System for receiving and illuminating a photoreactive medium

Abstract

The invention relates to a system (1, 10) for receiving and illuminating a photoreactive medium, comprising: a light source (2), a receiving compartment (11) having a zone in which the photoreactive medium is located, means for guiding the emitted light, and means for diffusing said light. The receiving compartment (11) is delimited between two walls (12, 13), the system (1) comprising a compartment adjacent (14) to the receiving compartment (11), separated from it by one of the walls (12) delimiting the receiving compartment (11), the adjacent compartment (14) containing a fluid medium forming means for guiding the emitted light towards a zone of the adjacent compartment in which are located the light diffusion means for diffusing the light through the wall (12) separating the two compartments towards the zone of the receiving compartment in which the photoreactive medium is located. Figure 1

Description

Title of the invention: System for receiving and illuminating a photoreactive medium

[0001] The present invention relates to a system for receiving and illuminating a photo-reactive medium. State of the art

[0002] Photons are the reactants in photochemical transformations in which molecules are produced, for example by direct light absorption, photocatalysis, or photopolymerization, or degraded, for example, during water purification by light. The light used is usually delivered with adjustable energy and flux from inexpensive light sources, such as light-emitting diodes (LEDs). It is also known that many chemical and enzymatic reactions can be influenced, altered, and / or regulated by light.

[0003] The use of photochemistry is therefore becoming increasingly important in research and development laboratories. Indeed, photons are environmentally friendly reagents, and the field of photocatalysis is experiencing significant development.

[0004] On the one hand, this implies a significant demand for photoreactors and photobioreactors with satisfactory illumination control in terms of intensity and uniformity of the applied light. On the other hand, it generates a strong demand for the development of in situ chemical and mechanistic analysis tools such as, for example, NMR tubes compatible with in situ illumination.

[0005] As already mentioned, it is recognized that many chemical and enzymatic reactions are modulated by light. Therefore, the combination of illumination of a sample of a photoreactive medium and analytical tools such as high-resolution NMR spectroscopy, measurement of luminescence (fluorescence, phosphorescence), and absorbance is recognized as a very interesting technique.

[0006] Photons are still not considered easy reagents for users to implement, as difficulties arise in terms of reproducibility and large-scale scaling up. The yield of photochemical reactions depends locally on the intensity of the light received. Uniformly illuminating a light-absorbing photoreactive medium is a challenge due to the attenuation of light intensity along the direction of its propagation; that is, the part of the medium near the illuminated part is exposed to the the highest light intensities while the rest is exposed to lower intensities or even no light at all.

[0007] Means of in situ illumination of a sample placed within an NMR spectrometer have been proposed, comprising systems using an optical fiber to guide light (from a laser or a light-emitting diode (LED)) to an area located just above the sample to be studied. This leads to a significantly non-uniform distribution of light intensity in the sample (for example, one area is illuminated while another is not).

[0008] Systems proposing in situ illumination of the sample with an optical fiber extending into the sample itself have also been studied, but this leads to distortion of the magnetic field and difficulties in calibrated the sample and suppressing the solvent signal, as well as possible contamination of the sample and, again, a non-uniform light distribution (Feldmeier, C., Bartling, H., Riedle, E., & Gschwind, RM (2013). Easy quantum yield determination via NMR actinometry. Organic letters, 20(8), 2156-2159; Ji, Y., DiRocco, DA, Kind, J., Thiele, CM, Gschwind, RM, & Reibarkh, M. (2019) LED based NMR illumination device for mechanistic studies on photochemical reactions—Versatile and simple, yet surprisingly powerful. Journal of Magnetic Resonance, 232, 39-44)

[0009] It has also been proposed to use a glass insert containing an optical fiber and a UV probe, thus enabling the insertion of these optical means within a light-absorbing solution in an NMR spectroscopy context. The end of the glass insert, containing the end of the optical fiber, can thus be introduced directly into the sample. The end of the insert is shaped to promote light scattering in all directions through the sample. However, no results have been reported on such a configuration regarding its ability to deliver uniform illumination within an absorbing solution without flux or agitation (Combination of illumination and high resolution NMR spectroscopy: Key features and practical aspects, photochemical applications, and new concepts. Progress in Nuclear Magnetic Resonance Spectroscopy, 114, 86-134).

[0010] Similarly, glass rods have been proposed to form light-guiding means inserted into tubes containing a light-absorbing solution in a photoreactor (Negishi, N., He, F., Matsuzawa, S., Takeuchi, K., & Ohno, K. (2006). Wave-guide type photoreactor for water purification. Comptes Rendus Chimie, 9(5-6), 822-828.).

[0011] Photobioreactors that rely on light (as the energy source for photosynthesis) are widely used nowadays for bioproduction by Microalgae. These photobioreactors are considered important elements for the bioeconomy.

[0012] Thus, a photobioreactor can be proposed with planar waveguides as described in the following publications: Sun, Y., Liao, Q., Huang, Y., Xia, A., Fu, Q., Zhu, X., & Zheng, Y. (2016). Integrating planar waveguides doped with light scattering nanoparticles into a flat-plate photobioreactor to improve light distribution and microalgae growth. Bioresource technology, 220, 215-224; Simultaneous enhancement of Chlorella vulgaris growth and lipid accumulation through the synergy effect between light and nitrate in a planar waveguide flat-plate photobioreactor. Bioresource technology, 243, 528-538). Such polymethyl methacrylate (PMMA) and / or polycarbonate (PC) plates include an LED bar mounted on a plate edge as well as reflective elements, making this system complex.

