine)palladium

Triphenylphosphine (PPh3) and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) are harmful at extremely low concentration, thus it is essential to explore selective and sensitive method to detect them. Based on Staudinger reaction mechanism, here we present a simple excited-state intramolecular proton transfer (ESIPT) fluorescent probe (HBTN) for detection of PPh3 and Pd(PPh3)4 over other phosphine derivatives with differential channel fluorescence (enol and keto emission). The reaction-typed fluorescence probe with novel Staudinger reaction mechanism offers very low autofluorescence and a large signal-to-noise response signal, which is beneficial for the sensitive detection of PPh3 and Pd(PPh3)4. Moreover, HBTN can be used for efficiently detecting Pd(PPh3)4 residues in highly purified products and imaging PPh3 in living cells.

Monitoring Cellular Responses to Infection with Fluorescent Biosensors.

Fluorescent biosensors are chemically or genetically encoded reporters of cellular processes, signaling pathways, or biomolecule concentration, whose output is quantified using fluorescence microscopy or fluorescence spectrometry. These biosensors can detect the target activity or metabolites via mechanisms including conversion between nonfluorescent and fluorescent forms, changes in reporter intensity, changes in the intensity ratio across fluorescence channels, alterations to the subcellular localization of the bioreporter, and by fluorescence resonance energy transfer. Here, we describe the use of a chemical photoconverting biosensor, and genetically encoded localization and ratiometric biosensors, for monitoring the cellular and signaling processes involved in pathogen-induced apoptosis and the resulting destruction of the pathogen. While this study uses biosensors to monitor responses to infection, these approaches can be readily translated to other cellular systems and other fluorescent biosensors.