Symmetrical bis-styrylindolium-based fluorescent compounds having two-photon absorption properties, method for producing said compounds and method and use as mitochondria identifiers

Abstract

The present invention discloses symmetrical bis-styrylindolium-based fluorescent compounds having two-photon absorption properties of general formula (3): (3) Wherein R = H, Me, MeO, CN, CO2H, Cl, Br, I, NO2; and the aryl group (Ar) is a diphenyl, triphenyl, naphthalene, anthracene, thiophene, dithiophene, furan, pyrrole. The present invention also relates to the formulations of the products of general formula (3) that are acceptable for use as mitochondria identifiers in living cells, preferably applying two-photon excitation having a wavelength of between 700 and 850 nm. The invention further relates to the method for producing said compound.

Description

Fluorescent compounds based on symmetrical bistyryl indoliums with two-photon absorption properties, preparation procedure of said compounds and procedure and use as mitochondrial identifiers. TECHNICAL SECTOR The present invention relates to the synthesis of symmetrical fluorescent compounds based on bistyryl indolium chromophores. These compounds have the ability to absorb two photons, with the unique feature that the synthesized molecules can be applied in the field of bioimaging, as they specifically recognize mitochondria in living cells. STATE OF THE PRIOR ART Mitochondria are the subcellular organelles responsible for managing the cell's energy supply. Within mitochondria, adenosine triphosphate (ATP) is generated through the action of various proton pumps to eliminate the excess protons produced in this process. Therefore, the double membrane surrounding these organelles is polarized, presenting a mitochondrial membrane potential of around 180 mV. This membrane potential facilitates the passage of charged molecules, allowing the selective accumulation of positively charged species within mitochondria. Furthermore, mitochondria are one of the most important sources of reactive oxygen species (ROS) at the cellular level. This function means that mitochondria can undergo gradual oxidative damage, which is often associated with aging, cancer, and neurodegenerative diseases.Access to easy and rapid methods that allow for selective labeling and visualization of mitochondria with fluorescent compounds allows for the study of their morphology and monitoring of their evolution in cells, tissues, and organs. The introduction of two-photon fluorescence microscopy (2PM) in the 1990s marked a turning point in the field of bioimaging. One of the main advantages of two-photon absorption processes is the ability to excite at wavelengths within biological windows. The relative transparency of tissues at these wavelengths makes it possible to image tissues between 500 and 1000 pm, a considerably greater depth than that achieved with confocal microscopy. (approximately less than 100 pm). Whether the goal is to obtain information about biological structures and / or processes or to study photoinduced processes in a given environment, 2PM is definitely established as an extremely useful tool. The use of excitation wavelengths in the near-infrared (NIR) not only allows imaging at greater depths but also facilitates the in vivo monitoring of biological processes due to the low phototoxicity of the light used. These reasons therefore justify the growing importance of 2PM in bioimaging tasks. In most cases, these compounds are organic and generally small in structure. Although the compounds initially used were those traditionally employed in conventional single-photon excitation microscopy, given the characteristics of 2PM and its potential, studies began on the development of new fluorophores specifically for 2PM applications, and thus, in turn, design guidelines for new compounds with these specific characteristics. The design of these compounds is essentially based on the theory of the two-photon absorption process and, specifically, on the dipolar approximation to it.Thus, the compounds that have traditionally been considered most effective for two-photon absorption are those that present symmetric structures that combine different electron donor and acceptor groups through various conjugated systems, thus existing multiple possible combinations (e.g., DKD, AKA, DAD, ADA, DKAKD, AKDKA, etc.). In general terms, these new fluorophores specially designed for 2P bioimaging can be classified into two categories: 1) 2P fluorophores designed to label subcellular compartments and 2) 2P fluorophores designed to monitor subcellular processes or chemical species within