Molecular constructs for multiphoton fluorescence microscopy imaging

Abstract

The present disclosure generally relates to a molecular construct for multiphoton fluorescence microscopy imaging. The molecular construct includes a two-photon absorption probe (2PAP) having a first, non-fluorescent configuration (2PAP-C) and a second, fluorescent configuration (2PAP-CL) linked to a photochromic molecule that can reversibly change from a first colored isomeric form (C) to a second colorless isomeric form (CL). The first colored form (C) can be isomerized to a second colorless isomeric form (CL) by absorption of two photons by the two-photon absorption probe (2PAP). The present disclosure also relates to a method for analyzing target structures using the molecular construct with a multiphoton microscope. Furthermore, the present disclosure relates to antibodies tagged with the molecular construct and the use of the molecular construct for imaging target structures.

Description

[Technical Field]

[0001] This disclosure generally relates to a molecular construct for multiphoton fluorescence microscopy imaging. This molecular construct comprises a two-photon absorption probe (2PAP) linked to a photochromic molecule having a first non-fluorescent configuration (2PAP-C) and a second fluorescent configuration (2PAP-CL), and capable of reversibly changing from the first chromogenic isomer (C) to the second colorless isomer (CL). The first chromogenic configuration (C) can be isomerized to the second colorless isomer (CL) by absorption of two photons by the two-photon absorption probe (2PAP). This disclosure also relates to a method for analyzing target structures using multiphoton microscopy with this molecular construct. Furthermore, this disclosure relates to antibodies tagged with this molecular construct and the use of the molecular construct for imaging target structures. [Background technology]

[0002] Multiphoton microscopy imaging enables highly high-resolution visualization of various structures and biological samples. Such imaging allows for the visualization and analysis of tissue morphology and physiology at the cellular level deep within the tissue, both in vivo and ex vivo.

[0003] The most common multiphoton fluorescence imaging technique is two-photon microscopy, which utilizes two near-infrared photons as excitation sources. Two-photon excitation is a fluorescence process in which a phosphor, or fluorescent probe, is excited by the simultaneous absorption of two photons.

[0004] Two-photon absorption probes enable penetration into deep tissues, efficient photodetection, and reduced photobleaching, making this technology useful for a variety of applications, including bioimaging, disease diagnosis, and monitoring.

[0005] Two-photon excitation is a nonlinear optical process that requires simultaneous excitation by two photons with wavelengths longer than the emitted light. Two-photon excitation microscopes typically utilize near-infrared (NIR) excitation light, such as that emitted by a laser, to excite fluorescent dyes. For each excitation, two photons of the NIR light are absorbed. Because this process depends on the simultaneous absorption of two photons, the resulting fluorescence emission changes with the square of the excitation intensity.

[0006] This indicates that when a two-photon absorption probe is applied to multiphoton microscopy, the light intensity shows a quadratic decrease with distance from the focal point, and that the two-photon absorption probe emits light and the fluorescence intensity is 1 / z 4 This means that it changes as (where z is the distance from the focal point). Here, z is the distance from the focal point. In multiphoton processes using 3-photon and 4-photon absorption probes, the fluorescence intensity is 1 / z, respectively. 6 and 1 / z 8 It can change as follows.

[0007] Therefore, the use of three-photon and four-photon absorption probes can significantly increase the spatial resolution of the imaged sample. However, the use of three-photon and four-photon absorption probes in multiphoton imaging applications typically requires extremely high light intensity. This is because the probability of simultaneous absorption of three and four photons by the probe is low, and the excitation wavelengths are difficult to access. For this reason, the use of three-photon and four-photon probes is limited in multiphoton microscopy applications.

[0008] Regardless of the molecular probe used in two-photon microscopy, the process is always constrained by the fundamental laws of optics. That is, the emission intensity is secondarily dependent on the intensity of the excitation light, and 1 / z 4This results in resolution. The secondary dependence on these molecules stems from the two-photon absorption mechanism, where two photons are absorbed by the molecule such that the sum of their photon energies corresponds to the energy of a two-photon-allowed electronic transition. Because two photons must strike the molecule simultaneously, the probability (and rate) of photon absorption for each individual molecule is secondarily dependent on the photon flux.

