Background Autofluorescence Research Articles

Novel Endoplasmic Reticulum-Targeted Luminescent Probe for Visualization of Carbon Monoxide in Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a major hepatic dysfunction commonly caused by hepatotoxic drug overdose, resulting in a considerable number of fatalities worldwide. Recent studies have highlighted the regulatory and hepatoprotective effects of carbon monoxide (CO) during the liver injury process. However, precisely tracking the dynamic changes in the composition of CO in DILI is still a great challenge. In this work, leveraging the innovative "quencher-insertion" strategy, a unique endoplasmic reticulum (ER)-targetable lanthanide complex-based luminescence probe, ER-ANBTTA-Eu3+/Tb3+, has been developed for the selective and accurate monitoring of CO fluxes in live cells and laboratory animals. The new probe is composed of three covalently linked functional moieties: the terpyridine polyacid-Eu3+/Tb3+-mixed chelates as the long-lived luminophore, a p-toluenesulfonamide moiety as the ER-anchoring motif, and an allyloxy-nitrobenzyl ether moiety as the CO-specific recognition unit. Upon reaction with CO in the presence of Pd2+ ions, the Tsuji-Trost reaction leads to the cleavage of the allyloxy-nitrobenzyl group from the Eu3+/Tb3+-mixed chelates, which results in the restoration of Tb3+ emission at 538 nm and the attenuation of Eu3+ emission at 688 nm, leading to a dramatic increase of the I538/I688 ratio. In addition to the exceptional response sensitivity and selectivity toward CO, ER-ANBTTA-Eu3+/Tb3+ also exhibits the outstanding ER-locating capability, which allows the probe to be used for imaging of CO in the ER of live cells. Using this probe, combined with the time-gated luminescence imaging mode, the exogenous and endogenous CO in ER of live cells were monitored without the interference of background autofluorescence. Moreover, the upregulation of hepatic CO in DILI mice was successfully visualized. The results suggested the potential of ER-ANBTTA-Eu3+/Tb3+ for deeply exploring the functions of CO in DILI pathogenesis.

Water-Soluble Chitosan-Europium Hybrid Sensor for Singlet Oxygen Detection.

The ability to effectively monitor singlet oxygen (1O2) with fluorescence probes in biological systems is severely restricted mainly by the background autofluorescence of these systems. Though the application of lanthanide complexes as 1O2 monitors successfully resolves this problem with time-gated luminescence measurements, the insolubility of these complexes in an aqueous medium heavily limits their application in biological systems. Here, we present a water-soluble 1O2 sensor based on a chitosan-europium hybrid material. A procedure for the modification of chitosan to expand its solubility to neutral and basic pH, while maintaining its free active amine groups, is described. These are then coupled covalently to a europium-based probe for the detection of 1O2. The resulting hybrid sensor is readily soluble in water across the pH scale and efficiently signals the presence of 1O2 at physiological pH, with the characteristic Eu3+ emission at 611 nm yielding up to a 15-fold increase in emission intensity and a decay time of 332 μs. Being of particular interest for time-gated measurements, this long decay time, coupled with the biocompatibility of chitosan, describes a material with potential biological applications, where 1O2 plays a vital role.

Near-Infrared Unimolecular Chemiluminescence Probes for Deep-Tissue Imaging.

Chemiluminescence (CL) imaging has emerged as a promising optical imaging technique due to minimal background autofluorescence and being excitation-free. However, the emission of most chemiluminescent probes was concentrated in the visible light region, which limited the tissue penetration. Although some NIR chemiluminescence probes have been reported based on the chemiluminescence resonance energy transfer (CRET) strategy, the energy loss was inevitable. Thus, it is crucial to develop near-infrared (NIR) unimolecular probes with direct chemiluminescence. Herein, we propose a strategy of increasing conjugation for designing and synthesizing novel NIR chemiluminescence unimolecular probes that consist of luminol, electron acceptor, π-bridge, and electron donor. Luminol was conjugated to the unimolecular backbone to produce direct NIR chemiluminescence. Notably, the direct CL mechanism of probes was investigated. Compared with CRET-based chemiluminescence, this direct CL was more advantageous to immediately convert the chemical energy into chemiluminescence, avoiding energy degradation. Furthermore, the corresponding nanoparticles with great biosafety were prepared by self-assembly with amphiphilic DSPE-PEG. Especially, TTBL@PEG-NPs with NIR-I emission were successfully used in the sensitive in vivo chemiluminescence imaging of various inflammation models, such as peritonitis, ear swelling, and colitis. This study paves the way for the design of NIR unimolecular chemiluminescence probes and deep-tissue imaging.

