Method for determining luminescence kinetic information of reversibly photo-convertible luminescent species

Abstract

The invention relates to a method for determining luminescence kinetic information of at least one reversibly photo-convertible luminescent species, comprising: - illumination of the sample with at least a first and second excitation light, the intensities of the first and second excitation lights being periodically modulated at different frequencies, - detection of the luminescence light and extraction, for a plurality of modulation frequencies of the first excitation light, of an intensity amplitude of the luminescence light, - determination of a kinetic information of the species from the luminescence amplitude. The invention also relates to another method for determining luminescence kinetic information of such species, comprising:- illumination of the sample with at least a first excitation light, the intensity of the first excitation light being periodically modulated, - detection of the luminescence light and extraction of an average luminescence intensity, - determination of a kinetic information of the species from the average luminescence intensity.

Description

Method for determining luminescence kinetic information ofreversibly photo-convertible luminescent species Field of the invention

[0001] The present invention relates to a method for determiningluminescence kinetic information of at least one reversibly photo-convertible luminescent species in a sample.

[0002] The field of the invention is luminescence imaging in micro- andmacro format, and biological and / or biochemical analysis.

[0003] In the present document, the term ‘species’ indicates a chemicalspecies such as a molecule, a molecular ion, or a complex. A ‘reversibly photo-convertible species’ (or ‘photo-switchable’ or ‘photo-activatable’) is a specieshaving at least two distinct states, with different luminescence properties, andcapable of commutating from one state to the other reversibly under the effectof light. Examples of reversibly photoactivatable luminescent species are the‘Dronpa’ protein and the ‘Spinach - DFHBI’ complex (‘Spinach’ being an RNAaptamer and DFHBI a fluorogenic probe). These species may, in particular, beused as labels or markers.

[0004] Luminescence includes fluorescence and phosphorescence.Background

[0005] Imaging photoactive light-emissive systems intervenes in multipletechnical fields and applications such as fluorescence and phosphorescencebioimaging, sensing films for active packaging and mapping oxygenation,encryption and anti-counterfeiting, dye-assisted surgery, or process control of silicon wafers and solar cells.

[0006] The obtained images generally carry spectral information. The timeresponse of the system to illumination enables for providing complementaryinformation for additional selectivity. However, imaging kinetics is demandingsince most lifetimes of photo-activated states are in a short ns-µs range. The optical setups for luminescence lifetime imaging have accordingly beenequipped with sophisticated detectors endowed with high-frequencymodulation, or fast gating, in order to overcome the frequency / time (kHz / ms)limitation of standard cameras.

[0007] Several techniques have been developed to discriminateluminophores, and in particular fluorophores, using dynamical, or temporal,information characterizing their absorption-fluorescence emission photocycles.

[0008] In reversibly photoswitchable fluorophores (RSFs), illumination drivesseveral photocycles including photochemical and thermal steps, which implya wide range of relaxation times (µs to s), thereby enabling discrimination attimescales compatible with real time observations of biological phenomena. Several protocols such as OPIOM and Speed OPIOM have exploited the time response of the fluorescence to light variations for imaging reversibly photoswitchable species that may not be spectrally discriminated.

[0009] OPIOM (Out-of-Phase Imaging after Optical Modulation), described in[Querard 2015] and in WO 2015 / 075209, is a method in which a samplecontaining a reversibly photoswitchable species is illuminated with aperiodically modulated light wave. It has been shown that the component of the fluorescence intensity emitted by the fluorophores at the same frequency and in phase quadrature with respect to the excitation wave exhibits a peak for particular, species-dependent, values of the illumination intensity and modulation frequency, called resonance conditions.

[0010] A variant of OPIOM, Speed OPIOM, is described in [Querard 2017]and in WO 2018 / 041588. This protocol implements two-wavelengthillumination, thereby achieving higher acquisition frequencies.

[0011] However, the OPIOM and Speed OPIOM protocols do not allow forretrieving a kinetic information on reversibly photoswitchable species usingstandard optical detectors or cameras when fast photoswitching times areinvolved.Summary of the invention

[0012] A purpose of the present invention is to overcome at least one of thedrawbacks of the known techniques.

[0013] A purpose of the present invention is to propose a method fordetermining luminescence kinetic information of at least one reversibly photo- convertible luminescent species, wherein the method may be carried out using a standard camera.

[0014] Another purpose of the invention is to propose a method fordetermining luminescence kinetic information of at least one reversibly photo-convertible luminescent species, enabling wide-field luminescence lifetimeimaging on simple optical setups with standard cameras.

[0015] Still another purpose of the invention is to propose a method fordetermining luminescence kinetic information of at least one reversibly photo- convertible luminescent species, going beyond the discrimination of one species from another.

[0016] At least one of these aims is achieved by a method according to afirst aspect of the invention. The method for determining luminescence kineticinformation of at least one reversibly photo-convertible luminescent species in a sample, the method comprising the steps of: a) illumination of the sample with at least a first and a secondexcitation light, the intensity of the first excitation light beingperiodically modulated at a frequency f1, and the intensity of the second excitation light being periodically modulated at afrequency f2, wherein f1 ≠ f2, the excitation lights being adapted to promote a luminescence light emission from the at least one luminescent species, b) detection, with an optical detector, of the luminescence light, theintensity of the luminescence light being modulated at a frequency difference Δf = f1 - f2, for a plurality of modulationfrequencies, f1, of the first excitation light,c) extraction, for a plurality of modulation frequencies, f1, of thefirst excitation light, of an amplitude of the intensity of theluminescence light, d) determination of a kinetic information of the at least onereversibly photo-convertible luminescent species from the dependence of the extracted luminescence amplitude on the modulation frequency f1 of the first excitation light.

[0017] The method according to the first aspect of the invention is referredto as HIOM (Heterodyne Imaging under Optical Modulation). In the HIOMprotocol, low angular-frequency modulated amplitudes of the luminescencefrom a species under periodic illumination of two excitation lights, modulatedat two fundamental high angular frequencies, are acquired at low acquisitionfrequency. Kinetic information, such as a map of luminescence lifetime, maybe retrieved from the dependence of the extracted luminescence amplitudeon one of the modulation frequencies of the excitation lights.

[0018] In the present document, the term “excitation light” refers to anexcitation light beam from an excitation light source.

[0019] According to an embodiment, the average intensities of the first andthe second excitation light, the modulation frequency, f1, of the first excitationlight, and the frequency difference, Δf, may be chosen to approach themaximum of^ the phase-quadrature luminescence amplitude when themodulation periods of the first and the second excitation lights areoffset by 0 or ^, ^the in phase luminescence amplitude when the modulation periodsof the first and the second excitation lights are offset by ^ / 2 or 3^ / 2.

[0020] These amplitudes are measured using a heterodyne detectiontechnique.

[0021] The first excitation light may have a wavelength λ1, and the secondexcitation light may have a central wavelength λ2. The wavelengths may beequal, λ1 = λ2, or different, λ1 ≠ λ2.