[0013] Characterizing the spectral distribution of the light source and measuring the light intensity at the level of a sample or a photoreactor is very important. It is also important to be able to use photoreactors that provide efficient, uniform illumination in order to optimize photocontrol and enable access to reliable kinetic information.

[0014] The controlled implementation of photochemical reactions in diverse and multiple technical fields therefore requires the ability to illuminate a light-absorbing medium in the most uniform way possible.

[0015] To remedy this, it has been proposed, in particular, to create agitation or flow within the photoreactive medium in order to obtain an "average" exposure of this medium to light. However, such an approach encounters several limitations. Indeed, when the illuminated medium or sample is very, or even too, reactive to light, agitation does not eliminate the heterogeneity of the impact of light on the entire medium in all its dimensions.

[0016] Furthermore, the illuminated medium may also be too sensitive to the shear resulting from agitation to exhibit normal behavior (e.g., in the case of a microalgae suspension). In addition, agitation under illumination within a liquid sample may be difficult to implement due to instrumental constraints or limitations related to the analytical devices used, such as in NMR spectrometers, sub-vacuum.

[0017] WO-A-2021250372 has proposed an illumination insert shaped to receive a sample and comprising a light guide portion for guiding light from a light source and a diffusion portion for diffusing the light received by the light guide portion towards the sample received in said illumination insert. This insert is in the form of a closed or open tube in which the sample is received either directly (closed tube) or in a tube (open tube). This insert includes, for this purpose, a wall surrounding the sample reception area, this wall allowing both the guidance of light from a source and the diffusion of this guided light towards the sample.

[0018] Generating the most uniform illumination possible within a highly absorbing medium with an optimal photon budget presents a very important challenge in multiple and diverse fields as mentioned above. Description of the invention

[0019] An objective of the present invention is to provide a system for receiving and illuminating a light-absorbing medium that can be used both to analyze a sample, for example in NMR spectroscopy, by measuring absorbance and / or fluorescence, and to create photo-(bio-)reactors.

[0020] To this end, the invention relates to a system for receiving and illuminating a photoreactive medium, said system comprising: at least one light source, at least one receiving compartment having at least one zone in which the photoreactive medium is located, means for guiding the light emitted by the light source and means for diffusing said light, characterized in that the at least one receiving compartment is delimited between two walls, the system comprising at least one compartment adjacent to the receiving compartment of the reactive medium, separated from it by one of the walls delimiting the receiving compartment, the compartment adjacent to the receiving compartment containing a fluid medium as means for guiding the light emitted by the light source,to guide the light towards at least one area of ​​the compartment adjacent to the area of ​​the receiving compartment containing the photoreactive medium, an area of ​​the adjacent compartment which also contains light-diffusion means arranged to diffuse the light through the partition separating the two compartments towards the area of ​​the receiving compartment containing the photoreactive medium.

[0021] Advantageously, the system according to the invention is simple to implement by providing two compartments separated by a partition, one receiving the photoreactive medium to be illuminated and the other the illumination light guided by a suitable fluid medium to constitute guiding means and preferably having a refractive index equal to the refractive index of the material of the partition between the compartments, the light guided by the fluid medium being diffused through the separating partition. Advantageously, this fluid medium is preferably a liquid medium such as, for example, dimethyl sulfoxide (DMSO) or any other suitable liquid.

[0022] Advantageously, the refractive index of the fluid medium and the refractive index of the material constituting the wall between the receiving compartment and said at least one adjacent compartment, are such that the difference between these refractive indices is less than 20%, preferably less than 10% and even more preferably the refractive indices are identical.

[0023] Preferably, the refractive index of the material constituting the wall between the receiving compartment and said at least one adjacent compartment is greater than that of the receiving compartment, i.e. the refractive index of the photoreactive medium.

[0024] The light source preferably consists of at least one light-emitting diode (LED) or a laser source positioned upstream of the light guiding means, the light of which is either directly emitted towards a compartment comprising a fluid medium as light guiding means or delivered to said compartment comprising a fluid medium as light guiding means, by an optical fiber connected to the light source at one end and the other end of which is immersed in the fluid medium to allow the propagation of light in the fluid medium.

[0025] It is thus possible to generate uniform illumination within a reactive medium such as a sample, thus enabling in situ illumination and therefore reliable in situ analysis as well.

[0026] Advantageously, the system according to the invention also allows the liquid medium to be used as a waveguide to retrieve in situ information on the absorbance and luminescence of the photoreactive medium. Advantageously, the fluid medium, as a light-guiding means, is suitable for transmitting the absorbance and / or luminescence of the photoreactive medium. The system includes means configured to detect said absorbance and / or luminescence, such as at least one additional optical fiber, connectable to devices for measuring absorbance and / or luminescence, in particular fluorescence and phosphorescence.