Wavefront shaping and imaging through a multimode hollow-core fiber

Multimode fibers recently emerged as compact minimally-invasive probes for high-resolution deep-tissue imaging. However, the commonly used silica fibers have a relatively low numerical aperture (NA) limiting the spatial resolution of a probe. On top of that, light propagation within the solid core generates auto-fluorescence and Raman background, which interferes with imaging. Here we propose to use a hollow-core fiber to solve these problems. We experimentally demonstrate spatial wavefront shaping at the multimode hollow-core fiber output with tunable high-NA. We demonstrate raster-scan and speckle-based compressive imaging through a multimode hollow-core fiber.

Multiplexed Shortwave Infrared Imaging Highlights Anatomical Structures in Mice

AbstractMultiplexed fluorescence in vivo imaging remains challenging due to the attenuation and scattering of visible and traditional near infrared (NIR‐I, 650–950 nm) wavelengths. Fluorescence imaging using shortwave infrared (SWIR, 1000–1700 nm, a.k.a. NIR‐II) light enables deeper tissue penetration due to reduced tissue scattering as well as minimal background autofluorescence. SWIR‐emitting semiconductor quantum dots (QDs) with tunable emission peaks and optical stability are powerful contrast agents, yet few imaging demonstrations exclusively use SWIR emission beyond two‐color imaging schemes. In this study, we engineered three high quality lead sulfide/cadmium sulfide (PbS/CdS) core/shell QDs with distinct SWIR emission peaks ranging from 1100–1550 nm for simultaneous three‐color imaging in mice. We first use the exceptional photostability of QDs to non‐invasively track lymphatic drainage with longitudinal imaging, highlighting the detailed networks of lymphatic vessels with widefield imaging over a 2 hr period. We then perform multiplexed imaging with all three QDs to distinctly visualize the lymphatic system and spatially overlapping vasculature networks, including clearly distinguishing the liver and spleen. This work establishes optimized SWIR QDs for next generation multiplexed and longitudinal preclinical imaging, unlocking numerous opportunities for preclinical studies of disease progression, drug biodistribution, and cell trafficking dynamics in living organisms.

PH-responsive persistent luminescence nanoprobes biosensor for autofluorescence-free determination of glucose level in human samples and fingerprint encryption.

Glucose level is an important indicator of diabetes, and maintaining an appropriate physiological concentration of glucose is important for human health. However, traditional optical sensors are interfered by the interference of strong background autofluorescence and natural responsive luminescence, which severely limits their application in complex biological samples. Herein, as a novel glucose biosensing probe, green-emitting Zn2GeO4:Mn2+, Eu3+ (ZGME) persistent luminescence nanoparticles (PLNPs) with pH stimulus-responsive was prepared by a facile one-pot hydrothermal method. We also investigated the pH stimulus-responsive luminescence behaviour of ZGME over a range of pH values from 2.8 to 8.0. Taking advantage of the interesting property that ZGME photoluminescence intensity has a pH response, within an extraordinarily narrow pH range of 5.0-6.5 for highly selectivity and sensitive determination of glucose level in human samples by acid-responsive quenching and persistent luminescent performance. The detection results show high accuracy of the measured values of glucose in serum with a wide detection range (2.5μg L-1-10mg L-1) and low detection limit (0.5μg L-1). Finally, the pH-responsive persistent luminescence also makes ZGME promising for high-level fingerprint information encryption. Hence, the established pH stimulation-responsive PLNPs-based biosensing probe offers excellent performance with high selective, accuracy and signal-to-noise ratio for detection of glucose level in human samples.

Organelle-Targeting Polymer Dots Exhibiting Thermally Activated Delayed Fluorescence for Subcellular Imaging.