[0009] In light of the above challenges, there is a need to provide an improved probe for multiphoton microscopy applications that combines the advantages associated with two-photon absorption probes and three-photon or four-photon absorption probes.

[0010] More specifically, there is a need to provide an improved two-photon absorption probe that can offer enhanced spatial resolution to the structure or sample being imaged. [Overview of the project] [Problems that the invention aims to solve]

[0011] In light of the above issues, one object of this disclosure is to provide some improvements to multiphoton probes for use in multiphoton microscopy imaging. In particular, there is a need to provide a two-photon absorption probe that can provide improved spatial resolution. [Means for solving the problem]

[0012] According to a first aspect of the present disclosure, a molecular construct for multiphoton fluorescence microscopy imaging, the molecular construct having a first non-fluorescent configuration (2PAP-C) and a second fluorescent configuration (2PAP-CL), wherein the molecular construct is - Photochromic molecules, - A two-photon absorption probe (2PAP) linked to a photochromic molecule, Includes, Photochromic molecules can reversibly change from a first colored isomer form (C) to a second colorless isomer form (CL). The first colored morphological form (C) is isomerized to a second colorless isomeric form (CL) by absorption of two photons by a two-photon absorption probe (2PAP), An absorption spectrum of the first colored isomeric form (C) overlaps with an emission spectrum of a two-photon absorption probe (2PAP), and an absorption spectrum of the second colorless isomeric form (CL) does not overlap with the emission spectrum of the two-photon absorption probe (2PAP), and a molecular construct is provided.

[0013] The molecular constructs of the present disclosure depend on excitation by two-photon absorption, but when used in two-photon microscopy, can provide the same spatial resolution as a four-photon absorption probe. In other words, the molecular constructs of the present disclosure combine the advantages of two-photon absorption probes (relatively low excitation intensity is required, a standard laser light source near 800 nm can be utilized for microscopy experiments, and the excitation light is in the center of an optical window where the penetration depth into tissue is maximized) and the advantages of four-photon microscopy (significantly improved spatial resolution such as less than 100 nm).

[0014] The molecular constructs of the present disclosure can achieve a spatial resolution equivalent to that provided by four-photon microscopy without requiring four-photon absorption, enabling a true paradigm shift in multiphoton microscopy. Instead, a two-photon absorption probe can be utilized, and imaging can be performed using a standard two-photon microscope apparatus and a laser as an irradiation source.

[0015] This enables a leap forward in the application of microscopy in biomedical situations such as, for example, the diagnosis and evaluation of diseases or other clinical situations.

[0016] Most users of multiphoton microscopy cannot enjoy the excellent resolution exhibited by higher-order excitation (three-photon or four-photon absorption). No matter how carefully a molecular probe for two-photon microscopy is designed, the experiment is based on the fundamental law of photophysics, that is, the emission intensity depends quadratically on the intensity of the excitation light, and 1 / z 4It is always subject to the constraint of providing a decomposition energy. The secondary dependence of these molecules is due to the mechanism of two-photon absorption, where two photons are absorbed by the molecule such that the sum of the photon energies corresponds to the energy of a two-photon allowed electronic transition. Since two photons need to hit the molecule simultaneously, the probability (and rate) of photon absorption for an individual molecule depends quadratically on the photon flux. The molecular constructs of the present disclosure present an innovative and unprecedented approach that avoids this constraint by complying with this law while also responding to the excitation light such that the concentration of the emissive chemical species also depends quadratically on the intensity of the excitation light.

[0017] The molecular constructs of the present disclosure include a two-photon absorption probe (2PAP) linked to a photochromic molecule, which may also be referred to as a “molecular photoswitch”. The two-photon absorption probe (2PAP) may be covalently linked to the photochromic molecule.

[0018] The photochromic molecule can take on a first colored isomeric form (C) and a second colorless form (CL).

[0019] The colored form can be isomerized to the colorless form by a one-photon process with visible light (vis). Furthermore, two-photon absorption, which is central to the function of the proposed design, induces the same process.

[0020] The absorption spectrum of the first colored isomeric form (C) overlaps with the emission spectrum of the two-photon absorption probe (2PAP), such that the emission from the two-photon absorption probe (2PAP) in the first configuration 2PAP-C is quantitatively quenched by the colored isomeric form (C) in a FRET reaction.

[0021] FRET (Fluorescence Resonance Energy Transfer) is a distance-dependent interaction between two phosphors. In FRET, a light source excites a donor phosphor, which then transfers its energy to an acceptor phosphor without emitting light. For an efficient FRET process to occur, the donor and acceptor phosphors must be in close proximity to each other. Furthermore, the emission spectrum of the donor phosphor must overlap with the absorption spectrum of the acceptor.

[0022] The FRET reaction not only quenches the emission of 2PAP but can also sensitize the excitation of the colored isomer (C) of the photochromic molecule. Since the fate of the colored isomer (C) does not depend on how it became excited, FRET sensitization isomerization can yield a second colorless isomer (CL) of the photochromic molecule. However, since the absorption spectrum of the second colorless isomer (CL) does not overlap with the emission spectrum of 2PAP, FRET does not occur. Therefore, in this isomer (2PAP-CL) of the molecular construct, 2PAP emits strong fluorescence.

[0023] In one embodiment, a two-photon absorption probe (2PAP) is coupled to a photochromic molecule such that the FRET efficiency of the molecular construct is at least 90%.

[0024] Therefore, the two-photon absorption probe (2PAP) is coupled to a photochromic molecule and positioned in sufficient spatial proximity to induce an efficient FRET process. This enables efficient FRET-induced isomerization from the first non-fluorescent configuration (2PAP-C) to the second fluorescent configuration (2PAP-CL).

[0025] "FRET efficiency" is the ratio of the number of molecules inactivated by FRET to the number of molecules excited.

[0026] The first non-fluorescent configuration (2PAP-C) is the thermodynamically stable form of the molecular construct.

[0027] In one embodiment, the second colorless isomeric form (CL) of the photochromic molecule is isomerized to the first colored isomeric form (C) by thermal isomerization. This is sometimes referred to as "negative photochromism". For most photochromic molecules (photoswitches), the reverse applies. That is, the colorless isomeric form is the thermally stable form.

[0028] In a "thermal" process, no photons are absorbed and the process occurs without the involvement of photoexcitation. Instead, the available thermal energy is sufficient to drive the isomerization reaction.

[0029] Thus, the first non-fluorescent configuration (2PAP-C) of the molecular construct is switched to the second fluorescent configuration (2PAP-CL) by photo-isomerization, i.e., FRET-induced photo-isomerization. The second fluorescent configuration (2PAP-CL) is switched to the first non-fluorescent configuration (2PAP-C) by thermal isomerization. This is an important feature of the molecular constructs of the present disclosure.

[0030] In one embodiment, the rate of isomerization from the second fluorescent configuration (2PAP-CL) to the first non-fluorescent configuration (2PAP-C) is faster than the rate of isomerization from the first non-fluorescent configuration (2PAP-C) to the second fluorescent configuration (2PAP-CL).

[0031] In other words, the thermal isomerization rate k therm is faster than the two-photon FRET-induced photo-isomerization rate k photo . Under this condition, the concentration of the fluorescent form (2PAP-CL) depends quadratically on the intensity of the excitation light. If this condition is not met, conventional two-photon behavior is observed as a result of the saturation of 2PAP-CL.

[0032] k therm is the two-photon FRET-induced photo-isomerization rate k photoIf the reaction is substantially faster than the excitation light intensity, the concentration of the phosphor morph (2PAP-CL) is secondarily dependent on the excitation light intensity. The emission intensity of these fluorescent chemical species is also secondarily dependent on the excitation light intensity. As a result, there is an overall fourth-order dependence of emission intensity on excitation intensity, i.e., I(emission)∝I(excitation). 4 This is brought about, and it is usually only observable with a four-photon microscope.

[0033] Therefore, improved spatial resolution and improved imaging techniques can be achieved, enabling significant improvements when applied to multiphoton microscopy analysis.