[0027] Furthermore, the system according to the invention is simple and economical to manufacture. The walls defining the compartment for receiving the photoreactive medium can simply be made of glass.

[0028] The fluid medium used as a light-guiding means has a refractive index equal to that of the material constituting the walls separating the receiving compartment of the photoreactive medium from the adjacent compartment comprising said medium as a light-guiding means. Thus, if the walls forming the compartment in which is the fluid medium forming the means of guiding the light, are made of glass the fluid medium has a refractive index equal to that of glass therefore equal to 1.5.

[0029] Preferably, the light diffusion means consist of micro and / or nano particles suspended in the fluid medium forming the light guidance means.

[0030] Alternatively, the diffusion means are formed by a rough surface obtained for example by frosting at least one face of the wall separating the receiving compartment of the photo-reactive medium and an adjacent compartment comprising the light guiding means.

[0031] Advantageously, the system according to the invention is scalable since it is easily conceivable to provide an additional compartment, also adjacent to the receiving compartment of the photo-reactive medium, separated from it by the other wall of said receiving compartment.

[0032] Thus, the system according to the invention comprises at least one additional adjacent compartment to the photo-reactive medium receiving compartment, separated from it by the other wall delimiting the photo-reactive medium receiving compartment, the additional adjacent compartment to the photo-reactive medium receiving compartment containing a fluid medium as means for guiding the light emitted by a light source towards at least one area of ​​the additional adjacent compartment opposite the area of ​​the receiving compartment in which the photo-reactive medium is located, area of ​​the additional adjacent compartment in which there are light-diffusion means arranged to diffuse the light through the wall separating the two compartments towards the area of ​​the receiving compartment in which the photo-reactive medium is located,The photoreactive medium receiving compartment is interposed between two adjacent compartments, each comprising a fluid medium as a guiding means and light diffusion means.

[0033] According to a first embodiment of the invention, the photoreactive medium receiving compartment is defined between two coaxial cylindrical walls, one inner wall having a smaller cross-section than the other, referred to as the outer wall. The inner wall defines the adjacent compartment containing the fluid medium, which serves as a means for guiding the light emitted by the light source and for diffusing the light. The cylindrical walls preferably have the same cross-sectional shape.

[0034] This embodiment advantageously allows the light guiding and diffusion means to be placed at the heart of the photo-reactive medium, which promotes uniform illumination of the photo-reactive medium.

[0035] The system further comprises a third coaxial cylindrical wall of larger cross-section forming the additional adjacent compartment.

[0036] Preferably, the coaxial cylindrical walls have a circular cross-section and are thus concentric, the receiving compartment being mainly annular in shape.

[0037] This embodiment of the invention is particularly advantageous because it allows illumination of the photo-reactive medium from its core.

[0038] According to a first configuration of this embodiment of the invention, the system comprises at least two tubes having a closed end, one of which has a diameter smaller than the diameter of the other, the tube with a smaller diameter, called the inner tube, being housed concentrically in the tube with a larger diameter, called the outer tube, the receiving compartment of the photo-reactive medium being formed by the space between the walls of the tubes, the adjacent compartment being delimited by the inner tube and comprising a fluid medium as means for guiding the light emitted by the light source and means for diffusing the light.

[0039] In a particularly advantageous embodiment of the system of the invention, the system comprises an additional tube in which the two inner and outer tubes are mounted, said additional tube defining with the outer tube an additional adjacent compartment to the receiving compartment of the photo-reactive medium, said additional adjacent compartment comprising a fluid medium as means for guiding the light emitted by a light source towards at least one area of ​​the additional adjacent compartment opposite the area of ​​the receiving compartment in which the photo-reactive medium is located, area of ​​the additional adjacent compartment in which are located the light-diffusion means arranged to diffuse the light through the wall separating the two compartments towards the area of ​​the receiving compartment in which the photo-reactive medium is located,The photoreactive medium receiving compartment is interposed between two adjacent compartments comprising a fluid medium as guiding means and light diffusion means.

[0040] Such a system structure allows for uniform illumination of the photoreactive medium. The photoreactive medium is thus placed in a receiving compartment which is radially illuminated from each of its faces.

[0041] A fourth tube defining a second receiving compartment for the photo-reactive medium can then be added, and so on to form a photoreactor.

[0042] According to a second embodiment of the invention, the system comprises at least three parallel flat walls, a receiving compartment for a photoreactive medium being defined between two successive walls and an adjacent compartment being defined between one of the walls of the receiving compartment of a photo-reactive medium and the third wall, said adjacent compartment comprising a fluid medium as means for guiding the light emitted by a light source towards at least one area of ​​the adjacent compartment opposite the area of ​​the receiving compartment in which the photo-reactive medium is located, area of ​​the adjacent compartment in which are the light diffusion means arranged to diffuse the light through the wall separating the two compartments towards the area of ​​the receiving compartment in which the photo-reactive medium is located.

[0043] The system preferably forms a parallelepiped-shaped receptacle having a base from which two lateral walls extend, between which parallel plane walls extend transversely.

[0044] Such an embodiment of the system according to the invention makes it possible in particular to produce a photo(bio)reactor.

[0045] According to a variant of this embodiment, the system according to the invention comprises an additional adjacent compartment, to the receiving compartment of the photo-reactive medium, separated from it by the other wall delimiting the receiving compartment of the photo-reactive medium, and defined by a fourth parallel flat wall, the additional adjacent compartment to the receiving compartment of the photo-reactive medium containing a fluid medium as means of guiding the light emitted by the light source towards at least one area of ​​the additional adjacent compartment opposite the area of ​​the receiving compartment in which the photo-reactive medium is located, area of ​​the additional adjacent compartment in which are the light diffusion means arranged to diffuse the light through the wall separating the two compartments towards the area of ​​the receiving compartment in which the photo-reactive medium is located,The photoreactive medium receiving compartment is interposed between two compartments comprising a fluid medium as guiding means and light diffusion means.

[0046] Thus, the system according to this embodiment comprises at least four parallel transverse walls defining a photoreactive medium reception compartment interposed between two adjacent compartments comprising respectively means for guiding the light emitted by the light source towards at least one area of ​​the wall separating each adjacent compartment from the photoreactive medium reception compartment, for example a sample, and means for diffusing the light guided towards said area of ​​the wall towards the area in which the sample is located.

[0047] The system proposed according to the invention thus makes it possible to meet two needs. The first concerns the manufacture of photo-(bio)-reactors with control of the satisfactory illumination in terms of intensity and uniformity of the applied light and the second the provision of a system usable in analytical tools, in which the light guide means can both transmit and recover the light from the light source, which allows both the illumination of a sample of photo-reactive medium and the recovery of information on the absorbance and luminescence of the sample.

[0048] Furthermore, for photo-(bio)-reactors, it is possible with the system according to the invention to improve the efficiency of illumination by means of adapted sequences of modulated illumination that can be applied with exclusive control of the intensity and uniformity of the modulated light.

[0049] Finally, the system according to the invention therefore offers the possibility of knowing the composition of a photo-reactive medium in real time, continuously and simultaneously with different phenomena observable in situ and observation tools such as NMR spectroscopy, absorbance and / or luminescence measurement using the reception and illumination system to recover these observable phenomena.

[0050] Advantageously, the system according to the invention is scalable since it is easily possible to provide an adjacent compartment on either side of a receiving compartment for the photoreactive medium. This allows the system to evolve, with a succession of compartments containing a fluid medium as a means of guiding the light (the so-called illumination compartment), then a receiving compartment for a photoreactive medium, then an illumination compartment, then a receiving compartment, then another illumination compartment, and so on. Furthermore, such a system is easy and economical to implement using walls made of simple materials such as glass, with a simple structure and manufacture, the guiding means being obtained by a liquid medium.

[0051] The invention will be well understood and other aspects and advantages will become clear upon reading the following description, given by way of example with reference to the examples and the accompanying drawing sheet on which the figures represent:

[0052] [Fig. 1] a longitudinal cross-sectional view of a first configuration of a first embodiment of the invention;

[0053] [Fig.2] an enlarged view of part A of [Fig.1];

[0054] [Fig.3] a cross-sectional view of a system according to [Fig.1];

[0055] [Fig. 4a] An initial image of the sample under illumination at 470 nm by fluorescence imaging of the first configuration; Constant light injection at 111 mW, scale bar: 10 mm, T = 293 K. Xexc = 470 nm; / .cm = 535 nm

[0056] [Fig. 4b] A graphical representation of the response time of normalized integrated fluorescence on the sample area, constant light injection at 111 mW

[0057] [Fig. 4c] A characteristic time map r(s) associated with the sample of [Fig. 4a], Constant light injection at 111 mW, scale: 10 mm, T = 293 K. Xexc = 470 nm; Àcm = 535 nm

[0058] [Fig. 4d] A normalized light intensity map I of the sample from [Fig. 4a], Constant light injection at 111 mW, scale: 10 mm. T = 293 K. Xexc = 470 nm; / .cm = 535 nm

[0059] [Fig.4e] a representation of the superposition of the calculated (dashed line) and experimental (solid line) light intensity dependence averaged along the radial axis with respect to the z-coordinate of the longitudinal axis,

[0060] [Fig.4f] a representation of the actual distribution of light intensity in the sample on the z-coordinate of the longitudinal axis

[0061] [Fig. 5a] An initial image of the sample under illumination at 405 nm by fluorescence imaging of the first configuration; Constant light injection at 102 mW, scale: 10 mm. T = 293 K. Xexc = 405 nm; / .cm = 535 nm

[0062] [Fig.5b] a graphical representation of the response time of the normalized fluorescence integrated on the sample area, constant light injection at 102 mW;

[0063] [Fig. 5c] A characteristic time map r(s), constant light injection at 102 mW, scale: 10 mm. T = 293 K. Xexc = 405 nm; / .cm = 535 nm

[0064] [Fig. 5d] A normalized luminous intensity map I, constant light injection at 102 mW, scale: 10 mm. T = 293 K. Xexc = 405 nm; / .cm = 535 nm

[0065] [Fig.6] a graphical representation of the light intensity at 470 nm (square) and at 405 nm (circle) as a function of the light power emitted by the optical fiber and delivered into the liquid tube,