Selective imaging of specific subcellular structures provides valuable information about the cellular microenvironment. Materials exhibiting thermally activated delayed fluorescence (TADF) are rapidly emerging as metal-free probes with long-lived emission for intracellular time-gated imaging applications. Polymers incorporating TADF emitters can self-assemble into luminescent nanoparticles, termed polymer dots (Pdots), and this strategy enables them to circumvent the limitations of commercial organelle trackers and small molecule TADF emitters. In this study, diblock copolymers comprised of a hydrophilic block containing organelle-targeting monomers and a hydrophobic TADF-active block were synthesized by ring-opening metathesis polymerization (ROMP). Oxanorbornene-based monomers incorporating morpholine and triphenylphosphonium groups for lysosome and mitochondria targeting, respectively, were also synthesized. ROMP by sequential addition yielded well-defined diblock copolymers with dispersities <1.28. To analyze the effect of tuning the hydrophilic corona on cellular viability and uptake, we prepared Pdots with poly(ethylene glycol) (PEG) and bis-guanidinium (BGN) coronas, resulting in limited and efficient cellular uptake, respectively. Red-emissive Pdots with BGN-based coronas and organelle-targeting functionality were obtained with quantum yields up to 12% in water under air. Colocalization analysis confirmed that lysosome and mitochondria labeling in live HeLa cells was accomplished within 2 h of incubation, affording Pearson's correlation coefficients of 0.37 and 0.70, respectively. The potential application of these Pdots for time-resolved imaging is highlighted by a proof of concept using time-gated spectroscopy, which effectively separates the delayed emission of the TADF Pdots from the background autofluorescence of biological serum.

Multiplexed Shortwave Infrared Imaging Highlights Anatomical Structures in Mice.

Multiplexed fluorescence in vivo imaging remains challenging due to the attenuation and scattering of visible and traditional near infrared (NIR-I, 650-950 nm) wavelengths. Fluorescence imaging using shortwave infrared (SWIR, 1000-1700 nm, a.k.a. NIR-II) light enables deeper tissue penetration due to reduced tissue scattering as well as minimal background autofluorescence. SWIR-emitting semiconductor quantum dots (QDs) with tunable emission peaks and optical stability are powerful contrast agents, yet few imaging demonstrations exclusively use SWIR emission beyond two-color imaging schemes. In this study, we engineered three high quality lead sulfide/cadmium sulfide (PbS/CdS) core/shell QDs with distinct SWIR emission peaks ranging from 1100-1550 nm for simultaneous three-color imaging in mice. We first use the exceptional photostability of QDs to non-invasively track lymphatic drainage with longitudinal imaging, highlighting the detailed networks of lymphatic vessels with widefield imaging over a 2 hr period. We then perform multiplexed imaging with all three QDs to distinctly visualize the lymphatic system and spatially overlapping vasculature networks, including clearly distinguishing the liver and spleen. This work establishes optimized SWIR QDs for next generation multiplexed and longitudinal preclinical imaging, unlocking numerous opportunities for preclinical studies of disease progression, drug biodistribution, and cell trafficking dynamics in living organisms.

Persistent Luminescence Nanoplatform for Autofluorescence-Free Tracking of Submicrometer Plastic Particles in Plant.

The uptake of plastic particles by plants and their transport through the food chain make great risks to biota and human health. Therefore, it is important to trace plastic particles in the plant. Traditional fluorescence imaging in plants usually suffers significant autofluorescence background. Here, we report a persistent luminescence nanoplatform for autofluorescence-free imaging and quantitation of submicrometer plastic particles in plant. The nanoplatform was fabricated by doping persistent luminescence nanoparticles (PLNPs) onto polystyrene (PS) nanoparticles. Cr3+-doped zinc gallate PLNP was employed as the dopant for autofluorescence-free imaging due to its persistent luminescence nature. In addition, the Ga element in PLNP was used as a proxy to quantify the PS in the plant by inductively coupled plasma mass spectrometry (ICP-MS). Thus, the developed nanoplatform allows not only dual-mode autofluorescence-free imaging (persistent luminescence and laser-ablation ICP-MS) but also ICP-MS quantitation for tracking PS in plant. Application of this nanoplatform in a typical plant model Arabidopsis thaliana revealed that PS mainly distributed in the root (>99.45%) and translocated very limited (<0.55%) to the shoot. The developed nanoplatform has great potential for quantitative tracing of submicrometer plastic particles to investigate the environmental process and impact of plastic particles.

