Rhodamine fluorescent dyes and their applications

Abstract

The present invention provides a rhodamine-based fluorescent dye and its application. The rhodamine-based fluorescent dye comprises a rhodamine-based dye molecule and a triplet quencher, cyclooctatetraene, covalently coupled to the rhodamine-based dye molecule. Because rhodamine can be applied to all living cells, the dye's scope of application is broadened. Furthermore, the spatial conformation of rhodamine allows it to further shorten the distance between the chromophore and the triplet quencher, thereby more effectively reducing phototoxicity. Therefore, the rhodamine-based fluorescent dye, in which the triplet quencher cyclooctatetraene is covalently coupled to the rhodamine-based dye molecule, can achieve low-phototoxicity imaging of various organelles and proteins at the living cell level.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fluorescence imaging, and in particular to a rhodamine fluorescent dye and applications thereof. Background Art

[0002] With the continuous innovation of microscopy technology, bioluminescence imaging technology is currently one of the important means of studying biological problems. From ordinary optical microscopes to fluorescence microscopes and then to confocal microscopes, people have increasingly higher requirements for the temporal and spatial resolution of imaging and the information contained. After super-resolution imaging technology won the Nobel Prize in Chemistry in 2014, many unknown structures and their interconnections were revealed at ultra-high resolution at the nanometer level, and the imaging field officially ushered in the era of super-resolution. The development of super-resolution fluorescence imaging relies on the coordinated optimization of microscope hardware and fluorescent dyes. For example: Stochastic Optical Resolution Microscopy (STORM) requires the fluorescent dye to be able to efficiently and multiple times emit light within a certain period of time; Stimulated Emission Depletion Microscopy (STED) requires a beam of extremely strong depleted light to achieve reduced resolution, which undoubtedly places higher requirements on the photostability of the dye.

[0003] Prior to this, due to limitations in instrumentation and dye development, much imaging data came from fixed biological samples. The future of super-resolution imaging will undoubtedly be long-term, wide-field imaging at the level of living cells and tissues. Whether it is STORM or STED, repeated excitation of dye molecules or the use of extremely strong loss light not only damages the dye itself, but is also a fatal blow to living biological samples. The damage to living cells, tissues, and other samples produced during the imaging process is called phototoxicity. For example, when observing the dynamic activities of mitochondria for a long time, the mitochondria often swell and become rounded as the observation time increases, and then rupture and induce cell apoptosis.

[0004] Phototoxicity is a common phenomenon, mainly caused by the energy transfer between the highly active species in the triplet excited state of the chromophore and the triplet oxygen molecules in the ground state of the environment through collision, thereby generating singlet oxygen and other reactive oxygen species (ROS). These reactive oxygen species react with biological macromolecules such as lipids, proteins, and DNA to destroy their normal physiological structure and function. The problem of phototoxicity undoubtedly puts forward a new dimension of requirements for fluorescent dyes. In addition to requiring fluorescent dyes to have anti-bleaching photostability, they are also required to have low phototoxicity to samples to match the biocompatibility of living samples. Therefore, the solution to reducing the phototoxicity of fluorescent dyes is to reduce the components of the triplet excited state of fluorescence.

[0005] The triplet state of fluorescent dyes is a key factor leading to dye photobleaching and phototoxicity. Therefore, reducing the lifetime of the triplet state and pulling the dye in the triplet state back to the ground state as much as possible is the only solution. Currently, the following technologies are mainly used:

[0006] 1. System deoxygenation: In a single-molecule system, in order to increase the photostability of the dye molecules, deoxygenation is usually used to inhibit the reaction of highly active triplet excited state dye molecules with oxygen.

[0007] 2. Adding triplet state quenchers (TSQs) to the imaging system: Triplet state quenchers collide with the triplet state of the dye molecule in the solvent, causing photophysical or photochemical quenching, thereby protecting the fluorophore in an intermolecular manner. Photophysical triplet state quenchers include cyclooctatetraene (COT), diphenylhexatriene, or nickel ions, all of which achieve photoprotection through energy transfer between the dye and the quencher. Photochemical triplet state quenchers require a reagent with both oxidizing and reducing capabilities. Examples include Trolox (TX), ascorbic acid, ferrocene, p-nitrobenzyl alcohol, nitrophenylalanine, and methyl viologen. The triplet state of the dye is quenched via a photoinduced electron transfer (PET) mechanism, forming a radical anion or cationic intermediate, which then undergoes a series of redox reactions to return to the dye's ground state.

[0008] 3. Self-repairing dyes: The existing technology is to covalently link the triplet quencher cyclooctatetraene (COT) inside the cyanine dye (mainly Cy5, Cy3) molecule. The triplet state of the dye will undergo intramolecular quenching with the triplet quencher, reducing the triplet-triplet energy transfer and realizing the self-repair ability of the dye molecule. This type of dye is called a self-repairing dye. Under deoxygenated conditions, the triplet lifetime of the Cy5-COT conjugate is about 0.15μs, while the triplet lifetime of Cy5 is about 110μs, and the photostability of Cy5-COT under aerobic conditions is about 5 times higher than that of Cy5. The two COT molecules are symmetrically connected at both ends of Cy3 and Cy5, and the resulting molecules reduce phototoxicity by 3 and 5 times, respectively.

[0009] However, the above-mentioned deoxygenation system is not suitable for live cell imaging. In the method of adding triplet quenchers to the imaging system, the quenching efficiency depends on the collision between molecules, so a higher concentration of triplet quencher is required in the system. Such a high concentration will cause cytotoxicity and poor biocompatibility. Self-repairing dyes also have certain defects, such as: (1) Lack of universality: COT is covalently linked to cyanine dyes with different parent nuclei, and the degree of triplet quenching of the dye molecules varies greatly, and some have no obvious improvement. (2) Limitations of live cell dyes: Cyanine dyes naturally carry a positive charge and will accumulate on the negatively charged inner membrane of mitochondria, making them unsuitable for imaging other organelles, proteins, and DNA.

[0010] Therefore, there is still a need to provide new solutions to reduce the phototoxicity of fluorescent dyes. Summary of the Invention

[0011] The main purpose of the present invention is to provide a rhodamine fluorescent dye and its application, so as to provide a new solution for reducing the phototoxicity of fluorescent dyes in the prior art.

[0012] To achieve the above object, according to one aspect of the present invention, a rhodamine fluorescent dye is provided. The rhodamine fluorescent dye includes a rhodamine dye molecule and a triplet quencher cyclooctatetraene covalently coupled to the rhodamine dye molecule.