cells. For these reasons, the development of new fluorescent compounds capable of absorbing two photons that allow for the specific, sensitive, and rapid labeling of mitochondria represents a significant advance in the field of bioimaging. For all the above reasons, the applicant for this patent identifies the need to develop new fluorescent compounds capable of absorbing two photons that allow for the specific, sensitive, and rapid labeling of mitochondria, which would be a significant advance in the field of bioimaging. DESCRIPTION OF THE INVENTION The present invention proposes fluorescent compounds that solve the problem detailed above and that have the property of absorbing two photons and are capable of selectively marking mitochondria in living cells, thus acting as identifiers and allowing the monitoring of mitochondria identified by the compounds of the general formula of the invention. Thus, the symmetrical fluorescent molecules synthesized in this invention and with two-photon absorption properties are the compounds of general formula (3) Where: R = H, Me, MeO, CN, CO2H, Cl, Br, I, NO2; and the aryl group (called “Ar”) can be diphenyl, triphenyl, naphthalene, anthracene, thiophene, dithiophene, furan, pyrrole; These compounds are prepared by the reactions shown in the following scheme (the conditions for the synthesis of each of the compounds are shown in example 1 detailed in the section “detailed description of preferred embodiments of the invention”): In general, the center of these molecules, which will act as an electron donor, called Ar, must be an aromatic di-aldehyde, where Ar = diphenyl, triphenyl, naphthalene, anthracene, thiophene, dithiophene, furan, pyrrole, and at each end they must have an electron acceptor unit which will be an indolium that may be substituted with R = H, Me, MeO, CN, CO2H, Cl, Br, I, NO2. As an example, when the aryl group is a diphenyl, the structural formula of compound (3) corresponds to compound A represented below: Compound A On the other hand, when the aryl group is a thiophene, the structural formula of compound (3) corresponds to compound B represented below: Compound B This invention has the following advantages: They allow obtaining two-photon images of mitochondria present in living cells. When using a 720 nm wavelength using two-photon excitation, the compounds of the present invention exhibit a strong fluorescence signal at the intracellular level. This allows for two-photon imaging. Simple synthesis and good applicability The fluorescent compounds synthesized in the present invention allow visualization of mitochondria and have the property of obtaining highly selective two-photon images. They present applications in obtaining biological images: The synthesized compounds recognize and fluorescently label mitochondria in living cells and have the ability to absorb two photons, allowing for intracellular imaging of these subcellular organelles. This allows their application in biological imaging and their potential to be added to the field of fluorescent biomarkers. These compounds have potential application value as additions to commercial mitochondrial markers, with the possibility of obtaining images using two-photon microscopy, opening up a new direction in the development of novel fluorescent markers for biological applications. It should be noted that the present invention also relates to the method of marking and identifying mitochondria of living cells using the compounds of formula (3), such that said method comprises the following steps: - Cultivation or treatment of living cells for at least 4 hours with a solution containing the compounds of formula (3). - Two-photon excitation with a wavelength between 700 and 850 nm. - Recording of fluorescence images using a fluorescence microscope coupled to a multiphoton laser where the labeled mitochondria are observed. Finally, the procedure for preparing compounds of formula (3) is detailed below, which consists of the following steps: Knoevenagel condensation between aromatic dicarboxaldehydes (1) and 1,2,3,3-tetramethyl-3H-indolinium iodide (2) using ethanol as solvent and adding 3A molecular sieve to the reaction. Preferably, said condensation is carried out in the presence of refluxing ethanol for a time of between 24 h and 36 h. Addition of methanol to dissolve the solid resulting from the previous step, obtaining a homogeneous solution together with the molecular sieve, Filtration on a layer of Celite to separate the molecular sieve, obtaining the homogeneous solution, Vacuum concentration of the homogeneous solution, Washing with diethyl ether and ethyl acetate, obtaining a