Dual Infrared 2‐Photon Microscopy Achieves Minimal Background Deep Tissue Imaging in Brain and Plant Tissues

AbstractTraditional deep fluorescence imaging has primarily focused on red‐shifting imaging wavelengths into the near‐infrared (NIR) windows or implementation of multi‐photon excitation approaches. Here, the advantages of NIR and multiphoton imaging are combined by developing a dual‐infrared two‐photon microscope that enables high‐resolution deep imaging in biological tissues. This study first computationally identifies that photon absorption, as opposed to scattering, is the primary contributor to signal attenuation. A NIR two‐photon microscope is constructed next with a 1640 nm femtosecond pulsed laser and a NIR PMT detector to image biological tissues labeled with fluorescent single‐walled carbon nanotubes (SWNTs). Spatial imaging resolutions are achieved close to the Abbe resolution limit and eliminate blur and background autofluorescence of biomolecules, 300 µm deep into brain slices and through the full 120 µm thickness of a Nicotiana benthamiana leaf. NIR‐II two‐photon microscopy can also measure tissue heterogeneity by quantifying how much the fluorescence power law function varies across tissues, a feature this study exploits to distinguish Huntington's Disease afflicted mouse brain tissues from wildtype. These results suggest dual‐infrared two‐photon microscopy can accomplish in‐tissue structural imaging and biochemical sensing with a minimal background, and with high spatial resolution, in optically opaque or highly autofluorescent biological tissues.

An activatable formaldehyde donor with high contrast bioluminescence to monitor its release

High signal-to-background ratio (SBR) monitoring of formaldehyde (FA) delivery in living systems is of great significance for the precise study of the physiological and pathological functions of FA. However, traditional FA donors mainly use fluorescent signals to monitor FA release, which hampers the high-performance bioimaging due to the undesirable background autofluorescence, leading to poor SBR and compromised imaging sensitivity. Herein, we present the first bioluminogenic donor B-FAD for esterase-activated FA release with high contrast bioluminescence imaging. By protecting the hydroxyl group of D-luciferin with acetoxymethyl ether group, B-FAD cannot be catalyzed by luciferase to emit light. In the presence of esterase, the ester bond of B-FAD breaks, producing FA and D-luciferin. B-FAD selectively and sensitively releases FA in the presence of esterase, accompanied by 34-fold bioluminescence enhancement. In addition, B-FAD has been successfully applied for FA delivery and bioluminescence imaging in living cells, with a SBR about 50 times higher than those obtained by fluorescence imaging. Therefore, in addition to providing an effective tool for imaging FA delivery in living cells, this work establishes a general approach for high contrast monitoring other reactive biological species release by easily changing the responsive unit on D-luciferin.

Designing SERS nanotags for profiling overexpressed surface markers on single cancer cells: A review

This review focuses on the chemical design and the use of Surface-Enhanced Raman Scattering (SERS)-active nanotags for measuring surface markers that can be overexpressed at the surface of single cancer cells. Indeed, providing analytical tools with true single-cell measurements capabilities is capital, especially since cancer research is increasingly leaning toward single-cell analysis, either to guide treatment decisions or to understand complex tumor behaviour including the single-cell heterogeneity and the appearance of treatment resistance. Over the past two decades, SERS nanotags have triggered significant interest in the scientific community owing their advantages over fluorescent tags, mainly because SERS nanotags resist photobleaching and exhibit sharper signal bands, which reduces possible spectral overlap and enables the discrimination between the SERS signals and the autofluorescence background from the sample itself. The extensive efforts invested in harnessing SERS nanotags for biomedical purposes, particularly in cancer research, highlight their potential as the next generation of optical labels for single-cell studies. The review unfolds in two main parts. The first part focuses on the structure of SERS nanotags, detailing their chemical composition and the role of each building block of the tags. The second part explores applications in measuring overexpressed surface markers on single-cells. The latter encompasses studies using single nanotags, multiplexed measurements, quantitative information extraction, monitoring treatment responses, and integrating phenotype measurements with SERS nanotags on single cells isolated from complex biological matrices. This comprehensive review anticipates SERS nanotags to persist as a pivotal technology in advancing single-cell analytical methods, particularly in the context of cancer research and personalized medicine.

