Molecular construct for multiphoton fluorescence microscopy imaging

Abstract

The present disclosure generally relates to a molecular construct for multiphoton fluorescence microscopy imaging. The molecular construct has a first, non-fluorescent configuration (2PAP-C) and a second, fluorescent configuration (2PAP-CL), and comprises a two-photon absorbing probe (2PAP) linked to a photochromic molecule that can be reversibly changed from a first colored isomeric form (C) to a second colorless isomeric form (CL). The first colored form (C) can be isomerized to the second colorless isomeric form (CL) upon absorption of two photons by the two-photon absorbing probe (2PAP). The present disclosure also relates to a method for analyzing a target structure in a multiphoton microscope utilizing the molecular construct. Furthermore, the present disclosure relates to an antibody tagged with the molecular construct, and to the use of the molecular construct for imaging a target structure.

Claims

1. A molecular construct for multiphoton fluorescence microscopy imaging, wherein said molecular construct has a first, non-fluorescent configuration (2PAP-C) and a second, fluorescent configuration (2PAP-CL), wherein said molecular construct comprises: a two-photon absorbing probe (2PAP) having an emission spectrum, and a photochromic molecule linked to the two-photon absorbing probe (2PAP), wherein said photochromic molecule has a first colored isomeric form (C) and a second colorless isomeric form (CL), wherein said first colored isomeric form (C) has a first absorption spectrum that overlaps the emission spectrum of said two-photon absorbing probe (2PAP) such that the first colored isomeric form (C) photoisomerizes to said second colorless isomeric form (CL) upon absorption of two photons by said two-photon absorbing probe (2PAP), wherein of said second colorless isomeric form (CL) has a second absorption spectrum that does not overlap said emission spectrum of said two-photon absorbing probe (2PAP), and wherein said second colorless isomeric form (CL) isomerizes to said first colored isomeric form (C) by thermal isomerization.

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

3. The molecular construct according to claim 1, wherein said first, non-fluorescent configuration (2PAP-C) is the thermodynamically stable form of said molecular construct.

4. The molecular construct according to claim 1, wherein the rate of the thermal isomerization from said second, fluorescent configuration (2PAP-CL) to said first, non-fluorescent configuration (2PAP-C) is faster than the rate of the photoisomerization from said first, non-fluorescent configuration (2PAP-C) to said second, fluorescent configuration (2PAP-CL).

5. The molecular construct according to claim 4, wherein the rate of the thermal isomerization rate from said second, fluorescent configuration (2PAP-CL) to said first, non-fluorescent configuration (2PAP-C) is at least 2 times faster than the rate of the photoisomerization from said first, non-fluorescent configuration (2PAP-C) to said second, fluorescent configuration (2PAP-CL).

6. The molecular construct according to claim 1, wherein said two-photon absorbing probe (2PAP) absorbs light of wavelengths of at least 700 nm.

7. The molecular construct according to claim 1, wherein said two-photon absorbing probe (2PAP) has a fluorescence quantum yield of at least 10%.

8. The molecular construct according to claim 1, wherein the absorption spectrum of said first colored isomeric form (C) and the emission spectrum of said two-photon absorbing probe (2PAP) have a spectral overlap integral of at least 1×10.sup.13mn.sup.4M.sup.−1cm.sup.−1.

9. The molecular construct according to claim 1, wherein said photochromic molecule has a thermal half-life (t.sub.1/2) of less than 20 seconds at room temperature.

10. The molecular construct according to claim 1, wherein said photochromic molecule absorbs light within the wavelength region of from 350 to 800 nm.

11. The molecular construct according to claim 4, wherein the rate of the thermal isomerization rate from said second, fluorescent configuration (2PAP-CL) to said first, non-fluorescent configuration (2PAP-C) is at least 10 times faster than the rate of the photoisomerization from said first, non-fluorescent configuration (2PAP-C) to said second, fluorescent configuration (2PAP-CL).

12. The molecular construct according to claim 4, wherein the rate of the thermal isomerization rate from said second, fluorescent configuration (2PAP-CL) to said first, non-fluorescent configuration (2PAP-C) is at least 50 times faster than the rate of the photoisomerization from said first, non-fluorescent configuration (2PAP-C) to said second, fluorescent configuration (2PAP-CL).

13. The molecular construct according to claim 1, wherein said two-photon absorbing probe (2PAP) absorbs light of wavelengths in the range of from 700 nm to 900 nm.

14. The molecular construct according to claim 1, wherein said two-photon absorbing probe (2PAP) has a fluorescence quantum yield of at least 30%.

15. The molecular construct according to claim 1, wherein said two-photon absorbing probe (2PAP) has a fluorescence quantum yield of at least 50%.

16. The molecular construct according to claim 1, wherein said photochromic molecule has a thermal half-life (t.sub.1/2) of less than 10 seconds at room temperature.

17. The molecular construct according to claim 1, wherein said photochromic molecule absorbs light within the wavelength region of from 450 nm to 700 nm.

18. An antibody tagged with the molecular construct according to claim 1.

19. A method for analyzing a target structure in a sample in a multiphoton microscope comprising the steps of: a) incubating the molecular construct according to claim 1 with the target structure to provide a fluorescently labeled target structure, b) irradiating said fluorescently labeled target structure with light in a wavelength range that enables two-photon absorption by said molecular construct such that a fluorescent signal is generated, and c) detecting and/or measuring said fluorescent signal.

20. The method according to claim 19, wherein said fluorescently labeled target structure is irradiated with light having a wavelength of at least 700 nm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The various aspects of the present disclosure, including its particular features and advantages, will be readily understood from the following detailed description and the accompanying drawings, in which:

(2) FIG. 1 schematically discloses the molecular construct of the present disclosure and its mode of action.