[0034] In some embodiments, the isomerization rate from the second fluorescent configuration (2PAP-CL) to the first non-fluorescent configuration (2PAP-C) is (k above). therm (as it is called) the isomerization rate from the first non-fluorescent configuration (2PAP-C) to the second fluorescent configuration (2PAP-CL) (k photo It is at least twice as fast as (as it is called), preferably at least 10 times faster, and more preferably at least 50 times faster.

[0035] The absorption of two photons by a two-photon absorption probe (2PAP) induces isomerization of photochromic molecules, thereby switching from the first (non-fluorescent) configuration (2PAP-C) to the second (fluorescent) configuration (2PAP-CL). Two-photon absorption is not limited to a specific range of light absorption; however, typically, light with wavelengths in the 700–900 nm range is used in two-photon microscopy.

[0036] Therefore, in some embodiments, the two-photon absorption probe (2PAP) absorbs light with a wavelength of at least 700 nm, preferably light with a wavelength in the range of 700 nm to 900 nm. This induces the absorption of two photons.

[0037] In some embodiments, the two-photon absorption probe (2PAP) has a fluorescence quantum yield of at least 10%, preferably at least 30%, and more preferably at least 50%.

[0038] As used herein, the term "fluorescence quantum yield" is the ratio of the number of photons emitted during fluorescence emission to the number of photons absorbed in a one-photon process. Thus, the quantum yield gives the probability that an excited state is deactivated by fluorescence emission rather than by another non-radiative mechanism.

[0039] In some embodiments, the absorption spectrum of the first colored isomer form (C) and the emission spectrum of the two-photon absorption probe (2PAP) are at least 1 × 10⁻¹⁶ 13 nm 4 M -1 cm -1 It has a spectral overlap integral.

[0040] Preferably, the spectral overlap integral is as high as possible to allow the fluorescence signal to be quenched in the FRET reaction (induced by two-photon absorption).

[0041] This reduces fluorescence "noise" and undesirable emission in 2PAP-C; in other words, this is the intended non-fluorescent configuration of the molecular construct.

[0042] In some embodiments, the photochromic molecule has a thermal half-life (t) of less than 20 seconds, preferably less than 10 seconds, and more preferably less than 1 second at room temperature. 1 / 2 This is because the concentration of the phosphor form (2PAP-CL) is secondarily dependent on the excitation intensity, and this is to ensure that the concentration of the phosphor form 2PAP-CL is always kept low.

[0043] In some embodiments, the colored form (C) of the photochromic molecule absorbs light in the wavelength range of 350–800 nm, preferably 450–700 nm.

[0044] The ability of photochromic molecules to absorb in these wavelength ranges enables efficient FRET from the two-photon absorption probe 2PAP to the first colored isomer form (C) of the photochromic molecule, and conventional lasers can be used as the excitation source.

[0045] In another embodiment, a method is provided for analyzing a target structure with a multiphoton microscope, comprising the following steps: a) To provide a fluorescently labeled target structure by incubating a molecular construct as described herein with a target structure, b) Irradiating a fluorescently labeled target structure with light in a wavelength range that enables two-photon absorption by a molecular construct so that a fluorescent signal is generated, and c) Detecting and / or measuring a fluorescent signal.

[0046] The target structures to be analyzed can be, for example, fixed or living cells, tissue samples, biological samples, such as bodily fluids, and various 3D structures.

[0047] Molecular constructs can be incubated with target structures by means known to those skilled in the art.

[0048] Typically, the target structure is irradiated with light having a wavelength of at least 700 nm. This causes two-photon absorption, followed by a series of events that trigger the molecular construct to fluoresce. Furthermore, this fluorescence is expected to provide a spatial resolution equivalent to that observed with a four-photon microscope.

[0049] The emitted fluorescence can be detected and / or quantitatively measured by means known to those skilled in the art. Therefore, the properties of the target structure can be analyzed in great detail with improved spatial resolution.

[0050] In another embodiment, antibodies tagged with molecular constructs as described herein are provided.

[0051] Such antibodies can be efficiently used to detect specific target regions in cells, tissues, or body fluids. Antibodies targeting specific antigens provide a useful approach that enables the molecular constructs of this disclosure to elucidate important information regarding the pathogenesis and biological pathways of specific diseases.

[0052] Preferably, the antibody is a monoclonal antibody.

[0053] In yet another aspect, the disclosure relates to the use of the molecular constructs described above for imaging target structures with a multiphoton microscope, such as a two-photon microscope.

[0054] Further features and advantages of this disclosure will become apparent upon consideration of the appended claims and the following description. Those skilled in the art will recognize that different features of this disclosure can be combined without departing from the scope of this disclosure to create embodiments other than those described below.

[0055] Various aspects of this disclosure, including their specific features and advantages, will be readily apparent from the following detailed description and accompanying drawings. [Brief explanation of the drawing]

[0056] [Figure 1] Figure 1 schematically discloses the molecular constructs of this disclosure and their modes of action. [Figure 2] Figure 2 is a performance plot illustrating the four-photon behavior of the molecular constructs of this disclosure. [Figure 3] Figure 3 shows examples of photochromic molecules that can be used in the molecular constructs of this disclosure. [Figure 4] Figure 4 schematically illustrates how a two-photon absorption probe (2PAP) can be coupled to an exemplary photochromic molecule. [Figure 5]Figure 5 schematically shows simulations of the four-photon performance of the two molecular constructs, where thermal isomerization from the second colorless isomer to the first colored isomer is present and absent, respectively. [Modes for carrying out the invention]

[0057] The Disclosure will be described more fully below with reference to the accompanying drawings illustrating currently preferred embodiments thereof. However, the Disclosure can be embodied in many different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided for thoroughness and completeness and fully convey the scope of the Disclosure to those skilled in the art.

[0058] Figure 1 schematically shows the molecular construct of this disclosure. The two-photon excitation process is shown by 2 × 800 nm photons.

[0059] 2PAP-C represents the first non-fluorescent configuration of the molecular construct, and 2PAP-CL represents the second fluorescent configuration. 2PAP-C is the thermodynamically stable form of the molecular construct.

[0060] The molecular construct includes a two-photon absorption probe (2PAP) linked to a photochromic molecule, which can take the form of a first colored isomer (C) and a second colorless isomer (CL).

[0061] Two-photon absorption probes (2PAPs) are typically covalently linked to photochromic molecules.

[0062] When two photons are simultaneously absorbed by 2PAP (which can occur, for example, by irradiating a molecular construct with a laser), 2PAP is excited to its lowest excited singlet state.

[0063] The absorption spectrum of C overlaps with the emission spectrum of 2PAP, and as a result, the emission from 2PAP in 2PAP-C is efficiently quenched by C in the FRET reaction. The FRET reaction not only quenches the emission of 2PAP but also sensitizes the excitation of C. Since the fate of C does not depend on how it reached its excited state, FRET sensitization isomerization results in CL. In this case, the absorption spectrum of CL does not overlap with the emission of 2PAP, so FRET does not occur. This suggests that in this isomer form of the molecular construct (2PAP-CL), 2PAP emits strong fluorescence.

[0064] Therefore, there are two effects of the intensity of the light used to excite 2PAP in the two-photon process (arbitrarily set to 800 nm in Figure 1). Firstly, the fluorescence intensity of 2PAP in individual fluorescent isomers (2PAP-CL) is secondarily dependent on the excitation intensity. Secondly, the concentration of the fluorescent isomer 2PAP-CL is also secondarily dependent on the excitation intensity. This is because the FRET sensitization isomerization rate from non-phosphor 2PAP-C to phosphorer 2PAP-CL depends on the rate at which 2PAP in 2PAP-C absorbs photons. This rate is secondarily dependent on the intensity of the excitation light.

[0065] Therefore, both the fluorescence intensity "per fluorescent molecule" and the concentration of fluorescent molecules are secondarily dependent on the excitation intensity. As a result, the fluorescence intensity as a whole exhibits a quartic dependence. This phenomenon occurs particularly when the thermal isomerization rate from the phosphor 2PAP-CL to the non-phosphor 2PAP-C is significantly faster than the isomerization from 2PAP-C to 2PAP-CL by two-photon FRET. In Figure 1, thermal isomerization is denoted as Δ.