[0022] In case of equal central wavelengths, it is possible to use two lightbeams of which the intensity is modulated at two different modulationfrequencies. It is also possible to use a single light beam of which the intensityis modulated at two different modulation frequencies. This latter modulationscheme may be achieved, for example, with a function generator.

[0023] It is also possible to implement more than two light beams to obtainmodulated excitation light at two frequencies.

[0024] As used herein, the term “wavelength” may designate the wavelengthof a narrow-band, or monochromatic, light source. It may also designate thecentral wavelength of a wide-spectrum light source.

[0025] According to an embodiment, the frequency difference, Δf, of theexcitation lights may be below 1000 Hz.

[0026] According to a second aspect of the same invention, it is proposed amethod for determining luminescence kinetic information of at least onereversibly photo-convertible luminescent species in a sample, the method comprising the steps of: a) illumination of the sample with at least a first excitation light, theintensity of the first excitation light being periodically modulated at a frequency, f,the first excitation light being adapted to promote a luminescencelight emission from the at least one luminescent species, b) detection of the luminescence light with an optical detector, theintensity of the luminescence light being an average intensity, for a plurality of modulation frequencies, f, of the at least firstexcitation light,c) extraction, for a plurality of modulation frequencies, f, of the atleast first excitation light, of the average luminescence intensity,d) determination of a kinetic information of the at least onereversibly photo-convertible luminescent species from the dependence of the average luminescence intensity on the modulation frequency, f, of the first excitation light.

[0027] The method according to the second aspect of the invention isreferred to as RIOM (Rectified Imaging under Optical Modulation). In the RIOM protocol, the luminescence from a species under periodic illuminationmodulated at fundamental high angular frequency is acquired at a single lowacquisition frequency. Kinetic information, such as a map of luminescencelifetime, may be retrieved from exploiting the dependence of the average luminescence intensity on the modulation frequency.

[0028] According to an embodiment, referred to as RIOM1, the illuminationstep may comprise the illumination of the sample with a first excitation light, the intensity of the first excitation light being periodically modulated at a frequency, f.

[0029] Alternatively, referred to as RIOM2, the illumination step maycomprise the illumination of the sample with a first excitation light and asecond excitation light, the intensity of the first excitation light beingperiodically modulated at a frequency, f, and the intensity of the secondexcitation light being constant.

[0030] Still alternatively, referred to as RIOM3, the illumination step maycomprise the illumination of the sample with a first excitation light and a second excitation light, the intensities of the first and second excitation lightsbeing periodically modulated at the same frequency, f, and in antiphase withrespect to each other.

[0031] In the case of illumination with a first and a second excitation light,the first excitation light has a wavelength λ1, and the second excitation lighthas a wavelength λ2, and these wavelengths may be equal, λ1 = λ2, ordifferent,

[0032] The average intensity of the at least first excitation light and themodulation frequency, f, of the at least first excitation light may be chosen toapproach the maximum of the amplitude of the frequency-induceddependence of the average luminescence intensity normalized by theluminescence intensity under the average intensity of the at least first excitation light.

[0033] In particular, in RIOM1, the average intensity of the first excitationlight and the modulation frequency, f, of the first excitation light may bechosen to approach the maximum of the amplitude of the frequency-induceddependence of the average luminescence intensity normalized by the average intensity of the at least first excitation light.

[0034] In RIOM2, the average intensities of the first and second excitationlights and the modulation frequency, f, of the first excitation light may bechosen to approach the maximum of the amplitude of the frequency-induceddependence of the average luminescence intensity normalized by theluminescence intensity under the average intensities of the first and secondexcitation lights.

[0035] In RIOM3, the average intensities of the first and second excitationlights and the modulation frequency, f, of the first and second excitation lightsmay be chosen to approach the maximum of the amplitude of the frequency- induced dependence of the average luminescence intensity normalized by theluminescence intensity under the average intensities of the first and secondexcitation lights.

[0036] The following embodiments may apply to the method according toboth the first and second aspect of the invention.

[0037] According embodiments of the invention, the determination of thekinetic information may comprise the extraction of a luminescence lifetime of the at least one luminescent species.

[0038] The method may further comprise imaging the luminescence lifetimeaccording to a field of view of the detector.

[0039] According to an embodiment, the determination of the kineticinformation may comprise the establishment of a Bode diagram linking theextracted luminescence signal to the modulation frequencies of the at least first excitation light.

[0040] The luminescence signal corresponds to the extracted amplitude ofthe luminescence intensity in the HIOM protocol and to the averageluminescence intensity in the RIOM protocol.

[0041] According to an embodiment, the determination of the kineticinformation may be realized using a fitting function comprising a ratio of twopolynomial functions, the polynomial functions each comprising a sum of polynomials of the luminescence lifetime and the modulation frequency of the at least first excitation light.

[0042] Preferably, the polynomials are of the order of 4 or less.

[0043] According to embodiments, the periodical modulation of theexcitation light(s) may be of sinusoidal form or square-wave form, or any other periodic form.

[0044] The at least one reversibly photo-convertible luminescent speciespresents a first and at least a second chemical state.

[0045] The reversibly photo-convertible luminescent species may be photo-convertible from the first to the at least second state upon illumination withthe first excitation light, and from the at least second to the first state throughthermal relaxation.

[0046] Alternatively, the luminescent species may be photo-convertible fromthe first to the at least second state upon illumination with the first excitationlight, and from the at least second to the first state upon illumination with thesecond excitation light.

[0047] Luminescence light is emitted at least by one of the chemical statesinvolved in the conversions.

[0048] According to another aspect of the same invention, it is proposed adevice for carrying out a method according to any one of the preceding claims, the device comprising -illumination means configured for carrying out method step a),- an optical detector configured for carrying out method step b),and -a data processing unit configured for carrying out method stepsc) and d).

[0049] According to yet another aspect of the same invention, it is proposedthe use of a method according to the first and / or the second aspect and / or adevice of the invention for reporting on the kinetic information of a physiological state of photosynthetic organisms. Description of the figures and embodiments

[0050] Other advantages and characteristics will become apparent onexamination of the detailed description of an embodiment which is in no way limitative, and the attached figures, where: -FIGURE 1 shows a diagrammatic representation of the principleimplemented with the present invention; -FIGURE 2 shows a diagrammatic representation of a device accordingto an embodiment of the invention; -FIGURE 3 shows a diagrammatic representation of a device accordingto another embodiment of the invention; and- FIGURES 4 to 9 show measurement results obtained with a methodaccording to an embodiment of the present invention.

[0051] It is well understood that the embodiments that will be describedbelow are in no way limitative. In particular, it is possible to imagine variants of the invention comprising only a selection of the characteristics described hereinafter, in isolation from the other characteristics described, if this selection of characteristics is sufficient to confer a technical advantage or to differentiate the invention with respect to the state of the prior art. Such a selection comprises at least one, preferably functional, characteristic without structural details, or with only a part of the structural details if this part alone is sufficient to confer a technical advantage or to differentiate the inventionwith respect to the prior art.

[0052] In the Figures, elements common to several Figures may keep thesame reference.