[0013] Furthermore, the rhodamine dye molecule is covalently coupled to the cyclooctatetraene via the bottom ring; preferably, the rhodamine dye molecule is covalently coupled to the cyclooctatetraene via the ortho position on the bottom ring.

[0014] Furthermore, the rhodamine dye molecules are selected from any one of the following: oxyrhodamine, carbon rhodamine or silicon rhodamine; preferably, the oxyrhodamine includes any one or more of the following: TMR, Rho110, MaP555, JF 503 、JF 525 、JF 536 、JF 549 、JF522 、JF 571 , 500R, 510R or 515R; preferably, the carborhodamines include any one or more of the following: 6-CPY, 5-CPY, CRhp, 580CP, JF 585 、JF 612 、JF 608 Or MaP618; Preferably, the silicon rhodamine class includes any one or more of the following: SiR, JF 626 、JF 629 、JF 630 、JF 635 、JF 646 、JF 669 or SiRh.

[0015] Furthermore, the singlet oxygen yield of the rhodamine fluorescent dye is 0.0014 to 0.018.

[0016] According to a second aspect of the present application, a fluorescence detection kit is provided, which comprises any one of the above-mentioned rhodamine fluorescent dyes.

[0017] According to a third aspect of the present application, a biomarker molecule is provided. The biomarker molecule includes a biomolecule and a fluorescent dye for marking the biomolecule, wherein the fluorescent dye for marking the biomolecule is any of the aforementioned rhodamine fluorescent dyes.

[0018] According to a fourth aspect of the present application, a bioluminescence imaging method is provided, wherein the method uses any of the aforementioned rhodamine fluorescent dyes to label cells.

[0019] Furthermore, the cells are living cells; preferably, the cells labeled with rhodamine-type fluorescent dyes are organelles within the cells, wherein the organelles include any one or more of the following: mitochondria, nuclei, cytoskeleton, Golgi, endoplasmic reticulum, lysosomes, endosomes, ribosomes or migratory bodies; preferably, the cells labeled with rhodamine-type fluorescent dyes are molecular markers within the cells, and the molecular markers are protein molecules, sugar molecules or nucleic acid molecules; preferably, the bioluminescence imaging method further includes placing the cells labeled with rhodamine-type fluorescent dyes under a microscope for imaging observation, and the microscope includes any one or more of the following: a confocal microscope, a stochastic optical reconstruction microscope or a stimulated emission depletion fluorescence microscope.

[0020] According to a fifth aspect of the present application, there is provided the use of any of the aforementioned rhodamine fluorescent dyes, or kits, or bioluminescence imaging methods in living cell imaging studies.

[0021] Furthermore, the imaging is performed under any one or more of the following microscopes: a confocal microscope, a stochastic optical reconstruction microscope, or a stimulated emission depletion fluorescence microscope.

[0022] The application of the technical solution of the present invention, on the one hand, broadens the scope of application of the dye because rhodamine can be applied to all living cells; on the other hand, the spatial conformation of rhodamine enables it to further shorten the distance between the chromophore and the triplet quencher, thereby more effectively reducing phototoxicity. Therefore, the rhodamine-based fluorescent dye of the present application, which covalently couples the triplet quencher cyclooctatetraene to the rhodamine-based dye molecule, can achieve low-phototoxicity imaging of various organelles and proteins at the living cell level. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0024] Figure 1 a to Figure 1 c shows the chemical structure of (a) TMR-TSQs; (b) TMR-TSQs at 520±10nm, 50mW / cm 2 (c) Absorption decay curve of DPBF over time under LED light, the slope of which reflects the singlet oxygen yield of each compound; (c) Absolute singlet oxygen yield table of TMR-TSQs.

[0025] Figure 2 A comparison of the phototoxicity of TMR-TSQs to Hela cells is shown.

[0026] Figure 3 a to Figure 3 f shows (a) the chemical structures of Rho110-COT, Rho123, SiR-COOMe, and SiR-COT, respectively; (b) a comparison of the phototoxicity of Rho110-COT and Rho123 on Hela cells; (c) a comparison of the phototoxicity of SiR-COOMe and SiR-COT on Hela cells; (d) a singlet oxygen yield graph of Rho110-COT and Rho123 under irradiation of a 520±10nm, 50mW / cm2 LED light; (e) a singlet oxygen yield graph of SiR-COOMe and SiR-COT under irradiation of a 620±10nm, 5mW / cm2 LED light, MB represents methylene blue, which is used as a quantitative reference; (f) the absolute singlet oxygen yield of each compound obtained by reference to the known singlet oxygen yields of Rho123 and MB, respectively.

[0027] Figure 4 a to Figure 4 c respectively show that the fluorescent dye of the present application also exhibits low phototoxicity when it is coupled with a protein tag to label protein molecules. Figure 4 a shows MaP555-COT-Halo and the reported compound MaP555-Halo labeling of the stably transfected cell line U2OS stably expressing H2B-Halo; Figure 4 b shows the DNA damage in the nuclear region of cells labeled with MaP555-COT-Halo and MaP555-Halo under the same illumination conditions. A small amount of XRCC1 punctate aggregation exists in the nuclear region of cells labeled with MaP555-COT-Halo, while obvious XRCC1 punctate aggregation distribution exists in the nuclear region of cells labeled with MaP555-Halo; Figure 4 c is Figure 4 b The quantitative analysis results showed that the aggregation of XRCC1 in the experimental group labeled with MaP555-COT-Halo (the number of aggregation points increased by about 5 times) was significantly less than that in the control group labeled with MaP555-Halo (the number of aggregation points increased by about 20 times).

[0028] Figure 5 It is shown that the fluorescent dye MaP555-COT-DNA of the present application also exhibits low phototoxicity when labeling nucleic acid molecules.

[0029] above Figure 4 and Figure 5 In the reference, MaP-COT-Halo refers to MaP555-COT-Halo, and MaP-COT-DNA refers to MaP555-COT-DNA. DETAILED DESCRIPTION

[0030] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0031] As mentioned in the background art, existing solutions for reducing the phototoxicity of fluorescent dyes have problems such as limited scope of application or poor biocompatibility. In order to improve this situation, this application provides a new improvement idea, which is detailed below.

[0032] In view of the above-mentioned shortcomings of the existing technology, this application applies the COT coupling method to rhodamine dyes to achieve low-phototoxicity imaging of various organelles and proteins at the living cell level. Among them, rhodamine dyes were selected as the parent dyes for the study for the following two main reasons:

[0033] First, rhodamine dyes are popular tools for live-cell imaging. Most fluorescent probes suitable for live-cell microscopy reported to date are based on rhodamine derivatives. The key is that rhodamine and its derivatives exist in a dynamic equilibrium between a non-fluorescent spirolactone form and a fluorescent zwitterion form. At first glance, this dynamic equilibrium appears to be disadvantageous, as it reduces the brightness of the fluorophore. However, the hydrophobic, non-fluorescent spirolactone structure is more cell-permeable than the zwitterion structure, and the binding of the probe to its target often shifts the equilibrium toward the fluorescent zwitterion, making the probe fluorescent and effectively reducing the background signal during imaging.