[0066] Figure 2 schematically illustrates the performance principle of the design of this disclosure. The general principle of this design is to subtly balance two-photon induced FRET photosensitization with thermal isomerization such that the emitted fluorescence intensity exhibits a fourth-order dependence on the excitation intensity. k1 and k -1 These are, respectively, the k mentioned above. photo , and k therm This corresponds to k1 / k -1When the value is close to zero (very low concentration of the phosphor 2PAP-CL), complete four-photon behavior (four-value dependence) is observed. This suggests that improved spatial resolution can be obtained using the molecular constructs of this disclosure.

[0067] Figure 5 shows a simulation of 4-photon performance versus irradiation time for two molecular constructs. One molecular construct is linked to a photochromic molecule that cannot be thermally isomerized, while the other molecular construct (dotted line) is linked to a photochromic molecule that can be thermally isomerized, and the thermal isomerization rate is faster than the photoisomerization rate, i.e., the isomerization rate from the first non-fluorescent configuration to the second fluorescent configuration.

[0068] As shown in Figure 5, molecular constructs in which photochromic molecules can isomerize from a second colorless isomer (CL) to a first colored isomer (C) via thermal isomerization consistently exhibit perfect four-photon behavior throughout the entire irradiation time. Therefore, the four-photon performance is maintained over a long period. Thermal isomerization keeps the concentration of the second fluorescent configuration of the molecular construct at a low level, preventing saturation. If the second fluorescent configuration (2PAP-CL) becomes saturated, the four-photon behavior is lost, and "conventional" two-photon behavior is observed. This is the case for molecular structures in which the photochromic molecule cannot thermally isomerize to the colored isomer (C). This is shown by the continuous decreasing lines in Figure 5.

[0069] The "four-photon behavior" in Figure 5 represents how far the two molecular constructs are from 2PAP-CL saturation. If the thermal isomerization process is efficient, the concentration of 2PAP-CL is very low (far from saturation), and the fluorescence behavior is very close to a perfect four-photon process. This is shown by the four-photon behavior close to 1.0. In contrast, four-photon behavior close to 0 means that the fluorescence intensity is described by the conventional two-photon behavior (fully saturated). Figure 3 shows examples of photochromic molecules, i.e., photoswitches, that can be used in the molecular constructs of this disclosure. These molecules satisfy the "reverse photochromism" feature of the molecular constructs of this disclosure. The isomerization scheme is also illustrated. 1 / 2 This indicates the thermal half-life and corresponds to the thermal isomerization of the photochromic molecule to its colored form at 25°C. λ max This indicates the maximum wavelength of the most redshifted absorption band in the colored isomer form (C). Possible and exemplary 2PAP derivatives are also shown in Figure 3.

[0070] It should be noted that the molecular constructs of this disclosure are not in any way limited to specific two-photon absorption probes, but can utilize any 2PAP that can be coupled to a photochromic molecule. Preferred 2PAPs for use in the molecular constructs of this disclosure have a fluorescence quantum yield of at least 10%, preferably at least 30%, and more preferably at least 50%.

[0071] For example, the two-photon absorption probe (2PAP) can be triphenylamine, fluorene, benzothiadiazole, stilbene, and / or cyanine derivatives.

[0072] Figure 4 schematically shows an exemplary molecular construct of this disclosure in which 2PAP is linked to a photochromic molecule.

[0073] This disclosure is by no means limited to the use of specific photochromic molecules. Any photochromic molecule having the ability to exhibit reverse photochromism can be used. That is, any photochromic molecule having the ability to be switched from a colorless isomer to a colored isomer by thermal isomerization can be used.

[0074] For example, the photochromic molecule can be a donor-acceptor Stenhouse adduct (DASA) unit or a phenoxyl-imidazolyl radical complex.

[0075] In a preferred embodiment, the photochromic molecule has a thermal half-life (t) of less than 20 seconds, preferably less than 10 seconds, and more preferably less than 1 second at room temperature. 1 / 2 ) has.

[0076] Terms, definitions, and embodiments in all aspects of this disclosure shall apply mutatis mutandis to other aspects of this disclosure.