(3) FIG. 2 is a performance plot illustrating the four-photon behavior of the molecular construct of the present disclosure.

(4) FIG. 3 illustrates examples of photochromic molecules that can be used in a molecular construct of the present disclosure.

(5) FIG. 4 schematically illustrates how the two-photon absorbing probe (2PAP) can be linked to an exemplary photochromic molecule.

DETAILED DESCRIPTION

(6) The present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which currently preferred embodiments of the present disclosure are shown. The present disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness, and fully convey the scope of the present disclosure to the skilled person.

(7) FIG. 1 schematically illustrates a molecular construct of the present disclosure. The two-photon excitation process is indicated by the 2×800 nm photons.

(8) 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.

(9) The molecular construct comprises a two-photon absorbing probe (2PAP) linked to a photochromic molecule, which can adopt a first colored isomeric form (C) and a second colorless isomeric form (CL).

(10) The two-photon absorbing probe (2PAP) is typically covalently linked to the photochromic molecule.

(11) When two photons are absorbed simultaneously by 2PAP, which may occur by e.g. irradiating the molecular construct with an irradiation source, such as laser, 2PAP is excited to the lowest excited singlet state.

(12) The absorption spectrum of C overlaps the emission spectrum of 2PAP resulting in that the emission from 2PAP in 2PAP-C is being efficiently quenched by C in a FRET reaction. The FRET reaction does not only result in quenching of the 2PAP emission, but it also sensitizes the excitation of C. As the fate of C does not depend on how it ended up in the excited state, FRET-sensitized isomerization to yield CL follows. The absorption spectrum of CL does not overlap the emission of 2PAP, and accordingly, FRET does not occur. This implies that in this isomeric form of the molecular construct (2PAP-CL), 2PAP emits intense fluorescence.

(13) Hence, the effect of the intensity of the light (arbitrarily set to 800 nm in FIG. 1) used to excite 2PAP in a two-photon process is twofold. First, the fluorescence intensity of 2PAP in each individual fluorescent isomer (2PAP-CL) depends quadratically on the excitation intensity. Second, the concentration of the fluorescent isomer 2PAP-CL also depends quadratically on the excitation intensity. This is because the rate of the FRET-sensitized isomerization from the non-fluorescent form 2PAP-C to the fluorescent form 2PAP-CL depends on the rate at which 2PAP in 2PAP-C absorbs photons. This rate depends quadratically on the intensity of the excitation light.

(14) Accordingly, both the fluorescence intensity “per fluorescent molecule” as well as the concentration of the fluorescent molecules depend quadratically on the excitation intensity. This results in an overall quartic dependence of the fluorescence intensity: I(em)∝I(exc).sup.4. Particularly, this applies if the thermal isomerization rate from the fluorescent form 2PAP-CL to the non-fluorescent form 2PAP-C is significantly faster than the two-photon FRET-induced isomerization from 2PAP-C to 2PAP-CL. In FIG. 1, the thermal isomerization is denoted A.

(15) In FIG. 2, the principles of the performance of the design of the present disclosure is schematically outlined. The general principle of the design is that the two-photon-induced FRET-sensitized photoisomerization is delicately balanced with thermal isomerization such that the emitted fluorescence intensity displays a quartic dependence on the excitation intensity. k.sub.1 and k.sub.−1 in FIG. 2 correspond to k.sub.photo, and k.sub.therm, discussed above, respectively. FIG. 2 shows that a perfect four-photon behavior (quartic dependence) is observed when k.sub.1/k.sub.−1 is close to zero (very low concentration of the fluorescent form 2PAP-CL). This implies that an improved spatial resolution can be obtained with the molecular construct of the present disclosure.

(16) FIG. 3 illustrates examples of photochromic molecules; i.e. photoswitches that can be used in the molecular construct of the present disclosure. These molecules fulfill the “negative photochromism” feature of the molecular construct of the present disclosure. The isomerization schemes are also illustrated. t.sub.1/2 denotes the thermal half-lives and correspond to the thermal isomerization to the colored form of the photochromic molecule at 25° C. λ.sub.max indicates the wavelength maximum of the most redshifted absorption band for the colored isomeric form (C). A potential, and exemplary 2PAP derivative is also shown in FIG. 3.

(17) It should be noted that the molecular construct of the present disclosure is by no means limited to a specific two photon absorbing probe, but any 2PAP that can be linked to a photochromic molecule can be utilized. A preferred 2PAP for use in the molecular construct of the present disclosure has a fluorescence quantum yield of at least 10%, preferably at least 30%, more preferably at least 50%.

(18) FIG. 4 schematically illustrates an exemplary molecular construct of the present disclosure, wherein 2PAP is linked to a photochromic molecule.

(19) The present disclosure is by no means limited to the use of a particular photochromic molecule. Any photochromic molecule having the ability to display negative photochromism can be used; i.e. any photochromic molecule having the ability to be switched from a colorless isomeric form to a colored isomeric by thermal isomerization can be used.

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

(21) Terms, definitions and embodiments of all aspects of the present disclosure apply mutatis mutandis to the other aspects of the present disclosure.

(22) Even though the present disclosure has been described with reference to specific exemplifying embodiments thereof, many different alterations, modifications and the like will become apparent for those skilled in the art.

(23) Variations to the disclosed embodiments can be understood and effected by the skilled addressee in practicing the present disclosure, from a study of the drawings, the disclosure, and the appended claims. Furthermore, in the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality.