Red Fluorescence Research Articles

Tuning Optical Properties of Carbon Dots by Co‐doping Strategy for Versatile Applications

AbstractCarbon dots (CDs) with tunable photoluminescence was successfully prepared via selective co‐doping of three elements, including nitrogen, phosphorus and sulfur element by green one‐step hydrothermal method. Nitrogen‐sulfur‐doped CDs (red NS‐CDs), which emitted red fluorescence under UV excitation, were used to detect iron ions in the linear range 1–50 μM, yielding detectable limits as low as 94 nM. The nitrogen‐phosphorus‐doped CDs (blue NP‐CDs) exhibited blue fluorescence with quantum yields up to 34.5 %. When the concentration is 800 μg/mL, it showed excellent anticancer effect in vitro. The nitrogen‐sulfur‐phosphorus‐doped CDs (green NPS‐CDs) emitting green fluorescence showed a low cytotoxicity at a high concentration of 800 μg/mL and were used for intracelluar imaging of NCl−H292 cells. This study demonstrated the photoluminescent characteristics of carbon dots (CDs) and highlighted the ability to modify their peak fluorescence emission wavelengths through the flexible combination of specific reagents under comparable hydrothermal conditions. Additionally, it provided valuable insights into the efficient methodologies for synthesizing CDs suitable for various application scenarios.

Self-calibrating probes constructed on a unique dual-emissive fluorescence platform for the precise tracking of cellular senescence

Human β-galactosidase (β-gal) is recognized as a crucial biomarker for evaluating senescence at the cellular and tissue levels in humans. However, tools to precisely track the endogenous β-gal are still limited. Herein, we present two novel self-calibrating β-gal probes 7a and 7b which were constructed on a unique green/red dual-emissive fluorescence platform. The two probes inherently exhibited a stable green fluorescence signal impervious to β-gal activity, serving as a reliable internal reference. They also displayed a progressively diminishing red fluorescence signal with the increasing of β-gal expression levels. The dual behavior endows them with self-calibration capacity and then renders excellently selective and sensitive for precisely monitoring β-gal activity. Notably, compared with E. coli β-gal, the two probes are more effectively response to A. oryzae β-gal homologous to human β-gal, indicating their unique species-selectivity. Furthermore, 7a was validated for its effectiveness in determining senescence-associated β-galactosidase (SA-β-gal) expression in senescent NRK-52E and HepG2 cells, underscoring its practical applicability in senescence research.

Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in Komagataella phaffii.

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast Komagataella phaffii, a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in K. phaffii. In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).

Intelligent detection of tetracycline by a rare earth multicolor fluorescent probe based on guanosine-5′-monophosphate

Long-term discharge of tetracycline (TC) in the environment exerts selective pressure on the bacteria, increasing resistance in sensitive bacteria and posing a serious threat to public health. Therefore, it is crucial to develop real-time detection of tetracycline. In this study, we reported a novel rare-earth fluorescent nanosensor FITC-GMP-Eu, based on the formation of guanosine-5′-monophosphate disodium salt (GMP) and fluorescein isothiocyanate (FITC), which enabled the real-time identification of tetracycline in the environment. When there was no TC in the environmental sample, the FITC-GMP-Eu nanosensor exhibited the green fluorescence of FITC due to the fluorescence quenching effect of water molecules on Eu3+. When the environmental samples contained TC, the red fluorescence of Eu3+ could be sensitized due to the diketone structure in the tetracycline molecule, which enabled the naked-eye visualized identification of TC. The nanosensor had high sensitivity (detection limit of 19 nM) and a wide detection range (0–30 μM) for TC. At the same time, the sensor was characterized by high visualization and anti-interference ability due to the strong fluorescence of both FITC and Eu complexes. In order to improve the practicality of the detection, this study also prepared a paper-based and sodium alginate gelated fluorescence detection platform with low background fluorescence. Combined with the common color analysis and RGB extraction procedures in mobile phones, real-time and on-site identification of TC on the surface of various fruit samples can be achieved.