[0077] While this disclosure has been described with reference to its specific exemplary embodiments, many different modifications, improvements, and so on will become apparent to those skilled in the art.

[0078] Those skilled in the art can understand and achieve variations of the disclosed embodiments by examining the drawings, this disclosure, and the appended claims when carrying out the disclosure. Furthermore, in the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude the plural. This disclosure also discloses embodiments as illustrated below. [Embodiment 1] A molecular construct for multiphoton fluorescence microscopy imaging, wherein the molecular construct has a first non-fluorescent configuration (2PAP-C) and a second fluorescent configuration (2PAP-CL), and the molecular construct is - Photochromic molecules, - A two-photon absorption probe (2PAP) linked to the aforementioned photochromic molecule, Includes, The aforementioned photochromic molecule can reversibly change from a first colored isomer form (C) to a second colorless isomer form (CL). The first colored form (C) can be isomerized to the second colorless isomer form (CL) by absorption of two photons by the two-photon absorption probe (2PAP). A molecular construct wherein the absorption spectrum of the first colored isomer (C) overlaps with the emission spectrum of the two-photon absorption probe (2PAP), and the absorption spectrum of the second colorless isomer (CL) does not overlap with the emission spectrum of the two-photon absorption probe (2PAP). [Embodiment 2] The molecular construct according to Embodiment 1, wherein the two-photon absorption probe (2PAP) is linked to the photochromic molecule such that the FRET efficiency of the molecular construct is at least 90%. [Embodiment 3] The molecular construct according to Embodiment 1 or 2, wherein the first non-fluorescent stereoconfiguration (2PAP-C) is a thermodynamically stable form of the molecular construct. [Embodiment 4] The molecular construct according to any one of Embodiments 1 to 3, wherein the second colorless isomer (CL) of the photochromic molecule can be isomerized to the first colored isomer (C) by thermal isomerization. [Embodiment 5] A molecular construct according to any one of Embodiments 1 to 4, wherein the isomerization rate from the second fluorescent configuration (2PAP-CL) to the first non-fluorescent configuration (2PAP-C) is faster than the isomerization rate from the first non-fluorescent configuration (2PAP-C) to the second fluorescent configuration (2PAP-CL). [Embodiment 6] The molecular construct according to Embodiment 5, wherein the isomerization rate from the second fluorescent configuration (2PAP-CL) to the first non-fluorescent configuration (2PAP-C) is at least twice as fast, preferably at least ten times faster, and more preferably at least fifty times faster, than the isomerization rate from the first non-fluorescent configuration (2PAP-C) to the second fluorescent configuration (2PAP-CL). [Embodiment 7] The molecular construct according to any one of Embodiments 1 to 6, wherein the two-photon absorption probe (2PAP) absorbs light with a wavelength of at least 700 nm, preferably light with a wavelength in the range of 700 nm to 900 nm. [Embodiment 8] The molecular construct according to any one of Embodiments 1 to 7, wherein the two-photon absorption probe (2PAP) has a fluorescence quantum yield of at least 10%, preferably at least 30%, and more preferably at least 50%. [Embodiment 9] The absorption spectrum of the first colored isomer form (C) and the emission spectrum of the two-photon absorption probe (2PAP) are at least 1 × 10⁻¹⁶ 13 nm 4 M -1 cm -1 A molecular construct according to any one of embodiments 1 to 8, having a spectral overlap integral. [Embodiment 10] The photochromic molecule has a thermal half-life of less than 20 seconds, preferably less than 10 seconds, and more preferably less than 1 second at room temperature (t 1 / 2 A molecular construct according to any one of Embodiments 1 to 9, having ). [Embodiment 11] The molecular construct according to any one of Embodiments 1 to 10, wherein the photochromic molecule absorbs light in the wavelength range of 350 to 800 nm, preferably 450 to 700 nm. [Embodiment 12] A method for analyzing target structures in a sample using multiphoton microscopy, including the following steps: a) Incubate a molecular construct described in any one of Embodiments 1 to 11 with a target structure to provide a fluorescently labeled target structure. b) Irradiating the fluorescently labeled target structure with light in a wavelength range that enables two-photon absorption by the molecular construct so that a fluorescent signal is generated, and c) Detecting and / or measuring the fluorescence signal. [Embodiment 13] The method according to Embodiment 12, wherein the labeled target structure is irradiated with light having a wavelength of at least 700 nm. [Embodiment 14] An antibody tagged with a molecular construct described in any one of Embodiments 1 to 11. [Embodiment 15] Use of a molecular construct according to any one of Embodiments 1 to 11 for imaging a target structure with a multiphoton microscope.