[0053] Figure 1 is a schematic illustration of the principle of a method fordetermining luminescence kinetic information of at least one reversibly photo-convertible luminescent species in a sample according to aspects of thepresent invention.

[0054] The method comprises the illumination of a sample S by at least oneexcitation light. The sample S comprises at least one reversibly photo-convertible species, or label, P. Under the effect of light, the species can switchbetween at least two different chemical states, a first state 1 and at least asecond state 2. The two chemical states possess a different brightness.

[0055] According to a first aspect, referred to as HIOM, the sample S isilluminated with a first excitation light of which the intensity I1 is periodically modulated at a frequency f1, or angular frequency ω1, and a second excitationlight of which the intensity I2 is periodically modulated at a frequency f2, orangular frequency ω1, wherein f1 ≠ f2. In the example represented in Figure 1, the excitation lights are modulated sinusoidally and in antiphase with eachother. According to alternative embodiments of the invention, the modulation periods of the first and the second excitation lights may also be offset by 0, ϖ / 2 or 3ϖ / 2. The excitation lights may also be periodically modulated accordingto other than the sinusoidal scheme, such as by a square wave.

[0056] The wavelengths λ1, λ2 of the two excitation lights may be equal ordifferent.

[0057] The species P is photo-convertible from the first chemical state 1 tothe at least second chemical state 2 upon illumination with the first excitationlight I1, and from the at least second to the first state either through thermalrelaxation, or upon illumination with the second excitation light I2.Luminescence light (with a component modulated at angular frequency Δω indicated by SΔω(t)) is emitted at least by one of the chemical states involvedin the conversions; its measured intensity varies periodically at the differenceof the frequencies of the excitation lights.

[0058] The intensity of the luminescence light may be decomposed in an in-phase component being in phase with the first excitation-light modulation anda quadrature component being quadrature-delayed with respect to the firstexcitation-light modulation. The amplitudes of the in-phase and quadrature-delayed components of the luminescence S modulated at low angularfrequency Δω = ω1 – ω2 are imaged at low acquisition frequency.

[0059] Signals are measured for a plurality of modulation frequencies ω1 ofthe first excitation light at constant Δω. In the example represented in Figure1, a map of the luminescence lifetime ^ is retrieved from exploiting thedependence of the quadrature-delayed S component on ω1 at constantangular frequency Δω.

[0060] The frequency difference, Δf, is preferably below 1000 Hz.

[0061] According to a second aspect of the invention, referred to as RIOM,the sample S is illuminated with an excitation light of which the intensity I isperiodically modulated at a frequency f, or angular frequency ω. In theexample represented in Figure 1, the excitation light is modulatedsinusoidally. According to alternative embodiments, the sample may beadditionally illuminated with a second excitation light of which the intensitymay be periodically modulated at the same frequency f, or angular frequencyω, and which is in antiphase with the first excitation light, or of which theintensity is constant.

[0062] As with the first aspect, the excitation lights may also be periodicallymodulated according to other than the sinusoidal scheme, such as by a square wave.

[0063] The species P is photo-convertible from the first chemical state 1 tothe at least second chemical state 2 upon illumination with the (first)excitation light I1, and from the at least second to the first state through thermal relaxation, or upon illumination with the second excitation light I2. The average value of the luminescence light (indicated by S0) is emitted at least by one of the chemical states involved in the conversions. The average luminescence S0is imaged at low acquisition frequency.

[0064] Signals are measured for a plurality of modulation frequencies ω ofthe excitation light. In the example represented in Figure 1, a map of theluminescence lifetime ^ is retrieved from exploiting the dependence of S0 onω.

[0065] In the examples represented in Figure 1, the first, thermodynamicallymore stable state is indicated by 1, and the at least second, thermodynamically less stable state is indicated by 2.

[0066] In the following, a theoretical analysis of the dynamic behaviour of areversibly photo-convertible luminescent species P illuminated with light ofintensity ^(^) involving two components ^^(^)and ^^(^) at wavelengthsandλ^, respectively, will be provided.

[0067] It is assumed that the dynamic behaviour of the luminescent speciesP may be reliably described by the two-state exchange ^^^(^)^^^(^).

[0068] In the reaction (1), the thermodynamically most stable state 1 isphotochemically converted to the thermodynamically less stable state 2 atrate constant ^^^(^) = ^^^,^^^(^) + ^^^,^^^(^) from which it can relax back to theinitial state 1 either by a photochemically- or a thermally-driven process atrate constant ^^^(^) = ^^^,^^^(^) + ^^^,^^^(^) + ^^^^ , where ^^^,^^^(^), ^^^,^^^(^), ^^^,^^^(^), and ^^^^ are respectively the photochemical and the thermal contributions of the rate constants. ^^^,^and ^^^,^are the molecular actioncross-sections for photoisomerization from state 1 to state 2 and vice versa,respectively, when illuminated with wavelength λ^, and ^^^,^ and ^^^,^ are themolecular action cross-sections for photoisomerization from state 1 to state2 and vice versa, respectively, when illuminated with wavelength λ^.

[0069] In that case, the molecular action cross-sections forphotoisomerization ^^^,^and ^^^,^(at λ^), ^^^,^and ^^^,^(at λ^), and the thermal rate constant ^^^^ fully define the behavior of the photo-convertible probe. Inthe following, λ^and λ^can be either different or identical. Similarly, thecomponents of illumination ^^(^) and ^^(^) can as well more favorably promotethe photoconversion of the state 1 to the state 2, or the one of the state 2 tothe state 1.

[0070] We assume that the system is closed and either uniformly illuminatedor that it can be considered homogeneous at any time of its evolution. The evolutions of the concentrations 1 (concentration of P in state 1) and 2 (concentration of P in state 2) may be written, using the two-state exchange (1):

[0071] In the following, the response of the luminescence emission from thereversibly photo-convertible luminescent species P will be analyzed when it issubmitted to various periodic light modulation situations.

[0072] The principle of the theoretical analysis, which is common to allconsidered periodic light modulations herein, is first considered.

[0073] For this, it is considered that the reversibly photo-convertibleluminescent species P is submitted to periodically modulated illumination. Tobe the most general possible, the illumination is assumed to involve twocomponents: a periodic illumination I1(t) at wavelength λ1 with average light intensity ^^^and a periodic illumination I2(t) at wavelength λ2 with average light intensity ^^^.

[0074] In the most general case, it may be written^(^) = ^^(^) + ^^(^) (4)^^(^) = ^ ^^[1 + αℎ^(^)] (5)^^(^) = ^ ^^[1 + αδ ℎ^(^)], (6)with δ = 0 or 1. In equations (5) et (6), α and αδ measure the amplitude of light modulation, and h1(t) and h2(t) designate periodic functions.