[0066] [Fig.7a] a time-dependent representation of the 19F-{ 'H]-NMR signals of the peak integrals at -74.28 and -74.12 ppm,

[0067] [Fig.7b] a time-dependent representation of 19F-{'H]-NMR signals associated with NPy-E,

[0068] [Fig.7c] a time-dependent representation of 19F-{ 'H]-NMR signals associated with Py-E under illumination at 405 nm of 250 pM NPy-E in deuterated acetonitrile: 20mM pH = 8.5 Tris buffer (9:1) (v:v) at 5.4 103 Em 2.s 1 of light intensity;

[0069] [Fig. 8] a graphical representation of the evolution over time of the normalized transmittance at 470 nm (circles) and fluorescence emission at 535 nm (disks), under constant illumination at 470 nm of 10 pM of Dronpa-2 in 10 mM Tris / 140 mM NaCl buffer pH 7.4 to 5.4 103 E.m2.s 1 luminous intensity, Markers: experimental data; solid lines: monoexponential fits with the SI equation;

[0070] [Fig.9a] a longitudinal cross-sectional view of a system according to a second configuration of the first embodiment of the invention;

[0071] [Fig.9b] a cross-sectional view of a system according to [Fig.9a];

[0072] [Fig. 10a] a graphical representation of the time response of normalized fluorescence on the sample area when internal (solid line) or external (dotted line) illumination is applied when 470 nm illumination for fluorescence imaging is implemented for the second configuration of a system according to the invention;

[0073] [Fig. 10b] a map representing the characteristic time during interior illumination;

[0074] [Fig. 10c] a map representing the characteristic time during illumination from the additional external adjacent compartment. The light intensity in Figures 10a, 10b, and 10c is adjusted to obtain equal characteristic time values ​​from the mono-exponential fitting of the curves in a. Here, 23 ± 0.5 and 21 ± 0.3 s were obtained respectively for internal illumination in [Fig. 10b] and external illumination in [Fig. 10c]. Scale: 10 mm in [Fig. 10b], c. T = 293 K. Xexc = 470 nm; / .cm = 535 nm.

[0075] [Fig. 1Od] a representation of the inverse of the constant time characteristic averaged along the radial axis with respect to the z-coordinate of the longitudinal axis for illumination from the inner adjacent compartment (solid line) or from the outer adjacent compartment (solid line),

[0076] [Fig. 11] a perspective view of a system according to a second embodiment of the invention;

[0077] [Fig. 12] a perspective view of a variant of the system of the second embodiment of the invention;

[0078] [Fig. 13] A graphical representation of the HPLC monitoring of the photochemical reaction and the photochemical yield of the phototransformation of NPy-E to Py-E after illumination of an NPy-E solution at 405 nm. Time evolution of the composition of 2 pM NPy-E in 2 mM pH = 8.4 Trizma buffer / ACN (1 / 9; v / v) under light at 405 nm with an intensity of 6.8 × 10⁴ Em 2.s⁻¹. Markers: caged concentrations of pyranine NPy-E (squares) and photoliberated concentrations of pyranine Py-E (circles) extracted from the peak areas in the HPLC chromatograms; solid lines: mono-exponential fit giving 5592 and 4417 s⁻¹ for the characteristic times of NPy-E and Py-E, respectively. T = 293 K Examples

[0079] Example 1

[0080] The system 1, according to a first configuration of the first embodiment of the invention, comprises a receiving compartment 11 for a sample E of a photoreactive medium having a zone ZI in which the sample E is received. This receiving compartment 11 is delimited between two walls 12, 13, preferably coaxial and of circular cross-section. In the example shown, these two walls 12, 13 are concentric and define, for one, a compartment with a first diameter DI and for the other, a compartment with a second diameter D2 greater than the first diameter D1. The space between the two walls 12, 13 forms the receiving compartment 11 (see [Fig. 3]).

[0081] In a preferred embodiment, these two walls 12, 13 are formed of two tubes 12a and 13a of different diameter, one 12a of a first diameter Dl being mounted in the other 13a which has the second diameter D2 greater than the first diameter DL. Such tubes of the NMR tube type have undergone a stripping of the lower part over 4 cm using 400 grit silicon carbide sandpaper until the surface is rough and visually uniform.

[0082] The space delimited by the wall 12 of the tube 12a defines a compartment 14 adjacent to the sample E receiving compartment 11. This compartment 14 forms an illumination compartment comprising a fluid medium such as a liquid like deuterated dimethyl sulfoxide (DMSO), constituting a means of guiding the light emitted by a light source. The refractive index of DMSO is 1.48, which is very close to 1.5, the refractive index of the glass constituting the walls 12, 13 of the tubes 12a, 13a.

[0083] The liquid forms a liquid tube 15 which constitutes the means for guiding the light emitted from a light source 2 towards at least one area Z2 of the wall 12 which separates the two compartments 11 and 14. The compartment 14 also includes means for diffusing the light guided by the liquid tube 15 towards the area ZI of the compartment 11 in which the sample E is located.

[0084] As can be seen in [Fig.1], the two tubes 12a, 13a can be held by centering rings 16a, 16b, allowing the two tubes 12a, 13a to be held apart from each other to form the receiving compartment 11 of the sample E.