Claims

1. A molecular construct for multiphoton fluorescence microscopy imaging, wherein the molecular construct has a first non-fluorescent configuration (2PAP-C) and a second fluorescent configuration (2PAP-CL), and the molecular construct is - Photochromic molecules and, - A two-photon absorption probe (2PAP) linked to the aforementioned photochromic molecule, Includes, The aforementioned photochromic molecule can reversibly change from a first colored isomer form (C) to a second colorless isomer form (CL). The first colored isomer (C) can be isomerized to the second colorless isomer (CL) by absorption of two photons by the two-photon absorption probe (2PAP). The absorption spectrum of the first colored isomer (C) overlaps with the emission spectrum of the two-photon absorption probe (2PAP), while the absorption spectrum of the second colorless isomer (CL) does not overlap with the emission spectrum of the two-photon absorption probe (2PAP). The second colorless isomer (CL) of the photochromic molecule can be isomerized to the first colored isomer (C) by thermal isomerization. The first non-fluorescent stereoconfiguration (2PAP-C) is a thermodynamically stable form of the molecular construct. A molecular construct in which the isomerization rate from the second fluorescent configuration (2PAP-CL) to the first non-fluorescent configuration (2PAP-C) is at least twice as fast as the isomerization rate from the first non-fluorescent configuration (2PAP-C) to the second fluorescent configuration (2PAP-CL).

2. The molecular construct according to claim 1, wherein the two-photon absorption probe (2PAP) is linked to the photochromic molecule such that the FRET efficiency of the molecular construct is at least 90%.

3. The molecular construct according to claim 1 or 2, wherein the isomerization rate from the second fluorescent configuration (2PAP-CL) to the first non-fluorescent configuration (2PAP-C) is at least 10 times faster, more preferably at least 50 times faster, than the isomerization rate from the first non-fluorescent configuration (2PAP-C) to the second fluorescent configuration (2PAP-CL).

4. The molecular construct according to any one of claims 1 to 3, wherein the two-photon absorption probe (2PAP) absorbs light with a wavelength of at least 700 nm, preferably light with a wavelength in the range of 700 nm to 900 nm.

5. The molecular construct according to any one of claims 1 to 4, wherein the two-photon absorption probe (2PAP) has a fluorescence quantum yield of at least 10%, preferably at least 30%, and more preferably at least 50%.

6. The absorption spectrum of the first colored isomer form (C) and the emission spectrum of the two-photon absorption probe (2PAP) are at least 1 × 10⁻¹⁶ １３ nm ４ M －１ cm －１ A molecular construct according to any one of claims 1 to 5, having a spectral overlap integral.

7. The photochromic molecule has a thermal half-life (t) of less than 20 seconds, preferably less than 10 seconds, and more preferably less than 1 second at room temperature. １／２ A molecular construct according to any one of claims 1 to 6, having )

8. The molecular construct according to any one of claims 1 to 7, wherein the photochromic molecule absorbs light in the wavelength range of 350 to 800 nm, preferably 450 to 700 nm.

9. A method for analyzing target structures in a sample using multiphoton microscopy, including the following steps: a) Incubating a molecular construct according to any one of claims 1 to 8 with a target structure to provide a fluorescently labeled target structure, b) Irradiating the fluorescently labeled target structure with light in a wavelength range that enables two-photon absorption by the molecular construct so that a fluorescent signal is generated, and c) Detecting and / or measuring the fluorescence signal.

10. The method according to claim 9, wherein the labeled target structure is irradiated with light having a wavelength of at least 700 nm.

11. An antibody tagged with a molecular construct according to any one of claims 1 to 8.

12. Use of a molecular construct according to any one of claims 1 to 8 for imaging a target structure with a multiphoton microscope.

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