[0075] The following theoretical analysis notably enables us to cover the caseof the absence of illumination at wavelength λ2 (by making ^^^ = 0) or the caseof non-modulated illumination at wavelength λ2 (by setting δ = 0). Weintroduce the notations ^^ = ^ ^^ + ^^^(7) ^^^^ = ^ ^^^,^ + ^^^^,^(8)where^^^^,^ = ^^^,^^^^(12) ^^^^,^ = ^^^,^^^^(13)Equations (4), (5), and (6) are used to express the rate constants

[0076] The relaxation time ^^^^ of the reversibly photo-convertible luminophore when it is submitted to illumination at constant light intensity I0may be written as

[0077] 10 and 20 are the concentrations of P in state 1 and 2 at the associatedphoto-stationary state reached after ^^^^ : 1^ = Ptot-, where ^ ^^ ^^^^^=^^^^and the total concentration in reversibly photo-convertible label P, Ptot = 1 +2.

[0078] Upon expanding the concentration expressions by introducing afunction ^(^)2= 2^ + ^^(^) (19)the system of differential equations governing the temporal evolution of theconcentrations of P in 1 and 2 is solved with equations (2) and (3) to yield^^ = α^σ^^,^ + σ^^,^^I^^τ^^^ (24) ^^ = ρ^^^ Δ^^^,^τ^^^ (25) ^^ = α^σ^^,^ + σ^^,^^^^^τ^^^ , (26)andρ^^^ = k ^^^ 1^ = k^^^2^(27)designate respectively the steady-state rate of reaction corresponding to equation (1) in the photo-stationary state, and the differences of the relative contributions of the average of the modulated light (^^^and ^^^ , respectively) todrive the transition from state 1 to state 2 or from state 2 to state 1,respectively.

[0079] After the relaxation time ^^^^ , a permanent regime is established inwhich f(^) is a continuous periodic function.

[0080] In the various embodiments of the invention, the modulation of theexcitation light may involve one (denoted ω) or at least two (denoted ^^ and^^) fundamental modulation frequencies.

[0081] In a first case, the Fourier series associated to ^(^) can be writtenwhere ^= ^τ ^^^ , (31)and ^^,^^^and ^^,^^^designate the amplitudes of the n-th components of the Fourier series.

[0082] In contrast, in a second case, the Fourier series associated to ^(^) canbe expressed aswhere θ^ = ω^τ^^^ (33) θ^ = ω^τ ^^^ , (34)and ^^ = ^^,^,^^^, ^^,^,^^^and ^^,^,^^^ correspond to the amplitudes of the zerothand {^, ^}-th components of the Fourier series.

[0083] Either the ^^,^^^and ^^,^^^, or the ^^, ^^,^,^^^, and ^^,^,^^^ terms can beextracted from equation (21) upon identifying the amplitudes of thecomponents of the same order (harmonic balance). The resulting set ofequations can then be transformed to explicit the amplitudes of the concentration modulations at all modulation frequencies. Thus, it is possibleto write eitherwhere 2^ = 2^ + αa^ (37)1^ = 1^ − αa^ (38)2^,^^^ = −1^,^^^ = ^^,^^^ (39)2^,^^^ = −1^,^^^ = ^^,^^^, (40)orwith ^^ = ^^,^,^^^.

[0084] In the following, the luminescence intensity will be derived.

[0085] Two cases may be considered:

[0086] In the case of phosphorescent species, their luminescence signal isproportional to the concentration of the triplet state 2 and the time evolutionof their phosphorescence intensity is governed by equations (35) and (41).

[0087] In the case of fluorescent species, reversibly photo-convertiblefluorophores, and the photosynthetic apparatus, the time evolution of their fluorescence signal involves terms which are products of concentrations andlight intensity. Hence, an observable ^^, i.e., a measurable property of aspecies, associated to the observation at the wavelength λ^with j = 1 or 2 isdefined:

[0088] Subsequently, fluorescence emission IF(t) may be extracted fromEq.(48):

[0089] With the temporal dependence of 1(t) and 2(t) given in equations(35) and (36), one obtainswithand

[0090] In contrast to the expressions of the amplitudes of the ^^(^) termswhich are generic, the expressions of the amplitudes of the ^F(^) terms varywith the temporal dependence of the illumination.

[0091] With the temporal dependences of 1(^)and 2(^) as given in equations(41) and (42), it may be writtenwithand

[0092] Again, whereas the expressions of the amplitudes of the ^^(^) termsare generic, the expressions of the amplitudes of the IF(t) terms vary with thetemporal dependence of the illumination.

[0093] The temporal dependencies of the modulated illumination will now beconsidered for different embodiments of the invention.

[0094] For both aspects of the present invention (RIOM and HIOM), twotypes of modulated illumination (sinusoidal and square wave) are considered.

[0095] For a sinusoidal modulation of the illumination, its temporaldependence may be written as: for RIOM: ^(^) = ^ ^^[1 + αℎ^(^)] + ^ ^^[1 + αδℎ^(^)] (59)ℎ^(^) = sin(ω^) (60)ℎ^(^) = sin(ω^ + φ) (61)with ^ = 0, or ^ = 1, and ^ = ^;for HIOM: ^(^) = ^ ^^[1 + αℎ^(^)] + ^ ^^[1 + αℎ^(^)] (62)ℎ^(^) = sin(ω^^) (63)ℎ^(^) = sin(ω^^ + φ) (64)

[0096] For a square-wave modulation of the illumination, its temporaldependence may be written as: for RIOM:^(^) = ^ ^^[1 + αℎ^(^)] + ^ ^^[1 + αδℎ^(^)] (65)with ^ = 0 or ^ = 1, and ^ = ^;for HIOM: ^(^) = ^ ^^[1 + αℎ^(^)] + ^ ^^[1 + αδh^(^)] (68)with ^ = 0 or ^ = −1.

[0097] The example of the square-wave modulation is given as arepresentative example of more complex modulations of the illumination.

[0098] Observables corresponding to the RIOM and HIOM aspects of theinvention will now be described.

[0099] According to embodiments of the invention, the RIOM observableSRIOM is the average value of the luminescence signal ^(^) (either global orfrom each analyzed pixel) from the reversibly photo-convertible luminophoreover an integer number k of periods T of modulated illumination. In practice,^ ^^ SRIOM= ^^∫^S(t)dt . (71)

[0100] This average value of the luminescence intensity is extracted usingknown measuring techniques.

[0101] According to embodiments of the invention, for ^ = ^ / 2 or ^ = −^ / 2,the HIOM observable SHIOMis the amplitude of the in-phase component of the luminescence signal ^(^), either global or from each analyzed pixel of thecamera, from the reversibly photo-convertible luminophore modulated at ^^ −^^angular frequency, this component being in phase with the first excitation- light modulation.

[0102] Alternatively, for ^ = 0 or ^ = ^, HIOM observable SH-OPIOM is theamplitude of the quadrature-delayed component of the luminescence signal^(^), either global or from each analyzed pixel, from the reversibly photo-convertible luminophore modulated at− ^^ angular frequency, thiscomponent being quadrature-delayed with respect to the first excitation-light modulation. H-OPIOM refers to “Heterodyne Out-of-Phase Imaging after Optical Modulation”.

