Method and apparatus for detecting a photochemically active chemical species in a sample

1 . A method for detecting a photochemically active chemical species in a sample, comprising the steps of:

a) illuminating the sample(S), according to an illumination sequence, with light (LB 1 ) of at least a first wavelength (λ 1 ) suitable to be absorbed by the chemical species triggering a first reaction (R 1 ) affecting at least one optical property of the chemical species and comprising at least one photochemically-activated step (PAS 1 ) and one thermally-activated step (TAS 1 ), wherein the illumination sequence is such that:

in at least a first time window (TW 1 , TW 1 ′) of the illumination sequence, a rate of the first reaction is limited by the photochemically-activated step; and in at least a second time window (TW 3 , TW 2 ′) of the illumination sequence, the rate of the first reaction is limited by the thermally-activated step;

b) measuring the evolution of the at least one optical property of the chemical species during the first and the second time windows;

c) determining, from said measuring, at least a first and a second time constants representing rate constants of the first reaction in the first and the second time windows, respectively; and

d) using the determined at least the first and second time constants for detecting the chemical species.

2 . The method according to claim 1 wherein:

step a) also comprises illuminating the sample with light (LB 2 ) of at least a second wavelength (λ 2 ) according to the illumination sequence, wherein light at the second wavelength is suitable to be absorbed either by the chemical species or by a product of the first reaction and to trigger a second reaction (R 2 ) affecting at least one optical property of the chemical species and comprising at least one photochemically-activated step (PAS 2 ) and one thermally-activated step (TAS 2 ), wherein the illumination sequence is such that:

in at least a third time window (TW 2 ) of the illumination sequence, a rate of the second reaction is limited by the photochemically-activated step; and in at least a fourth time window (TW 4 ) of the illumination sequence, the rate of the second reaction is limited by the thermally-activated step;

step b) also comprises measuring the evolution of the optical property of the chemical species during the third and the fourth time windows;

step c) also comprises determining, from said measuring, at least a third and a fourth time constants representing the rate constants of the second reaction in the third and the fourth time windows, respectively; the third and fourth time constants being used in step d), together with the first and second time constants, for detecting the chemical species.

3 . The method according to claim 1 wherein step b) comprises measuring fluorescence emission.

4 . The method according to claim 3 , wherein said photochemically active chemical species is chosen among a fluorescent protein and a reversibly photoswitchable fluorophore.

5 . The method according to claim 4 , wherein:

said or at least one said photochemically active chemical species is a reversibly photoswitchable fluorescent protein;

during at least a first time window (TW 1 ) of the illumination sequence, a light intensity level at the first wavelength (L) is kept sufficiently low for the rate of the first reaction to be limited by the photochemically-activated step; and

during at least a second time window (TW 3 ) of the illumination sequence, the light intensity level at the first wavelength is kept sufficiently high for the rate of the first reaction to be limited by the thermally-activated step.

6 . The method according to claim 2 , wherein:

said or at least one said photochemically active chemical species is a reversibly photoswitchable fluorescent protein;

during at least a first time window (TW 1 ) of the illumination sequence, a light intensity level at the first wavelength (I 1 ) is kept sufficiently low for the rate of the first reaction to be limited by the photochemically-activated step; and

during at least a second time window (TW 3 ) of the illumination sequence, the light intensity level at the first wavelength is kept sufficiently high for the rate of the first reaction to be limited by the thermally-activated step, and

during at least a third time window (TW 2 ) of the illumination sequence, a light intensity level (I 2 ) at the second wavelength is kept sufficiently low for the rate of the second reaction to be limited by the photochemically-activated step; and

during at least a fourth time window (TW 4 ) of the illumination sequence, the light intensity level at the second wavelength is kept sufficiently high for the rate of the second reaction to be limited by the thermally-activated step.

7 . The method according to claim 4 , wherein:

said or at least one said photochemically active chemical species is a fluorescent protein; during at least one first time window (TW 1 ′) of the illumination sequence, a light intensity level (I 1 ) at the first wavelength is kept sufficiently high for photochemically converting the chemical species into a non-fluorescent form, resulting in a progressive decrease in fluorescence intensity;

during at least one second time window (TW 2 ′) of the illumination sequence, following the first time window, the light intensity level (I 1 ) at the first wavelength is kept sufficiently low for allowing thermal recovery of the fluorescent intensity.

8 . The method according to claim 4 wherein:

said or at least one said photochemically active chemical species is a reversibly photoswitchable fluorescent protein;

during at least one first time window (TW 1 ) of the illumination sequence, the sample is illuminated by a first series of light pulses (LP 1 1 -LP 1 5 ) at the first wavelength, each pulse having a fluence sufficiently low for only a fraction of the chemical species to be converted through the photochemically-activated step of the first reaction, an interval (t d ) between two consecutive pulses being sufficiently long for allowing said fraction of the chemical species to be fully converted through the thermally-activated step of the first reaction, following said photochemically-activated step; and

during at least one second time window (TW 3 ) of the illumination sequence, the sample is illuminated by a second series of light pulses (LP 3 1 -LP 3 5 ) at the first wavelength, at least the first pulse of the series having a fluence sufficiently high for fully converting the chemical species through the photochemically-activated step of the first reaction, an interval (t d ) between two consecutive pulses being sufficiently short for sampling a subsequent further conversion of the chemical species through the thermally-activated step of the first reaction.