[0085] The light source 2 consists of a laser or at least one light-emitting diode (LED), to which is connected an optical fiber 21, for example 4 m long. The end of the optical fiber 21 is inserted into the compartment 14, referred to as the illumination compartment, and immersed in the liquid tube 15.

[0086] The wall 12 of the tube 12a includes a zone Z2 configured to allow lateral diffusion of light through the wall 12 towards the zone ZI of the receiving compartment 11 in which the sample E is located. This diffusion of light is carried out in a radial direction R from the illumination compartment 14 to the receiving compartment 11. The outer face of the wall 12, in this case the outer surface of the tube 12a, is a rough surface, preferably frosted, for example by sandblasting, on the area Z2 at the level of the area ZI in which the sample E is located in the receiving compartment 11.

[0087] In this first configuration, the width of the annular receiving compartment 11 provided between the two tubes 12a, 13a and therefore the thickness of the sample E allows to define an absorbance of the photo-reactive medium which allows to illuminate it in a uniform manner which limits the degradation of the light intensity on the radial axis.

[0088] Thus, tube 12a has a diameter DI of 3 mm, the external face of which is frosted over a height of 40 mm, corresponding to the NMR measurement area, and tube 13a has a diameter D2 of 5 mm. The width of the receiving compartment 11 is therefore 0.5 mm.

[0089] An absorbance of less than 0.2 is obtained at the excitation wavelength along the radial optical path. The factor 0.2 is the maximum absorbance value for which this configuration provides uniform illumination. Therefore, the concentration must be adjusted to meet this criterion based on the concentration and the molar absorption coefficient of the sample.

[0090] Quantitative characterization of the first configuration This first configuration was characterized quantitatively, using fluorescence absorption and emission spectroscopies, and NMR spectroscopy, to account for the illumination of a sample of photo-reactive medium contained in the receiving compartment 11 illuminated using a liquid with a refractive index equal to that of the glass constituting the tube 12a.

[0091] The analysis of the light intensity distribution of this configuration is based on the use of a fluorescent actinometer. Thus, a reversibly photoreactive molecule such as a fluorescent protein like Dronpa-2 is used, which changes from bright to dark under illumination at 470 nm and recovers its initial fluorescence under illumination at 405 nm (cf. Lahlou, A., Tehrani, HS, Coghill, I., Shpinov, Y., Mandai, M., Plamont, MA,... & Jullien, L. (2023). Fluorescence to measure light intensity. Nature Methods, 1-9.).

[0092] To this end, a 10 pM solution of Dronpa-2 in 10 mM Tris / 140 mM NaCl buffer at pH 7.4 is introduced into the receiving compartment 11 delimited between tubes 12a and 13a. It exhibits absorbances of 0.02 (at 470 nm [Fig. 4a]) and 0.03 (at 405 nm [Fig. 5a]) along the radial optical path R, respectively for the bright and dark states. The time evolution of the emission of Fluorescence at 535 nm was captured by a camera under sequential light input to illumination compartment 14 (liquid tube 15) with constant light at 470 nm ([Fig. 4b]) and 405 nm ([Fig. 5b]), respectively. The images were processed to extract the characteristic time of fluorescence change at each pixel of the sample area from a mono-exponential fit, Figures 4c and 5c.

[0093] The time map was converted into a light intensity map using the photoconversion cross sections of the Dronpa-2 at 470 nm ([Fig. 4d]) and 405 nm ([Fig. 5d]). This experiment was repeated over a range of constant light intensities at 470 and 405 nm. As shown in [Fig. 6], which illustrates the light intensity at 470 nm (square) and 405 nm (circle) as a function of the light power, the recovered light intensity depends linearly on the light power injected into the illumination compartment 14.

[0094] Figure 6 does not allow for the direct acquisition of the 3D distribution of light intensity in the system, since the signal from each image pixel integrates the contributions of multiple rays originating from various locations within the illuminated sample. To further process this data, an optical simulation of the experimental setup is used. The optical simulation was performed for both system configurations according to the first embodiment of the invention, and ray tracing simulations were carried out with them, using the non-sequential mode of the OpticStudio 18.9 optical design software (Zemax LLC, Kirkland, WA, USA).

[0095] A satisfactory agreement is obtained between the experimental and calculated images recorded by the camera, as illustrated in [Fig. 4e]. It is thus possible to collect the actual distribution of light intensity in the illuminated sample ([Fig. 4f]), which is robust with respect to the position of the light injection in the illuminated sample.

[0096] The extracted maps suggest that the light intensity is essentially uniform along the principal axis and the radius in the illuminated sample, with small deviations of approximately 30% of the average value over the entire tube, along the longitudinal axis and the radius, respectively. Therefore, this first variant of the first embodiment of the invention exhibits essentially uniform 3D illumination along the principal axis and the radius of the illuminated sample up to 1.2 x 10² and 5.4 x 10³ Em² s⁻¹ at 470 and 405 nm, respectively.