fluorescent compound based on symmetrical bistyryl indoliums with two-photon absorption properties of general formula (3). BRIEF DESCRIPTION OF THE DRAWINGS To complement the explanation of the invention and in order to help better understand its characteristics, a set of images is attached as an integral part of said description, where, for illustrative and non-limiting purposes, the following has been represented: Figure 1 shows a graph with the result of the normalized absorption and fluorescence spectra of compound A and compound B - preferred embodiments of the invention - at a concentration of 10 pM in acetonitrile, where the wavelength in nm is represented on the abscissa axis and the absorbance or Figure 2 shows a graph showing the dependence of the cross-section of compound A and compound B on the wavelength, where the wavelength in nm is represented on the abscissa axis and O2PA (GM) on the ordinate axis. Figure 3 shows a graph showing the dependence of the logarithm of the fluorescence intensity IF, ordinate axis, on the logarithm of the laser power, abscissa axis, for compounds A and B. Figure 4 shows an image of the fluorescence observed after 4 hours of incubation of compound A with HeLa cells. Figure 5 shows an image of the fluorescence observed after 4 hours of incubation of compound B with HeLa cells. Figure 6A represents the fluorescence image of compound A incubated with HeLa cells, while Figure 6A represents the fluorescence image of the commercial marker MitoTracker™ Red. Figure 7B represents the fluorescence image of compound B incubated with HeLa cells, while Figure 7B' represents the fluorescence image of the commercial marker MitoTracker™ Green. DETAILED EXPLANATION OF PREFERRED EMBODIMENTS OF THE INVENTION The following examples will serve to detail the synthesis process of two preferred embodiments of the invention (compounds A and B) [example 1], the verification of their behavior under one-photon excitation conditions [example 2], and the verification of their behavior under two-photon excitation conditions [example 3]. Also shown in this section are the results of the test by which compounds A and B are introduced into living cells [example 4], and finally the test to verify that compounds A and B label mitochondria [example 5]. The molecules synthesized in Example 1 were characterized by proton and carbon nuclear magnetic resonance (NMR) and high-resolution mass spectrometry. All of them were shown to possess good photophysical properties when excited under single-photon working conditions (see Figure 1, which corresponds to the result obtained in Example 2). They were also shown to have high cross-section values ​​when irradiated with two photons, i.e., they offer good two-photon absorption properties, as illustrated in Figures 2 and 3, which correspond to the result of Example 3. These compounds have been applied to detect mitochondria in living cells, allowing fluorescence images of these cellular organelles to be obtained using two-photon fluorescence microscopy, obtaining the results of example 4 represented in figures 4 and 5 for compounds A and B, respectively, which are similar or superior to the commercial markers MitoTracker™ Red, hereinafter MTR, and MitoTracker™ Green, hereinafter MTG, which need to be irradiated in the single-photon regime. At the same time, colocalization experiments have been carried out with respect to the MTR and MTG markers, thus demonstrating the ability of compounds A and B to recognize mitochondria, as illustrated in the results obtained from example 5 and which are represented in figures 6 and 7 for compounds A and B, respectively. Colocalization experiments have confirmed that mitochondria can be imaged in living cells using compounds A and B excited at a wavelength of 720 nm. The fluorescence signal obtained with these compounds when they have been irradiated with two photons has been strong and stable. This indicates that compounds A and B have the ability to perform two-photon imaging, which greatly reduces the damage that the cells studied may suffer, eliminates interference and signals corresponding to cellular autofluorescence, and increases the resolution of the images obtained. This invention has the following advantages: They allow obtaining two-photon images of mitochondria present in living cells. When using the 720 nm wavelength using two-photon excitation, the compounds show a strong fluorescence signal at the intracellular level. This allows for two-photon imaging. Simple synthesis and good applicability Compounds A and B are a new class of symmetrical fluorescent molecules capable of highly selective two-photon