[0103] The amplitudes of the in-phase and / or quadrature-delayedcomponents are extracted, for example, using lock-in detection of theluminescence signal or by analysis of the Fourier transform of theluminescence intensity.

[0104] As an example, the quadrature-delayed component of theluminescence signal with ^ = ^ is considered. In practice,

[0105] In the method according to aspects of the invention, resonanceconditions are determined in order to optimize the amplitudes of theobservables. Indeed, the luminescence intensity exhibits a maximum forparticular, species-dependent, values of the excitation illumination intensityand modulation parameters. These control parameters of the modulatedillumination drive the values of the terms^) for the RIOM aspect, andthe terms (^^^^ ,^^,^^) for the HIOM aspect.

[0106] The control parameters of the modulated illumination are tuned in 2-3 orders of magnitude-wide ranges. The mean light intensity / ies of the excitation light(s) must generate balanced proportions of the ground andphotoactivated states of the luminescence species. The frequency / ies of themodulated illumination must be in the range of the inverse of the photoactivation time. The resonant control parameters are obtained bymatching the mean light intensity / ies and frequency / ies of the modulatedillumination with simple functions of the rate constants associated with the photoactivation.

[0107] According to the RIOM aspect of the invention, the average intensityof the (first) excitation light and its modulation frequency f are chosen toapproach the maximum of the amplitude of the frequency-induceddependence of the average luminescence intensity normalized by theluminescence intensity under the average intensity of the at least firstexcitation light.

[0108] The resonance conditions are summarized in table S1.Modulated log10^^ ^^Luminophoreillumination ^. ^ range log10^^,^^^ ^^log10^Phosphorophores Sine-wave 0 – [- 0.72 ≤ 00.5;2.5] Fluorophores Sine-wave 0 – [-2;1] -0.20 ≤ 0Fluorophores Sine-wave 1 ^ [-2;0] -0.66 ≤ 0Phosphorophores Square-wave 0 – [-1;2] 0.54 ≤ 0Fluorophores Square-wave 0 – [-2;1] -0.21 ≤ 0Fluorophores Square-wave 1 ^ [-2;1] -0.56 ≤ 0Table S1: Resonance conditions of the RIOM observable.

[0109] According to the HIOM aspect of the invention, the average intensitiesof the first and the second excitation lights, the modulation frequency of the first excitation light f1 and the frequency difference Δf are chosen to approach the maximum of ^the phase-quadrature luminescence amplitude when the modulationperiods of the first and the second excitation lights are offset by 0 or ^, ^the in phase luminescence amplitude when the modulation periods ofthe first and the second excitation lights are offset by ^ / 2 or 3^ / 2.

[0110] The resonance conditions are summarized in table S2.Modulated log10^^ ^^Luminophoreillumination ^ ^ range log10^^,^^^ ^^log10^ Phosphorophores Sine-wave 1 ^ [-1;2.5] 0.64 ≤ 0Fluorophores Sine-wave 1 ^ [-3;1] -0.69 ≤ 0Phosphorophores Square-wave 1 ^ [-1;2] 0.54 ≤ 0Fluorophores Square-wave 1 ^ [-2;1] -0.56 ≤ 0Table S2: Resonance conditions of the HIOM observable

[0111] The method according to aspects of the present invention furthercomprises the determination of the characteristic time associated with thephoto-activation of the luminescent species P.

[0112] The amplitudes of the time varying terms contained in equations (49)and (53), and equations (54) and (58) depend on ^, and ^^ and ^^respectively.Hence, the investigation of the dependence of the observables of RIOM andHIOM on the applied angular frequency / ies is suitable to yield thecharacteristic time ^^^^ . However, this retrieval necessitates to identify appropriate fitting functions.

[0113] Once the time dependence of the applied modulated illumination isknown, it may be introduced into the master differential equation given inequation (21), in order to retrieve analytic expressions of the time varyingterms contained in equations (49) and (53), and equations (54) and (58) upontruncating the Fourier expansion (30) or (32) at any order. In principle, thehigher the order, the more precise the fitting function. However, the increasein the truncation order increases the number of fitting parameters as well.

[0114] The Fourier expansions (30) and (32) may be, for example, truncatedat first and second order for retrieving the fitting function sought for. Thischoice ensures both a satisfactory fit over a range of angular frequenciesdelivering significant values of the RIOM and HIOM luminescence signals andlimits the number of fitting parameters, thereby providing robustness of thefit.

[0115] Thus, two fitting functions are obtained allowing to retrieve thecharacteristic time ^^^^ from the dependence of the observables of RIOM and HIOM on the applied angular modulation frequency / ies:

[0116] In the RIOM protocol, a three floating-parameter fitting function isadopted, such as given in equation (73):with fitting parameters p1, p2, and p3.

[0117] In the HIOM protocol, another three floating-parameter fittingfunction is adopted, such as given in equation (74):

[0118] In equations (73) and (74), ^ = 1 and ^ = 1.85 with sine-wave andsquare-wave modulated illumination, respectively.

[0119] Fitting functions (73) and (74) comprise a ratio of two polynomialfunctions. Each polynomial function comprises a sum of polynomials of the luminescence lifetime ^^^^ and the modulation frequency ω of the least firstexcitation light. In this non-limitative example, the polynomials are of theorder of 4 or less.

[0120] According to embodiments of the method according to the invention,images of the HIOM and RIOM observables, respectively, are collected for a set of high illumination-modulation frequencies. For each pixel of an image, aBode diagram is obtained by linking the observable, i.e., the extractedluminescence amplitude component or the average luminescence intensity, to the modulation frequencies of the excitation light(s).

[0121] The appropriate fit function, as detailed above, or another treatmentsuch as a ratio of two values at two frequencies, is then applied to each pixelof the HIOM or RIOM image. A map of lifetimes or kinetic information isextracted from this signal processing, depending on the type of processing.

[0122] Examples of implementations of the method according to theinvention will be given below, after the description of devices configured to realize the method.

[0123] Figure 2 shows a diagrammatic representation and an optical layoutof a device that may be implemented in the present invention. In particular, the device of Figure 2 is configured to carry out the method according to embodiments of the invention.

[0124] The device according to the embodiment represented in Figure 2 isbased on an epi-fluorescence microscope.

[0125] The device comprises several light sources, namely light emittingdiodes (LEDs): an ultraviolet 405 nm LED LS1, two blue 470 nm LEDs LS2,LS3, and two green 540 nm LEDs LS4, LS5. Each LED is driven by a LED driver(not shown). Two of these LED drivers can be controlled by a waveformgenerator (not shown) in order to generate a desired intensity modulation of the excitation lights.

[0126] The light from each LED is collimated using a high numerical aperturecondenser lens L1-L5 (f = 16 mm). The light from the UV LED LS1 is filteredwith a bandpass filter OF1. For the blue and green LED pairs, respectively, thecollimated light beams from each LED LS2, LS3, LS4, LS5 are first combinedusing a 50:50 beam splitter BS1, BS2 before being filtered with acorresponding excitation filter OF2, OF3. The two resulting light beams are then combined with a dichroic mirror DM1 into a single beam which passesthrough a second dichroic mirror DM2 to mix with the collimated light beamcoming from the UV LED LS1. A focusing lens L (f = 75 mm) is used to focuslight at the back focal plane of an objective OB after being reflected by adichroic filter DF1. The objective OB may be, for example, a 10× (N.A. 0.5)or a 50× (N.A. 0.8) objective. The objective OB forms an illuminated area on the sample S, the size of the area depending on the magnitude of the objective.

[0127] The sample S may be a fluorescent sample, such as cells labelled withthe reversibly photoswitchable fluorescent protein Dronpa-2 or Platinumoctaethylporphyrin (PtOEP).

[0128] Fluorescence emission from the sample is collected with the objectiveOB, reflected by a mirror M, filtered by a band pass filter OF4 before beingrefocused onto a sensor of a camera CAM by another focusing lens L7 (f =150 mm).

[0129] In the example of Figure 2, the blue light sources LS2, LS3 areadapted to the excitation of fluorescence emission from Dronpa-2, and thegreen light sources LS4, LS5 are adapted to the excitation of fluorescenceemission from PtOEP.

[0130] Figure 3 shows a diagrammatic representation of another device thatmay be implemented in the present invention. In particular, the device of Figure 3 is configured to carry out the method according to embodiments of the invention.

[0131] The device according to the embodiment represented in Figure 3 is afluorescence macro-imager.

[0132] The device comprises two illumination paths and one imaging path,with separated optical axes.

[0133] The imaging path consists of a macroscope objective OB to collimatethe light originating from the sample plane in which a sample S is positioned,and an emission filter OF which may be selected depending on theluminophore, in order to pass only the luminescence light. The imaging pathconsists further of a camera objective COB (f = 50mm, f / 1.8D) to focus thecollimated light onto an image sensor of a camera CAM. The camera may bea greyscale global-shutter camera.

[0134] The two illumination paths each project excitation light onto thesample at an angle of 30° from the imaging path. The illumination paths areoffset by an azimuth angle of 40° from each other. The type and number ofLEDs present in the two illumination paths are modifiable and may be adapted to the envisaged application of the device.

[0135] According to a first example, and as represented in Figure 3, in a firstillumination path, the device comprises a blue LED LS6 (470 nm). The lightfrom the blue LED LS6 is collimated using a high numerical aperture condenserlens L6 before being filtered with a corresponding excitation filter OF6. Thelight is focused onto the small end of a tapered lightpipe LP using a plano-convex lens L9. The lightpipe LP is used to homogenize the input light, throughmultiple internal reflections, providing homogeneous light over the pipe’s exitface. The lens L10 used after the lightpipe LP is a matched achromatic doubletpair and conjugates the exit face of the lightpipe LP to the sample plane,forming an illumination area on the sample S. The other illumination armcomprises the same elements (470 nm LED LS7, condenser lens L7, filter OF7,plano-convex lens L11, and lightpipe LP2), in order to allow for higher lightintensities to be achieved.

[0136] In the first illumination path, a second, 405 nm LED LS8 of which thelight is collimated using another condenser lens L12, is coupled with anappropriate excitation filter OF8 and through a dichroic mirror DM.

[0137] The macro-imager according to the first example is in particulardesigned for measurements with a Dronpa-2 solution sample. In this case,the two wavelengths 470 et 405 nm are adapted to promote thephotoconversion from state 1 to state 2, and from state 2 to state 1,respectively.

[0138] According to a second example, the macro-imager has a firstillumination path as in the first example described with reference to Figure 3.The second illumination path comprises a blue 470 nm LED with acorresponding filter, and a green LED, used without excitation filter. A dichroicmirror is used to combine the light from the two LEDs in the secondillumination path.

[0139] The macro-imager according to the second example is in particulardesigned for measurements of the photosynthetic apparatus of Arabidopsisthaliana plants. In this case, the excitation is realized at 470 nm with one ortwo LEDs according to the RIOM or HIOM protocol, respectively.

[0140] The LEDs may be driven were driven in different ways, depending onthe type of sample to be measured.

[0141] For example, with Dronpa-2, high values of intensity of the excitationlights are requested. In this case, the LEDs are driven using an LED driver,and the LED outputs are modulated using a waveform generator.

[0142] In the case of the experiments with Arabidopsis thaliana, where lowerlight intensities are required, the LEDs may be driven directly with a waveformgenerator.

[0143] The macro-imager according to the embodiments described herein ispreferably placed in a dark box, in order to prevent environmental light fromreaching the sample(s) and interfering with measurements.

[0144] In the devices implementing the present invention, triggering of thecamera acquisition is synchronized with the start of the periodic modulation of the excitation light.

[0145] A data processing unit DPU such as a computer controls the operationof the waveform generation, in order to obtain the desired illumination conditions. The data processing unit DPU also receives the signals acquired by the camera CAM. The signals correspond to fluorescence light intensities. The DPU processes the signals, such as to extract the amplitudes or amplitude components used for determining kinetic information according to the present invention.

[0146] The method according to both the HIOM and RIOM aspects of theinvention can be implemented with either one of the devices described above.

[0147] The HIOM and RIOM protocols have been experimentally evaluatedwith several reversibly photo-convertible luminophores exhibiting luminescence lifetimes spanning a wide range, but too short to be directly imaged with a standard low-frequency camera. In the following, examples of the inventive methods implemented with the devices according to theexamples described above, i.e., the epifluorescence microscope and thefluorescence macro-imager, are described.

[0148] Figure 4 shows measurements carried out according to embodimentsof the method of the invention, on different types of samples.

[0149] Figures 4a - 4c show H-OPIOM measurements on fixed cells labelledat the nucleus with the fluorescent protein Dronpa-2, which, at neutral pH, reversibly photoswitches between a bright and a dark state upon blue and violet illumination. Average excitation light intensities at 470 and 405 nm are fixed to 6.4 and 4.0 W / cm2, respectively. Images at 12 Hz acquisitionfrequency are recorded upon sinusoidally modulating the 470 and 405 nmexcitation lights in antiphase with 100% duty cycle at frequencies departing from each other by 1 Hz in the [1 Hz; 100 Hz] range (Figure 4a). The dependence of the amplitude of the quadrature-delayed component of the fluorescence signal modulated at 1 Hz at each pixel on the angular frequencyof the 470 nm light modulation is fitted (Figure 4b). An H-OPIOM image of theDronpa-2 photoswitching time is then retrieved from the fit (Figure 4c). The H-OPIOM image is consistent with the expectation: <^> = 12.8 ± 0.5 ms overthe nucleus is found, whereas <^> = 9,5 ± 1 ms is anticipated from exploitingthe photoswitching information at 470 and 405 nm for Dronpa-2 ([Querard2017], [Chouket 2022]).