[0034] Second, the spatial conformation of rhodamine allows it to further shorten the distance between the chromophore and the triplet quencher. According to previous research structures, the rate of triplet-triplet energy transfer increases as the distance between the triplet quencher and the fluorescent dye decreases. Due to the planar sheet structure of cyanine dyes, the distance between the COT and the cyanine dye chromophore is relatively far. The bottom ring of rhodamine can rotate freely, and this torsion angle shortens the spatial distance between the bottom ring substituent and the conjugated chromophore. In addition, substitution at different positions of the bottom ring of rhodamine causes the spatial position of the triplet quencher group and the chromophore to change.

[0035] Therefore, in this application, N, N-tetramethylrhodamine (TMR) is first used as an example, and COT is coupled to the 3, 4, and 5 positions of the bottom ring of TMR in the form of an ester bond to obtain compounds o-TMR-COT, m-TMR-COT, and p-TMR-COT. In order to compare the improvement effect of different triplet quenchers on the phototoxicity of rhodamine dyes, o-TMR-NB ( Figure 1 a) A side-by-side comparison was conducted. The results showed that compared to the commercially available mitochondrial dyes tetramethylrhodamine methyl ester (TMRM) and tetramethylrhodamine ethyl ester (TMRE), the compounds o-TMR-COT, m-TMR-COT, and p-TMR-COT all exhibited lower singlet oxygen yields. Furthermore, the strategy of attaching COT to the carbon atom 3 of rhodamine reduced the singlet oxygen yield by approximately 6-fold compared to TMRM.

[0036] Furthermore, cell experiments revealed that TMRM reduced HeLa cell viability to 50% after approximately 2 minutes, and o-TMR-COT reduced HeLa cell viability to 50% after approximately 10 minutes. This is consistent with in vitro results on singlet oxygen production, demonstrating that attaching COT to the 3rd carbon of the rhodamine base ring can reduce phototoxicity by 5-6 times.

[0037] In order to further broaden the spectrum of low-phototoxic fluorescent dyes, the COT coupling strategy was applied to other rhodamine dyes in this application, and it was also found that the phototoxicity could be reduced.

[0038] Based on the above research results, the applicant has proposed a series of solutions of the present application. In a typical embodiment, a rhodamine fluorescent dye is provided, which includes a rhodamine dye molecule and a triplet quencher cyclooctatetraene covalently coupled to the rhodamine dye molecule.

[0039] Because rhodamine can be applied to all living cells, it broadens the dye's scope of application. Furthermore, rhodamine's spatial conformation allows it to further shorten the distance between the chromophore and the triplet quencher, thereby more effectively reducing phototoxicity. Therefore, the rhodamine-based fluorescent dyes described herein, which covalently couple the triplet quencher cyclooctatetraene to the rhodamine-based dye molecule, can achieve low-phototoxicity imaging of various organelles and proteins at the living cell level.

[0040] Rhodamine dye molecules, such as oxyrhodamine (such as TMR, Rho110, MaP555, JF 503 、JF 525 、JF 536 、JF 549 、JF 522 、JF 571 , 500R, 510R, 515R, etc.), carbo-rhodamines (such as 6-CPY, 5-CPY, CRhp, 580CP, JF 585 、JF 612 、JF 608 , MaP618, etc.), silicon rhodamine (such as SiR, JF 626 、JF 629 、JF 630 、JF 635 、JF 646 、JF 669 , SiRhp, etc.) (structural formula is as follows), its bottom ring (i.e., a benzene ring connected to a carboxylic acid group or a carboxylate group) can rotate freely, and the torsion angle can shorten the spatial distance between the bottom ring substituent and the conjugated chromophore. According to previous research results: the rate of triplet-triplet energy transfer increases with the shortening of the distance between the triplet quencher and the fluorescent dye, so the shortening of the distance helps to improve the oxidative quenching effect and is more helpful to reduce phototoxicity. Moreover, the substitution of different positions of the bottom ring of rhodamine changes the spatial position of the triplet quencher group and the chromophore. Therefore, in a preferred embodiment of the present application, it is covalently coupled to cyclooctatetraene through the bottom ring; more preferably, it is covalently coupled to cyclooctatetraene through the ortho position on the bottom ring. Experiments in this application have shown that covalently coupling cyclooctatetraene to the ortho position on the bottom ring has better low phototoxicity than at the para and meta positions.

[0041]

[0042]

[0043] In a preferred embodiment, the rhodamine dye molecules are selected from any one of the following: oxyrhodamine (TMR, Rho110, MaP555, JF 503 、JF 525 、JF 536 、JF 549 、JF 522 、JF 571 , 500R, 510R, 515R, etc.), carbo-rhodamines (6-CPY, 5-CPY, CRhp, 580CP, JF 585 、JF 612 、JF 608 , MaP618, etc.) or silicon rhodamine (SiR, JF 626 、JF 629 、JF 630 、JF 635 、JF 646 、JF 669 , SiRhp, etc.)

[0044] The singlet oxygen yield of the rhodamine fluorescent dye provided in this application is lower than that of existing self-repairing dyes of this type, ranging from 0.0014 to 0.018. This indicator can be used to test the performance of the rhodamine fluorescent dye of this application.

[0045] In a second exemplary embodiment of the present application, a biomarker molecule is provided, which is a biomolecule containing any of the aforementioned rhodamine-based fluorescent dyes. Such biomarkers are capable of fluorescence imaging under a microscope with relatively long exposure times and / or high illumination intensity. Specific biomolecules may be macromolecules such as proteins or nucleic acids. Such biomolecules may be distributed within cell organelles such as the cell membrane, cytoplasm, nucleus, or mitochondria.

[0046] In a third typical embodiment of the present application, a fluorescence detection kit is provided, which includes any of the above-mentioned rhodamine fluorescent dyes. Fluorescence labeling imaging using the dyes in the kit can achieve fluorescence imaging of various cellular submicroscopic structures at the living cell level.

[0047] In a fourth exemplary embodiment of the present application, a bioluminescent imaging method is provided, wherein the method uses any of the above-mentioned rhodamine-based fluorescent dyes to label cells. Using rhodamine-based fluorescent dyes to label cells for fluorescence imaging can achieve fluorescence imaging of various cellular submicroscopic structures at the living cell level.