absorption. These compounds have a simple synthesis route and are easily applied to cells, requiring no organic cosolvents for solubilization in water. This means they can be incubated directly with cells. Compounds A and B localize to mitochondria relative to the commercial markers MTR and MTG, with a Pearson colocalization coefficient between 0.8 and 0.9, allowing for efficient mitochondrial detection. Compounds A and B have applications in biological imaging Fluorescent compounds A and B based on compounds derived from symmetrical molecules derived from bistyryl indoles, allow the recognition of mitochondria in living cells and have the property of absorbing two photons, allowing the obtaining of intracellular images of these subcellular organelles. Example 1: General procedure for the synthesis of compounds A and B The bis(indolinium) derivatives (1-2) were prepared by a Knoevenagel type condensation between commercial aromatic dicarboxaldehydes (1 eq.) and 1,2,3,3- iodide. tetramethyl-3H-indole (2 eq.) using ethanol as a solvent and adding 3A molecular sieve to the reaction. Once the reaction was complete, methanol was added to completely dissolve the resulting solid, obtaining a homogeneous solution. The molecular sieve was then filtered along with the homogeneous solution through a layer or filter of Celite. The molecular sieve was retained on the Celite layer, and the homogeneous solution passed through the Celite layer. Finally, the reaction mixture was concentrated in vacuo. The resulting residue was washed with diethyl ether and ethyl acetate to obtain the desired product. Synthesis of Compound A: In a 20 mL tube, 4,4'-biphenyl dicarboxaldehyde (100 mg, 0.48 mmol) and 1,2,3,3-tetramethyl-3H-indolino iodide (289 mg, 0.96 mmol) were dissolved in ethanol (10 mL). Then, 3A molecular sieve was added. The tube was sealed and the mixture was heated under reflux for 48 h. After completion of the reaction, the crude product was worked up as indicated in the general procedure. Compound A was obtained as a red solid (30 mg, 8% yield). Compound A Below is a description of component A based on the distinct espectroscopic techniques and high resolution mass espectrometry: MRI- 1 H (400 MHz, (CD3)2SO, 8 ppm): 8.50 (d, J = 16.4 Hz, 2H, H 2alk ), 8.38 (d, J = 8.3 Hz, 4H, H 2Ph ), 8.13 (d, J = 8.3 Hz, 4H, H 3Ph ), 7.98-7.93 (m, 2H, H7), 7.93-7.90 (m, 2H, H4), 7.81 (d, J = 16.4 Hz, 2H, H 1aik ), 7.70-7.64 (m, 4H, H5& H6), 4.21 (s, 6H, NCH3), 1.84 (s, 12H, C(CH3)2). MRI- 13 C (101 MHz, (CD3)2SO, 8 ppm): 182.2 (C2), 152.4 (C 2alk ), 144.2 (C 3a ), 143.1 (C 4Ph ), 142.3 (C 7a ), 135.1 (There Ph ), 131.7 (C 2Ph ), 130.1 (C5), 129.5 (C6), 128.1 (C 3Ph ), 123.4 (C4), 115.8 (C7), 114.1 (Ci alk ), 52.8 (C(CH3)2), 35.1 (NCH3), 25.7(C(CH3)2). HRMS (ESI): mass calculated for C38H38N2 (M 2+ ): 261.1512, encontrada: 261.1516. Synthesis of compound B: In a 20 mcgL tube, 2,5-thiophenicarboxaldehyde (100 mg, 0.71 mmol) and 1,2,3,3-tetramethyl-3H-indolinoiodide (428 mg, 1.42 mmol) were dissolved in ethanol (10 mL). Molecular sieve 3A was then added. The tube was sealed and the mixture was heated under reflux for 48 h. After completion of the reaction, the crude product was worked up as directed in the general procedure. Compound B was obtained as a red solid (181 mg, 36% yield). Compound B The following is the description of compound B based on different spectroscopic techniques and high-resolution mass spectrometry: NMR- 1 H (400 MHz, (CD3)2SO, 8 ppm): 8.69 (d, J = 16.1 Hz, 2H, H 2aik ), 8.34 (s, 2H, H2th¡o), 7.99-7.93 (m, 2H, H7), 7.93-7.88 (m, 2H, H4), 7.71-7.64 (m, 4H, H5& H6), 7.52 (d, J = 16.1 Hz, 1 H, H 1aik ), 4.16 (s, 6H, NCH3), 1.81 (s, 12H, C(CH3)2). NMR- 13C (101 MHz, (CD3)2SO, 8 ppm): 181.2 (C2), 147.3 (Ci th ¡o), 144.3 (C 3a ), 143.8 (C 2alk ), 142.3 (C 7a ), 137.1 (C 2f O ), 130.3 (C5), 129.6 (C6), 123.4 (C4), 115.9 (C7), 114.8 (Ci alk ), 52.7 (C(CH3)2), 35.1 (NCH3), 25.6 (C(CH3)2). HRMS (ESI): mass calcd for C30H32N2S1 (M 2+ ): 226.1138, found: 226.1141. Example 2: Verification of the behavior of compounds A and B under single-photon excitation conditions. Compounds A and B have presented the absorption and fluorescence spectra shown in Figure 1 and whose photophysical data are found in Table 1. These spectra were obtained using acetonitrile as the solvent at a concentration of 10 pM in solutions containing oxygen. 1 cm path length quartz cuvettes were used. The fluorescence quantum yields (4>) were determined by comparison with cresyl violet (<|>f = 0.56, in ethanol). Table 1. Photophysical data of compounds A and B (10 pM in acetonitrile). abs (nm) [a] Compound em (nm) [b] [c] [YE' 1 cm' 1 )] A 434