[0150] Figures 4d – 4f show RIOM measurements on the same sample.Images at 12 Hz acquisition frequency are acquired upon applying 470 nmexcitation light sinusoidally modulated within the [1 Hz; 100 Hz] range around1.8 W / cm2) with 100% duty cycle, and constant 6.4 W / cm2 405 nm excitationlight (Figure 4d). After fitting the dependence of the average fluorescence signal on the angular frequency of the 470 nm light modulation at each pixel(Figure 4e), a RIOM image of the Dronpa-2 photoswitching time is retrieved.The RIOM image is again in line with the expectation (Figure 4f): <^> =8,2±1,4 ms over the nucleus is found, whereas <^> = 10±1 ms is computedfrom the Dronpa-2 photoswitching information.

[0151] A RIOM wide-field image of the Dronpa-2 photoswitching time is alsoobtained from a Dronpa-2 solution contained in micro-chambers of amicrofluidic device by using a fluorescence macro-imager. Figures 4g – 4ishow the corresponding measurements and results, similarly to Figures 4d -4f. In this case, <^> = 485±15 ms was found over the micro-chambers inRIOM whereas <^> = 545±50 ms was anticipated from photoswitchinginformation available for Dronpa-2 ([Querard 2017], [Chouket 2022]).

[0152] The measurements and results represented in Figure 4 are obtainedwith sinewave-modulated illumination of the excitation light(s). Sinewave modulation is the simplest periodical modulation to be implemented at anyfundamental frequency. When using LEDs as light sources, sinewavemodulation benefits from ease of correction for the generation of harmonicsover a wide range of frequencies.

[0153] Of course, the HIOM and RIOM aspects of the method of the inventionmay also be implemented with other periodically modulated illuminations.

[0154] As a representative example, and with reference to Figure 5, square-wave modulated illumination is implemented with both protocols. Signals of high amplitude and reliable fitting functions of their dependence on thefrequency of modulated illumination may be obtained.

[0155] Figures 5a -5f show equivalent measurements to those shown inFigures 4a -4f, carried out on fixed cells labelled at the nucleus with thefluorescent protein Dronpa-2, the measurements being carried out withsquare-wave modulated excitation illumination. Figures 5a – 5c relate tomeasurements under the HIOM protocol, and Figures 5d – 5f relate tomeasurements under the RIOM protocol. HIOM and RIOM maps of the fluorescence photoswitching time are obtained in agreement with theexpectations: <^> = 8.8 ± 0.4 ms and 14.4 ± 0.2 ms is found, respectively,over the nucleus whereas <^> = 11.4 ± 0.2 ms was anticipated ([Querard2017], [Chouket 2022]).

[0156] Figure 6 shows measurements carried out according to embodimentsof the method of the invention, with luminophores endowed with shorterphotoactivation lifetimes with respect to Dronpa-2, in order to evaluate, in anexemplary manner, the applicability of the RIOM protocol and to determinethe shortest photoactivation lifetimes that could be imaged implementing the methods with the epifluorescence microscope and fluorescence macro-imager as described above.

[0157] In the example of Figure 6, the method according to the RIOMprotocol is implemented to image monodisperse polystyrene beads loadedwith a selected luminescent metal complexe under sinusoidally modulatedillumination in epifluorescence microscopy. Figures 6a and 6b show 12-Hzacquired images of the RIOM signal from Platinum octaethylporphyrin (PtOEP)under an excitation light at λ = 405 nm modulated at f = 100 Hz without andwith binning, respectively. As displayed in Figure 6, it is possible to retrievemaps of lifetimes of their triplet state in the [1 µs; 1 ms] range. In particular,the lifetime of triplet-state phosphorescence emitters is imaged with quantum yields as low as a few percent with some pixel binning (Figure 6c).

[0158] The method according to aspects of the present invention may alsobe used to image information on the physiological state of photosyntheticorganisms. In particular, the HIOM and RIOM protocols may be exploited toretrieve Bode diagrams and obtain images therefrom. Bode diagrams deliverkinetic fingerprints in the frequency domain equivalent to the signatures presently obtained in the time domain.

[0159] Figures 7 to 9 show Bode diagram retrieval in both HIOM and RIOMprotocols on leaves of wild type Arabidopsis thaliana (Columbia-0) over the3-393221 and 1-80000 Hz, respectively, modulation-frequency rangesrecovering the [1 ms; 1 s] time range usually exploited to acquire thecorresponding fluorescence response to constant illumination in the timedomain.

[0160] In Figures 7a-7e, wide-field macroscopic fluorescence imaging, suchas implemented with a device of the example represented in Figure 3, is usedto capture fluorescence emission at 690 nm of the photosynthetic apparatusof wild-type Arabidopsis thaliana (Columbia-0), under modulated illuminationand an interfering sunlight source, with and without a solution of an inhibitorof photosynthesis applied (3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU);200 µM, 2% EtOH). Image frames are acquired (with a 1 s exposure time)under a 1 Hz modulated illumination at 470 nm (I0 = 100 µE / m2s) with (Figure7a) and without (Figure 7b) DMCU treatment.

[0161] The corresponding Bode diagrams (Figures 7c and 7d) are retrievedfrom a set of fluorescence images corresponding to a plurality of modulationfrequencies. In the Bode diagrams, the markers correspond to response data,and the solid line is an eye guideline from applying a fitting function such asgiven in equation (73), with p1 = 0.0014, p2 = 0.32, p3 = 1.0, and ^ = 119 ±112 ms.

[0162] Figures 7e and 7f show image maps of the ratio of the response at 1Hz versus 20 Hz for the cases with (Figure 7e) and without DCMU treatment(Figure 7f).

[0163] Figure 8 shows maps of the ratio of the response at 1 Hz vs 4094 Hzof a full plant upon injection of a solution of DCMU into the soil, over the course of almost 22 hours. The maps are obtained from Bode diagrams as inFigures 7a-7f.

[0164] Image frames from 12 Hz acquired videos of leaves with (Figure 9a)and without (Figure 9b) DCMU treatment under two 470 nm excitation lightssinusoidally modulated and in antiphase, with I1 = 50 µE / m2s, f1 = 3 Hz; I2 =50 µE / m2s, f2 = 2 Hz.

[0165] The Bode diagrams retrieved from the images show the dependenceof the quadrature-delayed component of the fluorescence emission at 1 Hzwith (Figure 9c) and without (Figure 9d) DCMU treatment. In the Bodediagrams, the markers correspond to measured data, and the solid line is aneye guideline from applying a fitting function such as given in equation (74),with p1 = 1554, p2 = -6.64, p3 = 0.023, and ^ = 25 ± 2 ms.

[0166] Figures 9c and 9f show image maps of the ratio of the response at 1Hz versus 773 Hz for the cases with (Figure 9e) and without DCMU treatment(Figure 9f).