Development of a novel in vitro model for non-cavitated carious lesion formation reflecting carious lesion activity

BackgroundOral biofilms are a critical component in dental caries formation. However, current remineralization studies often overlook the impact of microbial factors. Therefore, a comprehensive clinically relevant assessment of caries is needed. This study aimed to develop a novel in vitro model capable of generating non-cavitated carious lesions that incorporates both mineral loss and microbial activity using quantitative light-induced fluorescence-digital (QLF-D) technology. MethodsA total of 44 artificial early carious lesions were formed using bovine incisors. The extent of fluorescence loss (ΔF) was analyzed using a QLF-D camera. Oral microcosm biofilms were then employed to construct 22 active and 22 inactive carious lesions. The red fluorescence emission rate (ΔR) and bacterial viability (RatioG/G+R) was measured using QLF-D camera and a live-dead bacterial assay, respectively. Independent t-tests were performed to compare ΔF, ΔR, and bacterial viability of artificial carious lesions according to their activity status. ResultsNo significant difference in ΔF between the lesions was found based on activity status (p = 0.361). However, the ΔR of active lesions was 1.82 times higher than that of inactive lesions, and the RatioG/G+R was 1.49 times higher in active lesions than in inactive lesions (both p < 0.001). ConclusionsThe significant differences observed in ΔR and RatioG/G+R between active and inactive lesions emphasize the importance of considering lesion activity status when evaluating the potential efficacy of remineralization agents. This study presents a novel in vitro remineralization assessment model that reflects carious lesion activity while controlling baseline mineral distributions of lesions.

Single Fluorescent Probe for Multiple Tasks: Illuminating Lipid Droplets and Lysosomes in Dual Channels and Distinguishing Autophagy and Apoptosis.

Lipid droplets (LDs) and lysosomes play key roles in autophagy and cell apoptosis, and the discriminative visualization of the two organelles and simultaneously of autophagy and apoptosis is very helpful to understand their internal relationships. However, fluorescent probes that can concurrently achieve these tasks are not available currently. Herein, we delicately fabricate a robust probe CAQ2 for multiple tasks: illumination of LDs and lysosomes in dual emission colors as well as discriminative visualization of cell apoptosis and autophagy. The probe exhibited both lipophilic and basic properties and displayed different emission colors in neutral and protonated forms; thus, LDs and lysosomes emitted blue and red fluorescence colors, respectively. Because of the lysosomal acidification during autophagy, CAQ2 detected autophagy with evidently enhanced red emission. Because of the lysosomal alkalization during apoptosis, CAQ2 imaged apoptosis with a drastically decreased red fluorescence intensity. With the robust probe, the autophagy under starvation and lipidless conditions was visualized, and the apoptosis induced by H2O2, ultraviolet (UV) irradiation, and rotenone treatment was successfully observed. The efficient detoxification of Na2S against rotenone treatment was successfully revealed.

A triphenylamine-based carbohydrazide hydrazone fluorescent probe for selective detection of hypochlorite and sensing acidic gases.

Hypochlorous acid and its hypochlorite are important reactive oxygen species in the body, and are involved in various physiological processes related to immunity; their rapid detection is of great significance. Here, we synthesized a fluorescent probe (TPAS) by condensation of 4-(diphenylamino)benzaldehyde, carbohydrazide, and salicylaldehyde, which can be used for the detection of ClO- in water and sensing of acidic gas in its solid state. The probe showed strong selective recognition of ClO- in acetonitrile and good tolerance to interference ions. There were good linear responses between the intensity of absorbance and fluorescence and the amount of ClO-. The TPAS solid and its paper strips can emit red fluorescence when exposed to volatile acidic vapours. After being treated with NH3, the red fluorescence can be restored to yellow. The response process of TPAS to ClO- and acid gases was characterized using nuclear magnetic resonance, electrospray ionisation mass spectrometry, transmission electron microscopy, and density functional theory calculations. Furthermore, it can be utilized in analyzing ClO- in commercially available bleaching products; the detection results were basically compatible with the labelled values. In addition, the probe is biocompatible and can be applied for imaging ClO- in zebrafish.

Time-traceable micro-taggants for anti-counterfeiting and secure distribution of food and medicines.

This study presents an innovative solution for the enhanced tracking and security of pharmaceuticals through the development of microstructures incorporating environmentally responsive, coded microparticles. Utilizing maskless photolithography, we engineered these microparticles with a degradable masking layer with 30 μm thickness that undergoes controlled dissolution. Quantitative analysis revealed that the protective layer's degradation, monitored by red fluorescence intensity, diminishes predictably over 144 h in phosphate-buffered saline under physiological conditions. This degradation not only confirms the microparticles' integrity but also allows the extraction of encoded information, which can serve as a robust indicator of medicinal shelf life and a deterrent to tampering. These findings indicate the potential for applying this technology in real-time monitoring of pharmaceuticals, ensuring quality and authenticity in the supply chain.

Split Gp41-1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma.

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41-1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N-terminal split intein along with the N-moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C-terminal of split intein, the C-moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans-splicing was verified by transfecting both N-terminal and C-terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.