[59900] 539 0.004 B 509

[45000] 561 0.003 [al Maximum absorption wavelength. [bl Maximum fluorescence wavelength. [cl Fluorescence quantum yield. Figure 1 corresponds to the normalized absorption and fluorescence spectra of compound A and compound B at 10 pM concentration in acetonitrile. Specifically, Figure 1 shows the wavelength (expressed in nm) on the abscissa axis, while the data obtained from the absorption and fluorescence (IF) spectra are represented on the ordinate axis. That is, Figure 1 shows the data for compound A and the data for compound B. This example allows us to predict that compounds A and B will be able to absorb 2 photons of wavelengths above 700 nm and also ensures that their cellular distribution can be followed or monitored by measuring the fluorescence they emit between 530-570 nm. Example 3: Verification of the behavior of compounds A and B under two-photon excitation conditions. The behavior of compounds A and B under two-photon excitation conditions is represented in Figures 2 and 3 and Table 2. Table 2. Photophysical behavior of compounds A and B under two-photon excitation (at a concentration of 10 pM in acetonitrile). C2PA (GM) [b] Compound em (nm) [a] [ ibs,2P (nm)] A 539 248

[0700] , 30

[0890] B 561 510

[0700] , 48

[0890] [al Maximum fluorescence wavelength. [bl Cross-section obtained under two-photon excitation conditions Figure 2 represents the dependence of the cross-section of compound A and compound B on the wavelength. Specifically, Figure 2 shows the wavelength (expressed in nm) on the abscissa axis, while the data obtained from the Cross-section (O2PA) for each compound A and B, and expressed in GM, are represented on the ordinate axis. Compounds A and B exhibit cross-sections greater than 100 GM between 710 and 730 nm (Figure 2) and also have sufficiently high cross-section values ​​up to 850 nm. This ensures that they can be used for labeling cell organelles using low-energy excitation wavelengths. In a two-photon process, the dependence of the fluorescence intensity on the laser power is quadratic. Confirmation that compound A and compound B absorb two photons is obtained from the representation of the logarithms of the fluorescence intensity with the laser power, as can be seen in Figure 3, which represents the dependence of the logarithm of the fluorescence intensity on the logarithm of the laser power. Thus, in Figure 3 the logarithm of the laser power is represented on the abscissa axis, while the logarithm of the fluorescence intensity (FI) is represented on the ordinate axis. The slopes of the lines obtained, and represented in figure 3, have values ​​of 1.8 and 1.9 respectively. Example 4: Test in which compounds A and B are introduced into living cells Obtaining fluorescence images The cells used are HeLa (standard cancer cells widely used in scientific research because they can be constantly cultured in the laboratory). Cells were cultured in complete medium (DMEM + 10% FBS + 1% Penicillin-Streptomycin + 2 mM L-Glutamine) at 37°C in a humidified environment with 5% CO2. DMEM stands for Dulbecco's Modified Eagle's Medium, a type of modified medium that is commercially prepared as a creamy white powder, while FBS stands for Fetal Bovine Serum. For fluorescence microscopy experiments, cells were grown to approximately 50% confluence in 8-well multiwell plates suitable for light microscopy. HeLa cells were treated for 4 hours with a complete medium solution containing compounds A or B of the present invention at a concentration between 5 and 10 pM. Fluorescence images were subsequently recorded using a Leica SP5 multiphoton microscope in an inverted configuration. Cell images were made using a PLAN APO NA 1.4 63x oil immersion objective. Emission was recorded between X em = 500 - 600 nm using as excitation source: Xexc, 2P = 720 nm, for compound A. The issue was recorded between X em = 570 - 650 nm using as excitation source: X ex c, 2P = 720 nm, for compound B. Figure 4 shows the fluorescence observed after 4 hours of incubation of compound A with HeLa cells (10 pM, / xc,2P = 720 nm, / m = 500-600 nm). To make the fluorescence figure obtained illustrative, Figure 4 is the result of inverting the black background color to white and converting it to grayscale, so that the fluorescence that was originally observed in green appears represented in gray. Thus, it is concluded that the fluorescence is localized in cellular organelles that, due to their morphology, can be identified as mitochondria. Similarly, superimposing Figure 4 with