[0048] Therefore, preferably, the cells are living cells; more preferably, the rhodamine-based fluorescent dyes of the present application label intracellular organelles, wherein the organelles include, but are not limited to, any one or more of the following: mitochondria, nuclei, cytoskeleton, Golgi apparatus, endoplasmic reticulum, lysosomes, endosomes, ribosomes, or migrasomes. In other preferred embodiments, the rhodamine-based fluorescent dyes label molecular markers on cells, specifically, molecular markers such as protein molecules, sugar molecules, or nucleic acid molecules.

[0049] It should be noted that the specific labeling principle of the rhodamine-type fluorescent dye with low phototoxicity in the present application when marking organelles is based on: the microenvironment of different organelles, such as different pH values, different charged properties, and different hydrophilicity. For example, the pH of lysosomes, early endosomes, and late endosomes is between 4.0-5.5, while the environment of other organelles is around pH=7.4; the inner and outer membranes of mitochondria have a potential difference between negative inside and positive outside; the endoplasmic reticulum, Golgi apparatus, and lipid droplets have a relatively hydrophobic environment. The specific labeling method is: by further coupling each organelle-specific group to the rhodamine-type fluorescent dye, the dye can specifically label one or several organelles. The following is an illustrative explanation using mitochondria, lysosomes, and endoplasmic reticulum as examples: positively charged rhodamine molecules can target the inner mitochondrial membrane to achieve mitochondrial localization; by coupling the drug molecule glibenclamide to the rhodamine dye, endoplasmic reticulum localization can be achieved by binding to the sulfonylurea receptors on the endoplasmic reticulum; by adding some weakly alkaline amines to the rhodamine molecules, the molecules can be aggregated in low-pH chambers to achieve acidic organelle localization, and by adjusting the alkalinity of different amines, specific localization of different acidic organelles can be achieved.

[0050] When used to label molecular markers, rhodamine fluorescent dyes are conjugated to the corresponding labeling molecules. For example, when labeling proteins, they can be conjugated to the tags carried on the protein, thereby labeling the protein. When labeling nucleic acids, they are conjugated to small molecules that specifically embed into the DNA molecule chain.

[0051] Because rhodamine fluorescent dyes have low phototoxicity, they are not only suitable for conventional fluorescence microscopy, such as ordinary optical microscopy or laser confocal microscopy, but also have advantages in application in super-resolution imaging microscopy, such as stochastic optical reconstruction microscopy or stimulated emission depletion fluorescence microscopy.

[0052] In a fifth exemplary embodiment of the present application, a method for using any of the aforementioned rhodamine fluorescent dyes, or the aforementioned kit, or any of the aforementioned bioluminescence imaging methods in live cell imaging studies is provided. Preferably, imaging is performed using any one or more of the following microscopes: confocal microscopy, stochastic optical reconstruction microscopy, or stimulated emission depletion fluorescence microscopy.

[0053] The beneficial effects of the present application will be further illustrated below with reference to specific embodiments.

[0054] Example 1

[0055] Using organic synthesis methods, an alcohol derivative of cyclooctatetraene (COT) was first synthesized for subsequent coupling with a rhodamine dye. The abbreviations and corresponding full names of the compounds or steps used in the following synthetic steps are as follows:

[0056] CDI N,N'-Carbonyldiimidazole

[0057] DMF N,N-dimethylformamide

[0058] THF Tetrahydrofuran

[0059] COT Cyclooctatetraene

[0060] DCM dichloromethane

[0061] MeOH methanol

[0062] PE petroleum ether

[0063] EA Ethyl acetate

[0064] MgSO4 magnesium sulfate

[0065] Na2SO4 sodium sulfate

[0066] TsOH p-Toluenesulfonic acid

[0067] n-BuLi n-butyllithium

[0068] HATU 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0069] TEA triethylamine

[0070] DDQ 2,3-Dichloro-5,6-dicyanobenzoquinone

[0071] EDCI.HCl 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0072] HOBT 1-Hydroxybenzotriazole

[0073] HPLC High-pressure liquid chromatography

[0074] CuBr2 Copper bromide

[0075] RT Room temperature

[0076] Mass

[0077] 1 H NMR nuclear magnetic resonance spectroscopy

[0078] Chloroform-d deuterated chloroform.

[0079] 1.1 Synthesis of alcohol derivatives of cyclooctatetraene (COT):

[0080]

[0081] 35.38 mg, 221 mmol of liquid bromine was dissolved in 150 ml of DCM and slowly added to a DCM solution of commercially available compound 1 (23 g, 221 mmol) at -78 ° C. After stirring for 1 hour, 300 mL, 30 mmol of potassium tert-butoxide was added dropwise. The reaction mixture was stirred at -60 ° C for 4 hours, then warmed to -10 ° C and dropped into ice water. The emulsion phase was cracked with a small amount of MgSO4, the organic phase was removed, and the aqueous phase was extracted with ether (3 x 200 mL). The extracted aqueous phase was dried over MgSO4, filtered, and concentrated to obtain a yellow oily substance (40 g, 99% yield, i.e., compound 2), which was used in the next step without further purification.

[0082] 1 H NMR (400MHz, Chloroform-d): δ6.21(s,1H),5.77-5.92(m,5H),5.62(s,1H).

[0083]

[0084] 40g of compound 2 was dissolved in 50mL of ultra-dry tetrahydrofuran and added dropwise to a pre-polished magnesium rod (15.7g, 655mmol) in an ice bath under argon protection. The reaction mixture was stirred at room temperature for 3 hours until a dark blue-green solution appeared. The resulting solution was cooled to -78°C, and then an excess of solid dry ice was added to generate the carboxylate derivative of COT. The reaction was then quenched with 250ml of aqueous solution and acidified to pH = 2 with 1M hydrochloric acid solution. The mixture was extracted with dichloromethane and saturated brine, and the organic phases were combined, dried over MgSO4, filtered, concentrated, and chromatographed on a silica gel column (petroleum ether:ethyl acetate = 4:1) to obtain compound 3 as a yellow oil (17.2g, 53% yield).

[0085] 1 H NMR (400MHz, Chloroform-d) 7.16 (s, 1H), 6.06-5.96 (m, 1H), 5.93 (d, J = 10.9Hz, 3H), 5.90-5.84 (m, 1H), 5.81 (s, 1H).

[0086]

[0087] 400 mg, 2.70 mmol of compound 3 was dissolved in 5 ml of MeOH solution and added dropwise to sulfonyl chloride at room temperature. After the addition was complete, the reaction mixture was heated to 60°C, stirred for 5 hours, and then dried to obtain 440 mg of crude product compound 4, which was used for the next reaction.