9 . The method according to claim 2 , wherein:

said or at least one said photochemically active chemical species is a reversibly photoswitchable fluorescent protein;

during at least one first time window (TW 1 ) of the illumination sequence, the sample is illuminated by a first series of light pulses (LP 11 -LP 15 ) at the first wavelength, each pulse having a fluence sufficiently low for only a fraction of the chemical species to be converted through the photochemically-activated step of the first reaction, an interval (t d ) between two consecutive pulses being sufficiently long for allowing said fraction of the chemical species to be fully converted through the thermally-activated step of the first reaction, following said photochemically-activated step; and

during at least one second time window (TW 3 ) of the illumination sequence, the sample is illuminated by a second series of light pulses (LP 31 -LP 35 ) at the first wavelength, at least the first pulse of the series having a fluence sufficiently high for fully converting the chemical species through the photochemically-activated step of the first reaction, an interval (ta) between two consecutive pulses being sufficiently short for sampling a subsequent further conversion of the chemical species through the thermally-activated step of the first reaction; and

during at least the third time window (TW 2 ) of the illumination sequence, the sample is illuminated by a third series of light pulses (LP 2 1 -LP 2 5 ) at the second wavelength, each pulse having a fluence sufficiently low for only a fraction of the chemical species to be converted through the photochemically-activated step of the second reaction, an interval between two consecutive pulses being sufficiently long for allowing said fraction of the chemical species to be fully converted through the thermally-activated step of the second reaction, following said photochemically-activated step; and

during at least the fourth time window (TW 4 ) of the illumination sequence, the sample is illuminated by a fourth series of light pulses (LP 4 1 -LP 4 5 ) at the second wavelength, at least the first pulse of the series having a fluence sufficiently high for fully converting the chemical species through the photochemically-activated step of the second reaction, an interval between two consecutive pulses being sufficiently short for sampling a subsequent further conversion of the chemical species through the thermally-activated step of the second reaction.

10 . The method according to claim 4 , wherein:

said or at least one said photochemically active chemical species is a fluorescent protein;

during a plurality of first time windows (TW 1 a -TW 1 e ), the sample is illuminated at the first wavelength with a fluence sufficiently high for fully converting the chemical species through the photochemically-activated step of the first reaction;

during a plurality of second time windows (TW 2 a -TW 2 e ), alternating with the first time windows, the sample is not illuminated at the first wavelength, the second time windows having varying durations suitable for sampling a subsequent further conversion of the chemical species through the thermally-activated step of the first reaction.

11 . The method according to claim 8 wherein steps a) and b) are carried out by light scanning microscopy.

12 . The method according to claim 1 wherein:

the sample is illuminated with a spatially inhomogeneous light intensity; and

step d) comprises using the determined time constants representing reaction rate constants limited by photochemically-activated steps for localizing said or at least one said chemical species within the sample.

13 . The method according to claim 1 wherein step d) comprises:

computing a plurality of multidimensional logarithmic distances between a vector formed by the time constants determined at step c) and each one of a plurality of predetermined vectors of time constants, each of said predetermined vectors representing a respective photochemically active chemical species; and

detecting one of said photochemically active chemical species when the corresponding multidimensional logarithmic distance is lower than a threshold representative of measurement uncertainties.

14 . The method according to claim 1 wherein step b) comprises separately measuring the evolution of said or each said optical property of the chemical species at a plurality of locations within the sample, step c) comprising determining said time constants for each one of said location and step d) comprising determining if said optical species is present at each one of said locations.

15 . An apparatus for carrying out a method according to claim 1 comprising:

at least one controlled light source (LS 1 ) configured for illuminating the sample with light (LB 1 ) of at least a first wavelength (λ 1 ) suitable to be absorbed by the chemical species triggering a first reaction (R 1 ) affecting at least one optical property of the chemical species and comprising at least one photochemically-activated step (PAS 1 ) and one thermally-activated step (TAS 1 ) and according to a predetermined illumination sequence comprising at least a first time window and at least a second time window corresponding to different illumination conditions such that

in at least the first time window (TW 1 , TW 1 ′) of the predetermined illumination sequence, a rate of the first reaction is limited by the photochemically-activated step; and

in at least a second time window (TW 3 , TW 2 ′) of the predetermined illumination sequence, the rate of the first reaction is limited by the thermally-activated step;

a light detector (LD) configured for measuring the evolution of the at least one optical property of the sample during said at least a first time window and at least a second time window; and

a data processing device (DPD) configured for determining, from said measuring, at least a first and a second time constants representing rate constants of the first reaction in the first and the second time windows respectively, and for detecting a photochemically active chemical species within the sample as a function of the determined at least the first and the second time constants.