the bright field confirmed that all fluorescence is localized within the cells; that is, no fluorescence was observed in either the nucleus or the cytoplasm of the cells (this figure is not included in this report because it is not sufficiently clear in grayscale). Figure 5 shows the fluorescence observed after 4 hours of incubation of compound B with HeLa cells (5 pM, / xc,2P = 720 nm, / m = 570-650 nm). The fluorescence is localized in cell organelles that, due to their morphology, are identified as mitochondria. To make the fluorescence figure obtained illustrative, Figure 5 is the result of inverting the black background color to white and converting it to grayscale, so that the fluorescence that was originally observed in purple appears represented in gray. By superimposing Figure 5 with the bright field, it was confirmed that all the fluorescence is located inside the cells, that is, no fluorescence was observed in either the nucleus or the cytoplasm of the cells (this figure is not included in this report because it is not clear enough in grayscale). Example 5: Test to verify that compounds A and B label mitochondria In this example, it is verified that compounds A and B, introduced into mitochondria as detailed in the test in example 4, label the mitochondria. To do this, the colocalization of compounds A and B with respect to the commercial marker: MitoTracker™ Red (MTR) (Thermofisher Scientific Cat. No. M22426) and MitoTracker™ Green (Thermofisher Scientific Cat. No. M7514) is determined. Compounds A and B were incubated in HeLa cells following the procedure described in Example 4. The commercial mitochondrial marker MTR was added to the cells incubated with compound A and fluorescence images of the MTR marker were obtained with a confocal microscope ( / xc p = 561 nm, / m = 640-680 nm); those of compound A were obtained in the two-photon excitation regime as described in Example 4. For compound B, the MTG marker was used ( / Uxc p = 561 nm, / m = 490-520 nm). The Pearson indices (a type of colocalization index) of compounds A and B have values ​​between 0.8-0.9 measured based on MTR and MTG, indicating that the compounds have successfully colocalized (=labeled / stained) in mitochondria. Figure 6A shows the fluorescence image of compound A incubated with HeLa cells (10 pM, / xc,2P = 720 nm, / m = 500-600 nm). Figure 6A' shows the fluorescence image of the commercial marker MitoTracker™ Red ( / Uxc p = 561 nm, / Um = 640-680 nm). By superimposing the images represented in Figure 6A and Figure 6A' it is possible to calculate the colocalization index (PCC index) which in this case corresponds to 0.8. The Pearson colocalization index is a measure of linear correlation between two data sets [how the colocalization index is obtained to compare fluorescence means in cells is explained in the article Cytometry A, 2010, 77(8), 733-42. doi: 10.1002 / cyto.a.20896]. The closer to 1 , the better the colocalization between the two fluorescence measurements being compared. In this case, it allows us to conclude that the colocalization between the fluorescence obtained from the commercial mitochondrial marker and compound A is good. Figure 6A shows, originally in green, the fluorescence obtained when HeLa cells were incubated with Compound A using two-photon excitation. Figure 6A shows, originally in red, the fluorescence obtained when HeLa cells were incubated with MTR using one-photon excitation. Figure 7B shows the fluorescence image of compound B incubated with HeLa cells (5 pM, / xc,2P = 720 nm, / m = 570-650 nm). While Figure 7B' shows the fluorescence image of the commercial marker MitoTracker™ Green ( / XC,IP = 561 nm, / Um = 490-520 nm). By superimposing the images represented in Figure 7B and 7B' it is possible to calculate the colocalization index (PCC index) which in this case corresponds to 0.9. Figure 7A shows, originally in magenta, the fluorescence obtained when HeLa cells were incubated with compound B and two-photon excitation was used. Figure 7B' shows, originally in blue, the fluorescence obtained when HeLa cells were incubated with MTG and one-photon excitation was used. The good Pearson indices obtained for the superposition of the fluorescence images, under two-photon excitation conditions, for compounds A and B, with respect to the commercial markers MTR and MTG allow us to conclude that both compounds A and B proposed as preferred embodiments of the present invention are selectively marking mitochondria and that they can be used under two-photon excitation conditions.