Red Fluorescence Enhancement and Optical Communication Performance of CuI Crystals Grown in HI Solution

AbstractThe photoluminescence (PL) of cuprous iodide (CuI) has drawn significant attention in the scientific community. In order to investigate the growth methods and environmental conditions on the fluorescence, we first prepared CuI via immersion of copper in hydroiodic acid. This method is beneficial for the growth of large grains (&gt; 20 μm) with intensive red fluorescence emission at 708 nm. The fluorescence has a high quantum yield (34 %), and superior stability. Then we studied the fluorescence behaviors in water. We observed fluorescence quenching, which is due to the energy transfer. Additionally, we studied the effect of temperature on the fluorescence. We found that the fluorescence intensity increased initially then decreased as the temperature increased. The underlying mechanism was analyzed in terms of fluorescence quencher (water) removal and thermal quenching. By controlling heating, we successfully obtained intensive and pure red fluorescence. Finally, we explored the potential application of the red fluorescent of CuI in optical communication. The results demonstrate bit error rates within permissible range of forward error correction, highlighting the superior performance of CuI in optical communication systems.

A Eu-MOF-Based Fluorescent Sensing Probe for the Detection of Tryptophan and Cu2+ in Aqueous Solutions.

Abnormal tryptophan (Trp) metabolism can be used as an important indicator of chronic hepatitis, paranoia, Parkinson's disease and other diseases. Deficiency or excessive accumulation of Cu2+ can cause diseases such as Wilson's disease and Alzheimer's disease. Eu-based metal-organic framework (Eu-MOF) was successfully prepared for fluorescence sensing of Trp and Cu2+ in an aqueous solution (pH = 7.4). Eu-MOF showed high selectivity and sensitivity for Trp and Cu2+ with detection limits of 0.22 µM and 0.09 µM and Ksv of 6.17 × 103M- 1 and 2.37 × 104M- 1 respectively. Trp and Cu2+ had overlapped UV absorption spectra with that of Eu-MOF and competed for the excitation light source. Trp also attenuated the antennae effect of organic ligands on Eu-MOF, thus quenching the red fluorescence of Eu-MOF. This study provides insights into the application of MOFs in bioanalysis and diagnostics.

Genetic Transformation of Potato without Antibiotic-Assisted Selection

The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. The development of strategies enabling the generation of marker-free transgenic plants presents an alternative to address public concerns regarding the safety of biotech crops. This study examined the capabilities of highly regenerative potato cultivars to develop transgenic plants without the presence of selective substances in their media. Internodal segments of in vitro potato plants were inoculated with the Agrobacterium strain AGL0 carrying plasmids, which contained the GFP or RFP gene driven by the CaMV 35S promoter to monitor the transformation process by observing in vivo green or red fluorescence. Despite the absence of selective pressure, inoculated explants demonstrated comparable or even higher transient expression compared to experiments based on antibiotic assistant selection. Consequently, under non-selective conditions, non-transgenic, chimeric, and fully fluorescent potato plantlets were concurrently developed. Among the five tested cultivars, the regeneration efficiency of non-chimeric transgenic plants varied from 0.9 (‘Chicago’) to 2.7 (#12-36-42) plants per 100 detached plantlets. Depending on the regenerative characteristics of potato varieties (early, intermediate, or late), a specific time interval can be determined when a blind collection of transgenic plantlets is more successful, streamlining the transformation procedure. The results indicate that the outlined procedure is simple and reproducible, consistently achieving the transformation efficiency of 7.3–12.0% (per 100 inoculated explants) in potato cultivars without selective pressure. The described transformation procedure holds the potential for obtaining cisgenic or intragenic potato plants with new valuable traits that do not carry marker genes.

A multifunctional Eu-MOF with multi-color and proton conduction switching and its anti-counterfeiting applications

Lanthanide metal-organic frameworks (Ln-MOFs) have exhibited remarkable capabilities in color change, luminescence, proton conduction through the strategic modification of metal nodes, organic ligands and channels or pores. By modifying the electron-deficient bipyridinium to contain carboxylic acid moieties and coordinating them with the luninescent Eu3+ ion, a unique and versatile Eu-MOF has been synthesized. Under UV light conditions, the complex shows an obvious color change from colorless to blue, which is due to the formation of viologen radicals after photoinduced electron transfer (PIET) process. As we all know, the changed color is caused by the alteration of absorption intensity, ultimately impacting its luminescence as well. Thus, the bright and strong red fluorescence of this complex is quenched upon continuous UV light irradiation, which may be attributed to intramolecular energy transfer from the excited state of the europium ion to the colored state of the CV ligand. Based on the control of multiple colors of the complex, the designed encryption model can realize multiple anti-counterfeiting of real numbers. In addition, the conductivity of this compound increases significantly after UV illumination, showing excellent photo-modulated proton conductivity. In short, our work has proposed a multifunctional bipyridinium-based Ln-MOF with photochromism, photo-switchable photoluminescence and proton conduction, which offers valuable insights for the creation of versatile switchable materials based on MOFs.