[0088]

[0089] Dissolve 440 mg (2.7 mmol) of compound 4 in 10 ml of THF and add it to 150 mg (4.05 mmol) of lithium aluminum hydride in an ice bath under nitrogen. Stir for 30 minutes, then warm to room temperature and continue stirring for 2 hours. Add 150 μl of water, 15% NaOH solution, and 450 μl of water in that order, and continue stirring for 5 hours. Filter, wash the filter cake with 20 ml of ethyl acetate, concentrate, and chromatograph on a silica gel column (petroleum ether:ethyl acetate = 20:1) to obtain 5 (260 mg, 72% yield) as a colorless oil.

[0090] 1 H NMR (400MHz, Chloroform-d) δ5.81~5.94 (s, 7H), 4.04 (d, J=5.6Hz, 2H).

[0091] Preparation of COT sulfonamide

[0092]

[0093] A magnetic stir bar and 0.4 g (~6 eq) of activated magnesium ribbon were added to a heat gun-dried glass flask. Under nitrogen, a 2 mL solution of 500 mg (1 eq) of bromocyclooctatetraene (Compound 2) (prepared according to a previously published method, Nat. Methods 2012, 9, 68) in anhydrous THF was added. After stirring at room temperature for 10-15 min, the color of the solution darkened to a dark green. Stirring was continued for 4 h to allow complete formation of the Grignard reagent, followed by cooling to -78°C. A 1-2 M solution of SO₂Cl₂ in dichloromethane (DCM) was added to another dry, nitrogen-protected round-bottom flask and cooled to -78°C. The THF solution of the Grignard reagent was drawn up using a syringe, and the SO₂Cl₂ solution was slowly added dropwise at -78°C. After the addition was complete, the mixture was stirred at -78°C for 15 min. The mixture was returned to room temperature and dried under vacuum to yield crude COTsulfonyl chloride as a yellow gel. This product was used directly in subsequent transformations without further purification. A solution of the crude sulfonyl chloride and 5 mL of a 1.5 M ammonia solution in THF were prepared by adding 5 mL of DCM. The mixture was cooled to -78°C, mixed, and stirred at this temperature for 1 hour. The solvent and excess ammonia were removed by rotary evaporation. The mixture was then purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 3:1) to afford 130 mg of COTsulfonamide as a brown oil, in a 25% yield (based on COT-Br).

[0094] 1 H NMR (400MHz, Methanol-d4) δ6.78 (d, J = 3.6Hz, 1H), 6.16 (dd, J = 11.3, 3.5Hz, 1H), 6.04 (d, J = 11.4Hz, 1H), 5.90 (m, 4H).

[0095] 1.2 Synthesis of compound o-TMR-COT

[0096] To a solution of 80 mg (0.21 mmol) of compound 6 in 3.0 ml of DMF, 118 mg (0.310 mmol) of HATU, 42.5 mg (0.42 mmol) of EA, and 41.6 mg (0.310 mmol) of compound 5 were added sequentially. After stirring at room temperature for 24 hours, the mixture was concentrated and eluted with silica gel column chromatography using a developing solvent of DCM:MeOH = 20:1 to obtain 36 mg of the product. This product was redissolved in 2.5 ml of DCM and slowly added dropwise to 25 ml of MTBE for recrystallization. The mixture was filtered and concentrated to obtain 20 mg of a red solid (yield: 19%).

[0097] 1H NMR(400MHz,Chloroform-d)δ8.27(d,J=7.6Hz,1H),7.81(t,J=7.5Hz,1H),7.74(t,J=7.5Hz,1H),7 .35(d,J=7.7Hz,1H),7.09(d,J=10.0Hz,2H),6.87(m,4H),5.74(m,7H),4.39(s,2H),3.31(s,12H).

[0098] 1.3 Synthesis of compound o-TMR-NB

[0099]

[0100] A 100 mg, 0.258 mmol, solution of compound 6 in 5.0 ml of DMF was added, followed by the addition of 147 mg, 0.356 mmol, of HATU, 78.3 mg, 0.774 mmol, of TEA, and 51.4 mg, 0.356 mmol, of commercially available compound 7. The mixture was stirred at room temperature for 18 hours. The product was concentrated and eluted with a silica gel column chromatography using a developing solvent of DCM:MeOH = 50:1 to obtain 40 mg of a red solid (yield: 41%).

[0101] 1 H NMR (400MHz, DMSO-d6) δ8.31(dd,J=7.7,1.5Hz,1H),8.06-8.01(m,2H),7.92(td,J=7.5,1.5Hz,1H),7.87(td,J=7.6,1.5Hz,1H),7.49(dd, J=7.4,1.4Hz,1H),7.25-7.19(m,2H),7.04(dd,J=9.5,2.4Hz,2H),6.97(d,J=9.4Hz,2H),6.80(d,J=2.3Hz,2H),5.08(s,2H),3.26(s,12H).

[0102] 1.4 Synthesis of compound m-TMR-COT

[0103]

[0104] 685 mg (5.00 mmol) of compound 8, 375 mg (2.5 mmol) of compound 9, 76 mg (76 mg), and 0.441 mmol of TsOH were dissolved in 15 ml of propionic acid solution and stirred at 65°C for 16 hours. The reaction solution was cooled to room temperature and added dropwise to 100 ml of 3 mol / L sodium acetate solution. The mixture was extracted with DCM. The combined organic phases were washed with saturated brine, dried over Na2SO4, filtered, and concentrated to obtain a residue. The residue was dissolved in 50 ml of a 3:2 solution of MeOH:DCM, followed by the addition of 295 mg (1.20 mmol) of DDQ. The mixture was stirred at room temperature for 2 hours and then concentrated in vacuo to obtain a residue. The residue was purified by silica gel column chromatography (DCM:MeOH = 50:1) to obtain a red solid (Compound 10, 100 mg, 10% yield).

[0105]

[0106] The same synthetic steps as those for compound o-TMR-COT were used to couple compound 10 with compound 5 to obtain compound m-TMR-COT.

[0107] 1 H NMR (300MHz, Acetonitrile-d3) δ8.27(d,J=7.9Hz,1H),8.01(s,1H),7.79(t,J=7.7Hz,1H),7.66(d,J=7.2Hz,1H),7. 25(d,J=9.5Hz,2H),6.94(dd,J=9.5,2.4Hz,2H),6.79(d,J=2.4Hz,2H),6.06-5.68(m,7H),4.75(s,2H),3.24(s,12H).

[0108] 1.5 Synthesis of compound p-TMR-COT

[0109]

[0110] It is similar to the compound m-TMR-COT and will not be described in detail.