Claims

CLAIMS 1. Fluorescent compounds based on symmetric bistyryl indoliums with two-photon absorption properties of general formula (3) in which: R = H, Me, MeO, CN, CO2H, Cl, Br, I, NO2; Ar = diphenyl, triphenyl, naphthalene, anthracene, thiophene, dithiophene, furan, pyrrole; 2. Compounds of formula (3) defined in claim 1 for use as mitochondrial identifiers in living cells.

3. Compounds of formula (3) defined in claim 1 for use in monitoring mitochondria identified by the compounds of formula (3).

4. Process for preparing compounds of formula (3), according to the compounds described in any of the preceding claims, comprising: Knoevenagel type condensation between aromatic dicarboxaldehydes (1) and 1,2,3,3-tetramethyl-3H-indolin iodide (2) using ethanol as solvent and adding 3A molecular sieve to the reaction, Addition of methanol to dissolve the solid resulting from the previous step, obtaining a homogeneous solution together with the molecular sieve, Filtration on a layer of Celite to separate the molecular sieve, obtaining the homogeneous solution, Vacuum concentration of the homogeneous solution, Washing with diethyl ether and ethyl acetate, obtaining a fluorescent compound based on symmetrical bistyryl indoliums with two-photon absorption properties of general formula (3).

5. Method for marking and identifying mitochondria of living cells using the compounds of formula (3) defined in claim 1, comprising the steps of: - Cultivation or treatment of living cells for at least 4 hours with a solution containing the compounds of formula (3). - Two-photon excitation with a wavelength between 700 and 850 nm. - Recording of fluorescence images using a fluorescence microscope coupled to a multiphoton laser where the labeled mitochondria are observed.

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