[0167] As can be seen in Figures 7 to 9, both H-OPIOM and RIOM protocolsenable to extract an image of Bode diagrams, which can be processed to retrieve a map of photoactivation lifetime with a simple ratio. Furthermore, they deliver a markedly different Bode diagram from a leaf conditioned with DCMU, a current inhibitor of photosynthesis blocking electron flow from photosystem II. Hence, H-OPIOM and RIOM are relevant to retrieve a specific kinetic fingerprint evidencing the presence of the latter herbicide in leaves. Furthermore, we could analyze the kinetics of DCMU spreading from the rootsto the leaves by recording a time series of RIOM images of A. thaliana plantin the presence of interfering ambient light. Figures 7 to 9 show that the Bodediagram can be used to demonstrate that DCMU smoothly progresses from the roots to the leaves at the 15 h time scale, which range is in line with previous measurements exploiting the fluorescence level as stress reporter. However, in contrast to the latter observable, the Bode diagram provides a kinetic fingerprint bringing more information on the stress source.

[0168] Hence, HIOM and RIOM emerged as powerful protocols for deliveringwide-field luminescence lifetime imaging on simple cheap optical setups without any expansive detectors endowed with high-frequency modulation or fast gating. They have here been already applied on several types of reversibly photoactivable luminescent systems but their theoretical principle is very general so as to enable for a wide variety of applications with LEDs up to 1 MHz frequency of light modulation. However, they will only deliver reliable observables when the control parameters of modulated illumination (mean light intensity / ies and frequency / ies) are matched with the photoactivation kinetics.

[0169] The methods and devices according to embodiments of the presentinvention may be implemented with any reversibly photo-convertible luminophores as long as the kinetic model given in equation (1) accounts for their dynamic behaviour under illumination at an appropriate time scale. Such luminophores may include fluorescent probes, phosphorescent probes,reversibly photo-convertible fluorophores, and the photosynthetic apparatus.

[0170] Of course, the invention is not limited to the examples detailed above.In particular, different modulation schemes and more than two excitation lights may be used.

[0171] References[Querard 2015]: J. Querard et al., Expanding discriminative dimensions for analysis and imaging, Chem. Sci., 2015, 6, 2968-2978[Querard 2017]: J. Querard et al., Resonant out-of-phase fluorescencemicroscopy and remote imaging overcome spectral limitations, Nat.Commun., 2017, 8, 969[Chouket 2022]: R. Chouket et al, Extra kinetic dimensions for labeldiscrimination, Nat. Commun., 2022, 13, 1482.

Claims

CLAIMS1. A method for determining luminescence kinetic information of at least onereversibly photo-convertible luminescent species (P) in a sample (S), themethod comprising the steps of: a) illumination of the sample (S) with at least a first and a secondexcitation light, the intensity of the first excitation light beingperiodically modulated at a frequency f1, and the intensity of thesecond excitation light being periodically modulated at a frequency f2, wherein f1 ≠ f2, the excitation lights being adapted to promote a luminescence light emission from the at least one luminescent species (P),b) detection, with an optical detector (CAM), of the luminescencelight, the intensity of the luminescence light being modulated at afrequency difference Δf = f1 - f2, for a plurality of modulationfrequencies, f1, of the first excitation light,c) extraction, for a plurality of modulation frequencies, f1, of thefirst excitation light, of an amplitude of the intensity of theluminescence light, d) determination of a kinetic information of the at least onereversibly photo-convertible luminescent species (P) from thedependence of the extracted luminescence amplitude on themodulation frequency, f1, of the first excitation light.

2. The method according to claim 1, wherein the average intensities of thefirst and the second excitation lights, the modulation frequency, f1, of thefirst excitation light, and the frequency difference, Δf, are chosen toapproach the maximum of^ the phase-quadrature luminescence amplitude when themodulation periods of the first and the second excitation lights are offset by 0 or ^,.^ the in phase luminescence amplitude when the modulation periodsof the first and the second excitation lights are offset by ^ / 2 or 3^ / 2.

3. The method according to any one of the preceding claims, wherein the firstexcitation light has a wavelength λ1, and the second excitation light has awavelength λ2, and wherein λ1 = λ2 or λ1 ≠ λ2.

4. The method according to any one of the preceding claims, wherein thefrequency difference, Δf, is below 1000 Hz.

5. A method for determining luminescence kinetic information of at least onereversibly photo-convertible luminescent species (P) in a sample (S), themethod comprising the steps of: a) illumination of the sample (S) with at least a first excitation light,the intensity of the first excitation light being periodicallymodulated at a frequency f, the first excitation light being adapted to promote a luminescencelight emission from the at least one luminescent species (P),b) detection of the luminescence light with an optical detector(CAM), the intensity of the luminescence light being an average intensity, for a plurality of modulation frequencies, f, of the atleast first excitation light, c) extraction, for a plurality of modulation frequencies, f, of the atleast first excitation light, of the average luminescence intensity,d) determination of a kinetic information of the at least onereversibly photo-convertible luminescent species (P) from thedependence of the average luminescence intensity on the modulation frequency, f, of the first excitation light.

6. The method according to claim 5, wherein the illumination step comprisesthe illumination of the sample (S) with a first excitation light, the intensity of the first excitation light being periodically modulated at a frequency, f.

7. The method according to claim 5, wherein the illumination step comprisesthe illumination of the sample (S) with a first excitation light and a secondexcitation light, the intensity of the first excitation light being periodicallymodulated at a frequency, f, and the intensity of the second excitation lightbeing constant.

8. The method according to claim 5, wherein the illumination step comprisesthe illumination of the sample (S) with a first excitation light and a second excitation light, the intensities of the first and second excitation lights being periodically modulated at the same frequency, f, and in antiphase with respect to each other.

9. The method according to claim 7 or 8, wherein the first excitation light hasa wavelength λ1, and the second excitation light has a wavelength λ2, and wherein λ1 = λ2 or λ1 ≠ λ2. 10.The method according to any one of claims 5 to 9, wherein the average intensity of the at least first excitation light and the modulation frequency, f, of the first excitation light are chosen to approach the maximum of theamplitude of the frequency-induced dependence of the average luminescence intensity normalized by the luminescence intensity under the average intensity of the at least first excitation light. 11.The method according to any one of the preceding claims, wherein the determination of the kinetic information comprises the extraction of a luminescence lifetime of the at least one luminescent species (P).12.The method according to the preceding claim, further comprising imaging the luminescence lifetime according to a field of view of the detector (CAM). 13.The method according to any one of the preceding claims, wherein the determination of the kinetic information comprises the establishment of a Bode diagram linking the extracted luminescence signal to the modulationfrequencies of the at least first excitation light. 14.The method according to any one of the preceding claims, wherein the determination step is realized using a fitting function comprising a ratio oftwo polynomial functions, the polynomial functions each comprising a sum of polynomials of the luminescence lifetime and the modulation frequency of the at least first excitation light. 15.The method according to claim 14, wherein the polynomials are of the order of 4 or less. 16.The method according to any one of the preceding claims, wherein the periodical modulation of the excitation light(s) is of sinusoidal form or square-wave form.17.A device for carrying out a method according to any one of the preceding claims, the device comprising -illumination means (LS1-LS8) configured for carrying out methodstep a), -an optical detector (CAM) configured for carrying out method stepb), and- a data processing unit (DPU) configured for carrying out methodsteps c) and d).Use of the method according to any one of the preceding claims forreporting on the kinetic information of a physiological state ofphotosynthetic organisms.

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