[0097] Optical simulation shows that 90% of the injected LED light was delivered to the rough (frosted) surface of the tube on its exterior during sample illumination. This estimate was confirmed by dividing the photon flux delivered to the frosted element of the inner tube 12a (as calculated by integrating the surface photon flux recovered with the Dronpa-2 actinometer) by the photon flux delivered to the frosted element of the inner tube 12a. on the lateral surface) by the photon flux delivered at the end of the optical fiber (estimated with a power sensor). This measurement was performed by injecting light at 470 nm with a power of 111 mW, which increases the photon flux to 4.2 x 10⁶ mol·s⁻¹ for the entire photon flux delivered laterally, assuming uniform delivery of the scattered photons on the rough external surface Z₂ of the 3 mm tube 12a. It was thus shown that the photon budget was close to optimal, at nearly 90% of photons.

[0098] The characterization of this first configuration having been demonstrated, it was used to carry out a series of photo-conversion studies with NMR spectrometry.

[0099] Photo-conversion study with the first configuration of the first embodiment of a system according to the invention

[0100] It is first verified that the light intensity measured by fluorescence imaging is consistent with a measurement made by NMR actinometry. An irreversible actinometer is thus developed for 19F-NMR spectroscopy, to be used in the regime of the highest accessible light intensities: caged NPy-E pyranine containing fluorine atoms.

[0101] Measurement Under constant illumination at 405 nm, of 250 pM NPy-E in deuterated acetonitrile: 20 mM pH = 8.5 Tris buffer (9:1) (v:v). (0.22 absorbance at 405 nm along the radial optical path), the 19F-{'H] NMR peaks of NPy-E at -74.22, -74.26 and -74.28 were replaced by peaks at -74.12, -74.18 and +74.22 from photo-released pyranine (Py-E) ([Fig.7a]). From the mono-exponential fitting of the integral of the 19F-{1H} NMR peaks at -74.28 and -74.12 ppm respectively associated with NPy-E and Py-E ([Fig. 7b]), 650 ± 51 s of identical release time were recovered. These were converted into luminous intensity using 0.29 m².mol⁻¹ as the measured value of its decaking cross-section at 405 nm. Thus, (5.9 ± 0.5) × 10³ E.m².s⁻¹ of luminous intensity was extracted from the NMR measurement, in agreement with the 5.4 × 10³ E.m².s⁻¹ recovered from the fluorescence imaging of Dronpa-2.

[0102] In this first configuration, the illumination compartment or liquid tube 15 supplies the sample E with excitation light. However, it also guides the backscattered excitation light, which relates to the transmittance of the sample and any luminescence emitted backscattered from the illuminated sample.

[0103] It is then possible to immerse a second optical fiber acting as a sensor in the liquid tube 15 by placing its end above the end of the illumination optical fiber and connecting its other end to a detector of light. We can then record the evolution over time of the transmittance and luminescence of the illuminated sample.

[0104] (i) 10 pM Dronpa-2 in 10 mM Tris / 140 mM NaCl buffer pH 7.4 under constant illumination at 470 nm. [Fig. 8] shows the increase in transmittance at 405 nm and the decrease in fluorescence at 535 nm. These changes are consistent with a higher absorbance and the disappearance of the brightness of Dronpa-2 in the dark state. We extracted tT = 1.3 ± 0.1 s and tF = 1.05 ± 0.05 s, values ​​consistent with the time characteristic of the mono-exponential fit. Then, we exploited the photoconversion cross section of Dronpa-2 at 470 nm to recover (5.0 ± 0.5)403 E.m2.s 1 for the light intensity which is in agreement with the applied 5.5403 E.m2.s 1;

[0105] This first configuration of the first embodiment of the invention is therefore validated as suitable for providing uniform illumination with an optimal photon budget in a light-absorbing solution provided that its absorbance along the radial optical path remains less than 0.2.

[0106] To overcome this limitation, it is proposed to implement a second configuration in order to allow light delivery from both sides of the sample.

[0107] Example 2

[0108] Thus, in figures 9a and 9b, this second configuration of the system is represented according to the first embodiment with which a photo-reactive medium of higher absorbance can be illuminated uniformly.

[0109] The system described in Figures 1 and 2 is thus surrounded by a third concentric wall 17 formed by a third outer tube 17a with a diameter D3 greater than the diameter D2 of the tube 13a. This third wall 17 defines an additional compartment 18 adjacent to the receiving compartment 11, said compartment 18 comprising a liquid such as deuterated dimethyl sulfoxide (DMSO) constituting means for guiding the light emitted by an optical fiber 21a.

[0110] The external surface of tube 17a is frosted in the same area Z3 as the frosted area Z2 of tube 12a. The space between the second tube 13a and the third tube 17a is filled with light from an optical fiber 21a connected to a light source, the end of the optical fiber 21a being positioned in the liquid element, above area Z3. This additional adjacent compartment, referred to as the illumination compartment 18, may also include additional optical fibers that allow for the recovery of absorbance and luminescence, for example, fluorescence.

[0111] As can be seen in [Fig. 1], the two tubes 12a, 13a can be held by centering rings 16a, 16b, allowing the two tubes 12a, 13a to be held spaced apart to form the sample receiving compartment 11 E, and visible on [Fig.9a], a centering ring 16c allows the two tubes 13a, 17a to be held together, spaced apart to form compartment 18.