[0111] 1 H NMR(300MHz,Chloroform-d)δ8.27(d,J=7.9Hz,1H),7.48(d,J=8.2Hz,1H),7.33-7.19(m,1H) ,6.92(dd,J=9.4,2.2Hz,1H),6.84(d,J=2.3Hz,1H),5.92(m,7H),4.81(s,2H),3.32(s,12H).

[0112] 1.6 Synthesis of compound Rho110-COT

[0113]

[0114] 100 mg, 0.272 mmol of commercially available compound 13 was dissolved in 5.0 ml of DMF solution, and 67 mg, 0.354 mmol, 47.9 mg, 0.354 mmol of EDCI-HCl, 47.9 mg, 0.354 mmol of HOBT, and 110 mg, 1.09 mmol of TEA were added in sequence. After stirring at room temperature for 15 minutes, 43.8 mg, 0.327 mmol of compound 5 was added. After stirring for 24 hours, the mixture was concentrated in vacuo and purified by reverse-phase HPLC (0.5% HCl) to give a red solid (8.0 mg, yield: 7%).

[0115] 1.7 Synthesis of compounds SiR-COOMe and SiR-COT

[0116]

[0117] 1.7.1 Synthesis of Compound 16

[0118]

[0119] 2.0 g, 10.0 mmol of compound 14 was dissolved in 30 ml of THF and added dropwise to 6.9 ml, 11.0 mmol of n-BuLi at -78°C under nitrogen. Stirring was continued for 1 hour, followed by the addition of 645 mg, 5.0 mmol of compound 15. Stirring was continued for 10 minutes. The reaction solution was slowly returned to room temperature and stirred for 2 hours. 10 ml of water was added to the reaction system, and the mixture was extracted with 30 ml of EA three times. The combined organic phase was dried, filtered, and concentrated in vacuo. The mixture was eluted by silica gel column chromatography (PE:EA = 20:1 to 10:1 developing solvent) to obtain a colorless oil (460 mg, yield: 31%).

[0120] 1.7.2 Synthesis of Compound 18

[0121] Compound 16 (50 mg, 0.167 mmol), compound 17 (75 mg, 0.501 mmol), and CuBr2 (3.5 mg, 0.017 mmol) were added to an autoclave and reacted at 120°C under argon for 6 hours. The mixture was slowly cooled to room temperature and purified by silica gel column chromatography (DCM:MeOH = 30:1) to obtain a blue solid (12 mg, yield: 17%).

[0122] 1.7.3 Synthesis of Compound 19

[0123]

[0124] 10 mg, 0.023 mmol of compound 18 was dissolved in 2.0 ml of ultra-dry DCM, followed by the addition of oxalyl chloride (148 mg, 1.17 mmol) and DMF (1.0 uL). The mixture was stirred at room temperature for 8 hours. The residue was concentrated in vacuo and redissolved in 2.0 ml of ultra-dry DCM and used directly in the next step without purification.

[0125] 1.7.4 Synthesis of SiR-COOMe Compound

[0126]

[0127] In an ice bath under nitrogen, 1.0 ml of the crude product, compound 19 (5.5 mg, 0.012 mmol), from the previous step was added to 1.0 ml of MeOH. After addition, the reaction system was slowly returned to room temperature, stirred for 30 minutes, and concentrated in vacuo to yield a residual solid. The product was isolated by silica gel column chromatography (DCM:MeOH = 10:1) to afford a blue solid (1.8 mg, 34% yield).

[0128] Mass:[M] + =443.25.

[0129] 1 H NMR (400MHz, Methanol-d4) δ8.25(dd,J=7.8,1.5Hz,1H),7.78(td,J=7.5,1.4Hz,1H),7.71(td,J=7.7,1.4Hz,1H),7.34(d ,J=2.8Hz,2H),7.30(dd,J=7.5,1.4Hz,2H),6.97(d,J=9.6Hz,2H),6.73(dd,J=9.6,2.8Hz,2H),3.62(s,3H),3.33(s,12H).

[0130] 1.7.5 Synthesis of SiR-COT Compound

[0131]

[0132] In an ice bath under nitrogen, 1.0 ml of the crude product, compound 19 (5.5 mg, 0.012 mmol), from the previous step was added to compound 5 (1.90 mg, 0.014 mmol). After the addition of the reactants, the reaction system was slowly returned to room temperature. After stirring for 3 hours, it was concentrated in vacuo to obtain a residual solid. The product was isolated by silica gel column chromatography (DCM:MeOH = 10:1) to obtain a blue solid (1.2 mg, 18% yield).

[0133] Mass:[M]+ =545.12.

[0134] 1 H NMR (400MHz, Acetonitrile-d3) δ7.97(dd,J=7.8,1.4Hz,1H),7.53(td,J=7.5,1.5Hz,1H),7.47(td,J=7.6,1.4Hz,1H),7.07(d,J=1.3Hz, 1H),7.03(d,J=2.8Hz,2H),6.70(d,J=9.6Hz,2H),6.41(dd,J=9.7,2.8Hz,2H),5.63-5.24(m,7H),4.17(s,2H),3.03(s,12H),0.36(s,6H).

[0135] 1.8 Synthesis of compound MaP555-COT-Halo

[0136]

[0137] Dissolve 25 mg of compound 20 in 1 mL of DMF, add 16 μL of triethylamine (2 eq) and 16 mg of potassium carbonate (2 eq). Cool the reaction to 0°C and add 7.5 μL of allyl bromide (1.5 eq). Warm the reaction system to room temperature and allow to react for 2 h before terminating. Wash the reaction solution with water and extract with DCM. The solvent is then dried and separated by silica gel column chromatography (DCM:MeOH = 10:1) to yield 25 mg of the desired product in a 90% yield.

[0138] 1 H NMR(400MHz,Chloroform-d)δ8.28(dd,J=8.0,1.4Hz,1H),8.11(d,J=7.8Hz,1H ),7.84(d,J=1.4Hz,1H),6.65(d,J=8.9Hz,2H),6.53(d,J=2.5Hz,2H),6.45(dd, J=8.9,2.5Hz,2H),5.98(ddt,J=16.6,10.4,5.9Hz,1H),5.36(dq,J=17.2,1.5H z,1H),5.27(dq,J=10.4,1.3Hz,1H),4.78(dt,J=6.0,1.3Hz,2H),3.03(s,12H).