Direct Transamidation of Thioamides with Amines via Acetophenone-Promoted Enamine Catalysis under Metal-Free Conditions.

Herein, we developed a highly selective, efficient, and simple method for direct transamidation of thioamides with amines, promoted by commercially available acetophenone under metal-/solvent-free conditions. The reaction tolerated a wide range of functional groups and substrates, including single- or double-thioamides, benzylamines, or alkyl/cycloalkyl-substituted aliphatic amines. The present protocol can be applied to gram-scale in good yields. In addition, the Pt-/Ni-complexes of double-transamidation products were obtained in good to excellent yields. The investigation of photophysical properties indicated that the fluorescence spectra of Pt-complexes showed an emission band centered at 550-750 nm and exhibited red fluorescence when irradiated by a UV lamp (365 nm).

Evidence of Heritability in Prebiotically Realistic Membrane-Bound Systems.

The vesicles of short chain amphiphiles have been demonstrated to grow and divide. Here, we explored whether vesicle populations show evidence of heritability. We prepared 1:1 decanoic acid:decylamine vesicles with or without a detergent and in either water or prebiotic soup, a mixture of compounds that might have been present on early Earth. The mixtures were subjected to transfer with dilution, where, after 24 h of incubation (one generation), we transferred 10% of the mix into a 90% volume of a fresh vesicle-containing solution. This was continued for 30 generations. Samples with a history of transfers were compared to no-transfer controls (NTCs), initiated each generation using the same solutions but without 10% of the prior generation. We compared the vesicle size distribution and chemical composition of the transfer samples and NTCs and compared their fluorescence signals in the presence of Nile Red dye. We observe changes in the vesicle size but did not detect differences in the chemical composition. In the samples with detergent and soup, we observed irregular changes in the Nile Red fluorescence, with a tendency for parent and offspring samples to have correlated values, suggestive of heritability. This last result, combined with evidence of temporal autocorrelation across generations, suggests the possibility that vesicles could respond to selection.

Selective fluoride detection based on modulation of red emissive carbon dots fluorescence by zirconium-alizarin complex: Application to Nile River water and human saliva samples

Fluoride is an essential ion for dental health that is optimally supplemented through drinking water, toothpastes, and other oral hygiene products. This work reports a selective, simple, and inexpensive fluorescent sesnor for fluoride quantification. The sensor exploits the red fluorescence of carbon dots coupled with the colorimetric response of a zirconium-alizarin complex. The zirconium-alizarin exhibits an absorption band at 530 nm that overlaps the carbon dot excitation peak, resulting in inner filter effect-based quenching of the 612 nm red emission. However, in the presence of fluoride, binding disrupts the zirconium complex, releasing free alizarin with an absorption blue shifted to 420 nm. This reduces spectral overlap and restores carbon dot fluorescence. The carbon dots were synthesized by a hydrothermal method and exhibited excellent fluorescence quantum yield (16.3 %). Structural and optical characterizations were performed using TEM, XRD, XPS, FTIR, UV–vis, and fluorescence techniques. Under optimized conditions, the probe provided selective turn-on fluorescence detection of fluoride with a linear response from 1.0 to 130.0 μM and limit of detection (S/N = 3) of 0.32 μM. Practical utility was demonstrated by accurate quantification of fluoride in Nile River water and human saliva samples. This dual nanoprobe strategy offers a rapid and cost-effective platform for fluoride analysis in support of healthcare and environmental monitoring applications.

Dual‐modality imaging for identifying thrombosis via platelet GPIIb/IIIa receptor targeted cyclic RGDfK microbubbles

AbstractBackgroundAcute thrombotic events play a major role in various cardiovascular diseases. Therefore, direct thrombus imaging can be proved beneficial for early diagnosis and prompt therapy of thrombosis. Our study investigated targeted dual‐modality cyclic arginine‐glycine‐aspartic micro bubbles (cRGD‐MBs) for direct imaging of thrombi by fluorescence and ultrasound.MethodscRGD‐MBs were prepared by mechanical vibration and chemical chelation methods.ResultsCoulter counter analysis demonstrated that the cRGD‐MBs were well dispersed, with diameters ranging from 1 to 3 μm. They emitted bright red fluorescence under an excitation wavelength of 660 nm. In vivo fluorescence and ultrasound imaging revealed that cRGD‐MBs accumulated at the site of thrombus in the carotid artery with significant fluorescence and ultrasonic signal.ConclusionsThis study showed that novel microbubble cRGD‐MBs were successfully synthesized, and that these could potentially be used as contrast agents for immediate diagnosis of acute thrombus in vivo.

Role of Structural Peculiarities of Flavonoids in Suppressing AGEs Generated From HSA/Glucose System.

The pathogenesis of diabetes is related to the amount of advanced glycation end products (AGEs) that are naturally generated from the attachment of glucose with tissue and circular proteins. Human serum albumin (HSA) is more susceptible to AGE occurrence than other circular proteins due to its sensitive sites and high abundance. Considering the location of hydroxyl groups in the structure of flavonoids, which play a major role in suppressing of AGEs generating pathways, the present study was conducted to compare the effect of the chemical peculiarities of five flavonoids: apigenin (AP), naringenin (NA), luteolin (LU), Quercetin (QU), and methylquercetin (MQ), in suppressing AGEs generated in the HSA/glucose system. The results showed that all used flavonoids are capable of quenching the fluorescence intensity of AGEs in vitro. Analytical methods including UV-visible spectroscopy, CD spectro-polarimetry, TNBS, DTNB, DNPH, Congo red assay, ThT, and ANS fluorescence were used to deeper analysis of flavonoid performance. The anti-AGE effects of flavonoids followed the order of LU > QU > MQ > AP > NA. Docking results showed that flavonoids are associated with glycation-prone lysines and arginine residues in the "Sudlow pocket" through non-covalent interactions. Hydroxylation at the C4' and the double bond between C2-C3 increase the antiglycation potential of used flavonoids, while methylation of the OH group at the C3 position decreases this effect. It was also found that hydroxylation at C3 can play a dual role in anti-glycation ability. These findings may introduce a new approach to the structure-inhibition relationship of flavonoids in the design of operative anti-glycemic agents.

Deriving photosystem-level red chlorophyll fluorescence emission by combining leaf chlorophyll content and canopy far-red solar-induced fluorescence: Possibilities and challenges

Solar-induced chlorophyll fluorescence (SIF) emitted from photosystem I (PSI) and photosystem II (PSII) is characterized by two peaks centered in the red and far-red spectral regions. SIF provides a unique remotely sensible signal to track plant photosynthetic dynamics. Compared with far-red SIF, red SIF (RSIF) is more strongly linked to PSII and thus with plant photosynthetic activity, but is subject to stronger reabsorption within leaves and canopies. This hinders the understanding and use of canopy RSIF observations (RSIFobs), which is only a small fraction of the total RSIF emitted by the photosystems (RSIFtotal). Deriving RSIFtotal from RSIFobs is still challenging due to retrieval uncertainty, limited availability of RSIFobs and spectral overlap with chlorophyll absorption. To address the challenges associated with deriving RSIFtotal, we propose an exploratory method framework that combines canopy far-red SIF observations (FRSIFobs) and leaf chlorophyll content (LCC) to derive RSIFtotal. We first downscale FRSIFobs from canopy to leaf, and then leverage LCC information to estimate RSIF at the leaf level. Finally, we incorporate LCC information in the subsequent downscaling of RSIF from leaf to photosystem. To evaluate our approach, we use ground-based observation data in three crop types (rice, wheat, and maize) and SCOPE model simulations. Our results demonstrate that the seasonal patterns of RSIFtotal show a close agreement with the seasonal patterns of gross primary production (GPP) and absorbed photosynthetic active radiation (APAR). More importantly, RSIFtotal slightly outperforms FRSIFobs in estimating GPP for the three crop types. Our study has also revealed a strong linear relationship between the escape probability of RSIFtotal (fesc_R) and the RSIFobs/FRSIFobs ratio affected by LCC. The simplicity and robustness of our approach, along with its potential application in satellite remote sensing, will contribute to the improvement of large-scale GPP estimation and photosynthetic phenology detection. Moreover, our investigation of fesc_R will contribute to a better understanding the physiological and non-physiological dynamics of RSIFobs.

Generation of Dopamine Transporter (DAT)-mCherry Knock-in Rats by CRISPR-Cas9 Genome Editing.

Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.