NIR-II fluorescence lateral flow immunosensor based on efficient energy transfer probe for point-of-care testing of tumor biomarkers

Fluorescence lateral flow immunoassay (LFA) has emerged as a powerful tool for rapid screening of various biomarkers owing to its simplicity, sensitivity and flexibility. It is noteworthy that fluorescent probe mainly determines the analytical performance of LFA. Due to the emission and excitation wavelengths are located in the visible region, most fluorophores are inevitably subject to light scattering and background autofluorescence. Herein, we reported a novel LFA sensor based on the second near-infrared (NIR-II) fluorescent probe with excellent anti-interference capability. The designed NIR-II probe was the Nd3+ and Yb3+ doped rare earth nanoparticles (RENPs) by employing Nd3+ as energy donor and Yb3+ as energy acceptor, which of the donor-acceptor energy transfer (ET) efficiency reached up to 80.7 %. Meanwhile, relying on the convenient and effective encapsulation strategy of poly(lactic-co-glycolic acid) (PLGA) microspheres to RENPs, the surface functionalized NIR-II probe (RE@PLGA) was obtained for subsequent bioconjugation. Benefiting from the optical advantages of NIR-II probe, this proposed NIR-II LFA displayed a good linear relationship ranging from 7 ng/mL to 200 ng/mL for the detection of α-fetoprotein (AFP), an important biomarker of hepatocellular carcinoma (HCC). The limit of detection (LOD) was determined as low as 3.0 ng/mL, which was of 8.3 times lower than clinical cutoff value. It is promising that LFA sensor based on this efficient RENPs probe provides new opportunities for high sensitive detection of various biomarkers in biological samples.

Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.

Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes. This method has proven to be stable, convenient, and cost-effective. Notably, for the mRNA detection in SKBR3 cells, the lanthanide probe group exhibited 2.5 times higher luminescence intensity and detected 3 times more signal points in cells compared with the Cy3 group. Furthermore, we successfully applied the probe to image HER2 mRNA molecules in breast cancer FFPE tissue sections, achieving a 2.7-fold improvement in sensitivity compared to Cy3-based probes. These results emphasize the potential of time-resolved smFISH as a highly sensitive method for nucleic acid detection, free of background fluorescence interference.

Tuning optical properties of aromatic near-infrared fluorescent switch through engineering functional heterocycle on donor moiety

Near-infrared (NIR) emissive fluorophores with donor−acceptor−donor (D−A−D) structures are attractive for deep tissue in vivo bioimaging and sensing, owing to their low background autofluorescence and deep penetration depth. In this study, we constructed a series of nitric oxide (NO)-responsive fluorescent switches with D−A−D structure, in which functional heterocycles act as donor moiety, resulting in the modification of fluorescence wavelengths from NIR-I (750–950 nm) to NIR-II (1000–1700 nm) region. Specifically, the NIR-I fluorescence can be achieved by using furan, thiophene and selenophene as the donor moiety. By using pyrrole as the donor moiety, NIR-II fluorescence can be achieved due to the strong N–H⋯N hydrogen bond interaction and tunable intramolecular charge transfer (ICT) process. Based on our findings, tailoring electronegativity of donor moiety could controllably tune the fluorescence wavelength from NIR-I to NIR-II region, proposing a practical design strategy for developing NIR-II fluorescent switches.

Recent advances in bioluminescent probes for neurobiology.

Bioluminescence is a popular modality for imaging in living organisms. The platform relies on enzymatically (luciferase) generated light via the oxidation of small molecule luciferins. Since no external light is needed for photon production, there are no concerns with background autofluorescence or photobleaching over time-features that have historically limited other optical readouts. Bioluminescence is thus routinely used for longitudinal tracking across whole animals. Applications in the brain, though, have been more challenging due to a lack of sufficiently bioavailable, bright, and easily multiplexed probes. Recent years have seen the development of designer luciferase and luciferin pairs that address these issues, providing more sensitive and real-time readouts of biochemical features relevant to neurobiology. This review highlights many of the advances in bioluminescent probe design, with a focus on the small molecule light emitter, the luciferin. Specific efforts to improve luciferin pharmacokinetics and tissue-penetrant emission are covered, in addition to applications that such probes have enabled. The continued development of improved bioluminescent probes will aid in illuminating critical neurochemical processes in the brain.