[0139]

[0140] 25 mg of compound 21 obtained in the previous step was dissolved in 5 mL of DCM, and 100 μL of phosphorus oxychloride (20 eq) was added. The mixture was refluxed at 40°C for 4 h. The solvent was dried under vacuum and dissolved in 3 mL of acetonitrile. 43 mg of COT sulfonamide (4.4 eq) and 65 μL of DIPEA (7 eq) were then added and reacted at 50°C overnight without further separation. The solvent was dried again and redissolved in 3 mL of (DCM:MeOH=5:1) solvent. 30 mg of 1,3-dimethylbarbituric acid (4.5 eq) and 25 mg of tetrakis(triphenylphosphine)palladium (0.5 eq) were added. The mixture was reacted for 4 h and separated by HPLC to obtain 3.8 mg of the product with a yield of 12%.

[0141] Mass:[M+H] + =596.21.

[0142]

[0143] 1 mg of the above-obtained molecule was dissolved in 1 mL of DMF, and 2 mg of Halo-NH2·HCl (4.6 eq), 1.5 mg of Carter's condensation agent (2 eq), and 6 μL of DIPEA (20 eq) were added. After reacting overnight, the reaction solution was separated by HPLC to obtain 34 μg of the final product with a yield of 3%.

[0144] Mass:[M+H] + =801.38.

[0145] 1.9 Synthesis of compound MaP555-COT-TMP

[0146]

[0147] 1 mg of the above-obtained molecule was dissolved in 1 mL of DMF, and 1 mg of TMP3-NH2·HCl (1 eq), 1.5 mg of Carter's condensation agent (2 eq), and 6 μL of DIPEA (20 eq) were added. After reacting overnight, the reaction solution was separated by HPLC to obtain 85 μg of the final product with a yield of 5%.

[0148] Mass:[M+H] + =1070.34.

[0149] It should be noted that the compound MaP555-COT-TMP in step 1.9 and the compound MaP555-COT-Halo in step 1.8 are both conjugated to protein tags to achieve protein labeling. The only difference is that the protein tags Halo and TMP are different. Therefore, when the two are linked to the same protein, the fluorescence effects are similar (see Examples 5 and Figure 4 a and 4b).

[0150] 2.0 Synthesis of the compound MaP555-COT-DNA

[0151]

[0152] Compound 24: 1 H NMR (400MHz, Methanol-d4) δ8.93(d,J=1.5Hz,1H),8.46(dd,J=7.9,1.7Hz,1H),7.57(d,J=7.9Hz,1H),7.14(d,J=9.5Hz,2H),7.06(dd,J=9.5,2.4Hz,2H),6 .99(d,J=2.3Hz,2H),6.15(ddt,J=16.2,10.9,5.7Hz,1H),5.49(dq,J=17.1,1 .7Hz,1H),5.36(dq,J=10.5,1.4Hz,1H),4.95(d,J=5.7Hz,2H),3.31(s,12H).

[0153] Compound 25: 1 H NMR (400MHz, Methanol-d4) δ8.53(d,J=1.5Hz,1H),8.40(d,J=7.8Hz,1H),7.52(s,1H),7.20-6.74(m,6H),3.24(s,12H),2.63(s,6H).

[0154] Compound 25 was synthesized using compound 23 as the starting material, which was completely consistent with the synthesis steps of compounds 20-22 in 1.8 and need not be repeated.

[0155]

[0156] 20 mg (0.038 mmol) of compound 26 was weighed and dissolved in 1 ml of DMF. K2CO3 (30 mg, 0.218 mmol) was added and stirred at room temperature for 30 minutes. Compound 27 (12.3 mg, 0.049 mmol) was added to the reaction solution and stirred at 60°C overnight.

[0157] The reaction solution was purified by HPLC (solution A: H2O + 0.1% TFA, solution B: ACN) to obtain 18 mg of the product with a yield of 75%.

[0158]

[0159] 1 mg of the above-obtained molecule was dissolved in 1 mL of DMF, and 1.6 mg of 28 (2 eq), 1.5 mg of Carter's condensing agent (2 eq), and 6 μL of DIPEA (20 eq) were added. After overnight reaction, the reaction solution was separated by HPLC to obtain 321 μg of the final product, MaP555-COT-DNA (the DNA in this structural formula refers to the small molecule Hoechst targeting DNA, i.e., compound 28. The small molecule Hoechst emits fluorescence by intercalating into the minor groove of DNA). The yield was 17.8%.

[0160] 1 H NMR (400MHz, Methanol-d4) δ8.42(d,J=1.7Hz,1H),8.36(d,J=1.7Hz,1H),8.21(dd,J=8.0,1.8Hz,1H),8.11(d,J=8.6Hz,2H),8.02(dd,J=8.5,1.7Hz,1H ),7.86(d,J=8.6Hz,1H),7.71(d,J=9.0Hz,1H),7.47(d,J=7.9Hz,1H),7.38( dd,J=9.0,2.3Hz,1H),7.31(d,J=2.4Hz,1H),7.18(d,J=8.5Hz,2H),6.98(d, J=9.4Hz,2H),6.89(dd,J=9.3,2.4Hz,2H),6.82(d,J=2.5Hz,2H),6.64(s,1 H),6.02-5.93(m,1H),5.91-5.84(m,1H),5.75-5.59(m,4H),4.21(t,J=5.7H z,2H),4.03-3.87(s,2H),3.75-3.62(m,2H),3.58(t,J=6.4Hz,2H),3.44-3. 34(m,2H),3.21(s,12H),3.16-3.08(m,2H),3.02(s,3H),2.01-1.89(m,4H).

[0161] Example 2 Singlet oxygen yield test

[0162] For the aforementioned coupling of COT at positions 3, 4, and 5 of the bottom ring of TMR in the form of an ester bond, the obtained compounds o-TMR-COT, m-TMR-COT, p-TMR-COT, and o-TMR-NB ( Figure 1 a) The singlet oxygen yield of the solution was measured in vitro. The singlet oxygen yield was quantified using 1,3-diphenylisopropylfuran (DPBF).

[0163] like Figure 1 b and Figure 1As shown in Figure c, compared to the commercially available mitochondrial dyes tetramethylrhodamine methyl ester (TMRM) and tetramethylrhodamine ethyl ester (TMRE), the compounds o-TMR-COT, m-TMR-COT, and p-TMR-COT all exhibited lower singlet oxygen yields. According to literature research, the singlet oxygen yield of TMRE is known to be 0.012. According to a reference method, the singlet oxygen yield of TMRM is 0.0087, while the absolute singlet oxygen yield of the compound o-TMR-COT is only 0.0014, which is 1 / 6 of that of TMRM. Therefore, it can be concluded that the strategy of attaching COT to the carbon atom 3 of rhodamine can reduce the singlet oxygen yield by approximately 6 times.

[0164] Example 3

[0165] This example uses HeLa (human cervical cancer cell) cells as an example to test the phototoxicity of this series of dyes.