Lanthanide-Complex-Enhanced Bioorthogonal Branched DNA Amplification.

Fluorescence in situ hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for in situ nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use.

Precise Non-invasive Imaging Mouse Model of Pancreatic Cancer: Very Narrow Band-width Laser Fluorescence Excitation of Green Fluorescent Protein Provides Ultra-bright Tumor Images With no Skin Autofluorescence.

Pancreatic cancer is a recalcitrant disease with 5-year survival of only 12%. Improved mouse models of pancreatic cancer are critical for discovery of effective therapeutics. Orthotopic mouse nude-mouse models of pancreatic cancer were established with the human pancreatic-cancer cell line Panc-1 expressing green fluorescent protein (GFP) by transplanting tumor fragments into the pancreas, using the procedure of surgical orthotopic implantation (SOI). Four weeks after establishment of the orthotopic models, the mice were imaged with the Analytik Jena UVP Biospectrum Advanced with a very-narrow-band-width excitation at 487 nm and peak emission at 513 nm. Non-invasive fluorescence imaging of the mice implanted with Panc-1-GFP showed a very bright tumor in the area of the pancreas and peritoneal cavity. The skin background autofluorescence was absent. When a laparotomy was performed on the mouse for open imaging, the tumor on the pancreas was clearly imaged. There was very clear concordance of the non-invasive image and the image obtained during laparotomy. A precise orthotopic mouse model of pancreatic cancer was developed in which there was high concordance between non-invasive and invasive fluorescence imaging due to the ultra-bright signal and ultra-low background using very-narrow-band-width laser fluorescence excitation. This model can be used for high-throughput in vivo screening for improved therapeutics for pancreatic cancer.

Monitoring Cell Proliferation by Dye Dilution: Considerations for Panel Design.

High dimensional studies that include proliferation dyes face two inherent challenges in panel design. First, the more rounds of cell division to be monitored based on dye dilution, the greater the starting intensity of the labeled parent cells must be in order to distinguish highly divided daughter cells from background autofluorescence. Second, the greater their starting intensity, the more difficult it becomes to avoid spillover of proliferation dye signal into adjacent spectral channels, with resulting limitations on the use of other fluorochromes and ability to resolve dim signals of interest. In the third and fourth editions of this series, we described the similarities and differences between protein-reactive and membrane-intercalating dyes used for general cell tracking, provided detailed protocols for optimized labeling with each dye type, and summarized characteristics to be tested by the supplier and/or user when validating either dye type for use as a proliferation dye. In this fifth edition, we review: (a) Fundamental assumptions and critical controls for dye dilution proliferation assays; (b) Methods to evaluate the effect of labeling on cell growth rate and test the fidelity with which dye dilution reports cell division; and. (c) Factors that determine how many daughter generations can be accurately included in proliferation modeling. We also provide an expanded section on spectral characterization, using data collected for three protein-reactive dyes (CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far Red) and three membrane-intercalating dyes (PKH67, PKH26, and CellVue® Claret) on three different cytometers to illustrate typical decisions and trade-offs required during multicolor panel design. Lastly, we include methods and controls for assessing regulatory T cell potency, a functional assay that incorporates the "know your dye" and "know your cytometer" principles described herein.

A rapid investigation of near-infrared (NIR) fluorescent switch-on probes for detection and in cellulo tracking of G-quadruplex and double-stranded DNA.

This review provides a comprehensive overview of the recent advancements in Near Infrared (NIR) fluorescence switch-on probes designed for the detection and in cellulo tracking of G-quadruplex and double-stranded DNA (dsDNA). G-quadruplexes, non-canonical DNA structures, play pivotal roles in regulating various biological processes, making them critical targets for therapeutic and diagnostic applications. The unique properties of NIR fluorescence probes, such as deep tissue penetration, minimal photodamage, and low autofluorescence background, offer significant advantages for bioimaging. We critically analyze the design strategies, photophysical properties, and binding mechanisms of various NIR fluorescence switch-on probes. Additionally, we discuss their efficacy and specificity in identifying G-quadruplexes and dsDNA within cellular environments. Key challenges and future directions for improving the sensitivity, selectivity, and biocompatibility of these probes are also highlighted. This review aims to underscore the potential of NIR fluorescence probes in advancing our understanding of DNA dynamics and their applications in biomedical research.