[0166] All compounds were diluted to a concentration of 250 nM in culture medium, and cells were incubated at 37°C in 5% carbon dioxide for 15 minutes for subsequent experiments. Using a high-content live-cell imaging system, cells in different wells were imaged in time series using a 561 nm laser, with a 10-second exposure time, capturing the time points of the gradient. One hour after imaging, propidium iodide (PI) staining was added, and the number of PI-positive signals was counted as the number of dead cells (A), while the number of cells before exposure was counted as B. This yielded the cell viability (B / A) / B*100%, an important indicator for evaluating dye phototoxicity.

[0167] The results of phototoxicity data analysis are shown in Figure 2 The survival rate of TMRM dropped to 50% after approximately 2 minutes, and that of o-TMR-COT dropped to 50% after approximately 10 minutes. This result is consistent with the singlet oxygen production rate in vitro. These two data confirm that attaching COT to the 3rd carbon of the rhodamine ring can reduce phototoxicity by 5-6 times.

[0168] Example 4

[0169] In order to further broaden the spectrum of the low phototoxic fluorescent dye of the present application, this example applies the COT coupling strategy to other rhodamine dyes to obtain a green fluorescent probe compound Rho110-COT with a similar structure (see 1.6 in Example 1). The commercial green mitochondrial membrane potential probe Rho123 can be used as a control compound for the compound Rho110-COT (e.g. Figure 3 and far-red fluorescent probe compounds SiR-COOMe and SiR-COT (1.7 in Example 1) (wherein compound SiR-COOMe is a control compound of compound SiR-COT, as shown in FIG. Figure 3As shown in a), the phototoxicity of the compounds Rho110-COT and SiR-COT was significantly reduced compared with the control compound by in vitro ROS level detection and intracellular phototoxicity detection. The phototoxicity of Rho110-COT and Rho123 was characterized by the changes in mitochondrial membrane potential (see Figure 3 b, 3c, 3d, 3e and 3f).

[0170] Example 5

[0171] Combining the COT coupling strategy with protein labeling technology can achieve low-toxicity imaging of specific proteins in living cells. Taking the highly abundant histone H2B in the cell nucleus as an example, the stable transgenic cell line U2OS stably expressing H2B-Halo was labeled with MaP555-COT-Halo and the reported compound MaP555-Halo. (See Figure 4 a) Because H2B is tightly wrapped around DNA, the generation of reactive oxygen species such as singlet oxygen around H2B will rapidly induce DNA damage. The DNA damage is characterized by the DNA repair protein human X-ray cross-complementing repair factor (XRCC1). When the DNA in the cell nucleus is undamaged or minimally damaged, XRCC1 is evenly distributed in the nuclear region or aggregates in very small amounts. When DNA damage is more severe, XRCC1 is clearly observed in the nuclear region in the form of punctate aggregates (see Figure 4 b).

[0172] By comparison, the aggregation of XRCC1 in the experimental group labeled with MaP555-COT-Halo (the number of aggregation points increased by about 5 times) was significantly less than that in the control group labeled with MaP555-Halo (the number of aggregation points increased by about 20 times). After replacing the protein tag of Halo-Tag with TMP-Tag, MaP555-COT-TMP also showed the property of less protein damage under light conditions. Figure 4 c.

[0173] In addition to ligands linked to protein tags, other marker molecules can also be linked. Taking nucleic acids as an example, the compound MaP555-COT-DNA was synthesized (commercially available SPY555-DNA was used as a control molecule). Using the above evaluation system, the same conclusion was obtained. Under the same labeling intensity and the same illumination conditions, MaP555-COT-DNA showed lower DNA toxicity (see Figure 5 ).

[0174] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0175] 1. The present invention can be applied to living cell imaging systems without the need for deoxygenation to improve the photostability and phototoxicity of the dye.

[0176] 2. This invention significantly improves the biocompatibility of imaging. On the one hand, the intramolecular covalent attachment of the triplet quencher increases the reaction rate and reduces the cytotoxicity of high concentrations of the triplet quencher. On the other hand, the effective collision of the triplet quencher with the dye more efficiently reduces the phototoxicity of the dye molecule.

[0177] 3. The present invention has a wide range of applications. This strategy can be applied to the specific labeling of various organelles and proteins within cells to achieve ultra-long-term super-resolution imaging.

[0178] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A rhodamine fluorescent dye, characterized in that The structural formula of the rhodamine fluorescent dye is selected from 2. The rhodamine fluorescent dye according to claim 1, characterized in that The singlet oxygen yield of the rhodamine fluorescent dye is 0.0014-0.

018.

3. A fluorescence detection kit, characterized in that: The invention comprises the rhodamine fluorescent dye according to claim 1 or 2.

4. A biomarker molecule, comprising a biomolecule and a fluorescent dye for marking the biomolecule, characterized in that: The fluorescent dye is the rhodamine fluorescent dye according to claim 1 or 2.

5. A bioluminescence imaging method for non-medical diagnostic purposes, characterized in that: Cells are labeled with the rhodamine fluorescent dye according to claim 1 or 2.

6. The bioluminescence imaging method for non-medical diagnosis purposes according to claim 5, characterized in that: The cells are living cells.

7. The bioluminescence imaging method for non-medical diagnosis purposes according to claim 6, characterized in that: The rhodamine fluorescent dye labels cells to label organelles in the cells, wherein the organelles include any one or more of the following: mitochondria, nucleus, cytoskeleton, Golgi, endoplasmic reticulum, lysosome, endosome, ribosome or migratoria.

8. The bioluminescence imaging method for non-medical diagnosis purposes according to claim 6, characterized in that: The rhodamine fluorescent dyes used to label cells are molecular markers in the cells, and the molecular markers are protein molecules, sugar molecules or nucleic acid molecules.

9. The bioluminescence imaging method for non-medical diagnosis purposes according to claim 6, characterized in that: The bioluminescence imaging method further comprises placing the cells labeled with the rhodamine fluorescent dye under a microscope for imaging observation, wherein the microscope comprises any one or more of the following: a confocal microscope, a stochastic optical reconstruction microscope, or a stimulated emission depletion fluorescence microscope.

10. Use of the rhodamine fluorescent dye according to claim 1 or 2, or the fluorescence detection kit according to claim 3, or the bioluminescence imaging method for non-medical diagnostic purposes according to any one of claims 5 to 9 in live cell imaging research for non-medical diagnostic purposes.

11. The use according to claim 10, characterized in that The imaging is performed under any one or more of the following microscopes: confocal microscope, stochastic optical reconstruction microscope or stimulated emission depletion fluorescence microscope.

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