[0112] In this embodiment, the decrease in light intensity from the lateral illumination of the so-called illumination compartment 14 is counterbalanced by the light guiding means (liquid of compartment 18) on the outer face of the receiving compartment 11 so that the photo-reactive sample is exposed to uniform illumination in 3 dimensions.

[0113] Thus, according to this second configuration of the present invention, it is possible to obtain uniform illumination by immersing the liquid-filled cylindrical tube 15, acting as a light-guiding means, in the light-absorbing solution and then immersing the assembly in a new liquid-filled cylindrical tube. When the absorption power of this solution along the radial optical path is greater than 0.2, this embodiment is found to be effective in creating uniform illumination over approximately 3.5 cm along the longitudinal axis of the tube 12a and over approximately 0.55 mm along the radial optical path.

[0114] This embodiment of the invention is therefore particularly advantageous, the illumination being applied not only from the core of the light-absorbing solution which constitutes the sample to be analyzed, by NMR for example, but also from the outer surface of the sample.

[0115] This second configuration of the first embodiment of the system according to the invention is evaluated.

[0116] Quantitative characterization of the second configuration

[0117] To characterize the distribution of the light intensity of this second configuration of the system according to the invention, we rely on the imaging of the temporal evolution of the fluorescence emitted at 560 nm during the photo-conversion of a solution of Dronpa-2 and an additional non-fluorescent dye acting as a strong light absorber at an excitation wavelength of 470 nm.

[0118] Thus, 230 pL of 10 pM Dronpa-2 and 525 pM ethyl orange in PBS at pH=7.4 are introduced into the receiving compartment between tubes 12a and 13a, the solution exhibiting an absorbance of 0.65 along the radial optical path.

[0119] In a first step, this sample is illuminated at maximum power under a 470 nm light delivered from the outer tube 17a only, and the average fluorescence over the sample area is adjusted monoexponentially as above to recover the time characteristic rout. In a second step, the sample is illuminated under a 470 nm light delivered from the inner tube 12a only, and the power is adjusted until the time characteristic rin extracted by the monoexponential treatment of the evolution of the average fluorescence as a function of time is close to rout. This light The internally injected fluorescence is maintained for subsequent experiments. Finally, the fluorescence evolution over time is studied by imaging during illumination of the sample from both sides at power values ​​identified during the first and second steps. The temporal evolution of the fluorescence signal as a function of external, internal, and dual illumination is then processed as before to extract an image of the characteristic time values ​​as well as a z-axis profile of the light intensity.

[0120] Figures 11 and 12 show a second embodiment of the system according to the invention, for forming photobioreactors.

[0121] Thus, the system comprises a parallelepiped-shaped receptacle having a base 101 from which extend two lateral walls 102, between which extend at least three parallel walls 103, 104, 105 transversely. Between two successive walls 103, 104, a receiving compartment 110 for a photoreactive medium is defined. An adjacent compartment 111 is then defined between one 104 of the walls of the receiving compartment 110 and the last wall 105. This adjacent compartment 111 comprises a liquid as a means of guiding the light emitted by a light source, thus forming a liquid light-guiding plate. The light is thus guided towards at least one area of ​​the adjacent compartment 111 opposite the area of ​​the receiving compartment 110 in which the photoreactive medium is located.This area of ​​the adjacent compartment 110 also includes the light diffusion means arranged to diffuse light through the wall 104 separating the two compartments 110, 111 towards the area of ​​the receiving compartment 110 in which the photoreactive medium is located.

[0122] According to the variant shown in [Fig.1 1], the system includes a fourth parallel transverse wall 106 delimiting with the other wall 103 of the receiving compartment 110 an additional adjacent compartment 112.

[0123] This additional adjacent compartment 112 contains a liquid as a means of guiding the light emitted by a light source, towards at least one area of ​​the additional adjacent compartment 112 opposite the area of ​​the receiving compartment 110 in which the photo-reactive medium is located, area of ​​the additional adjacent compartment in which are the light diffusion means arranged to diffuse the light through the wall 103 separating the two compartments 110, 112 towards the area of ​​the receiving compartment 110 in which the photo-reactive medium is located, the receiving compartment 110 of the photo-reactive medium being interposed between the two compartments 111 and 112 comprising respectively a liquid as a means of guiding.

[0124] The light source(s) are positioned upstream of light guiding means.

[0125] For larger photoreactors, LEDs can be placed to shine directly into the tube or liquid plate and not lose light due to the inefficiency of LED-fibre optic coupling with which a third of the light is lost.

[0126] Materials and methods used

[0127] Synthesis of PyE and NPy-E:

[0128] The synthesis of the PyE and NPy-E compounds was carried out according to the following schemes: [CHEM1] NPy-S 3 [Chem. 2] NFy-E

[0130] 4,5-Dimethoxy-2-nitrobenzyl bromide and trisodium 8-hydroxypyrene-1,3,6-trisulfonate sold by Acros Organics are used. ¹H and ¹³C NMR spectra were recorded at 300 K on a Bruker AM 300 spectrometer; chemical shifts are reported in ppm with protonated solvent as internal reference (¹H, CHD2SOCD3 in CD3SOCD3 2.50 ppm, CHD2COCD3 in CD3COCD3 2.05 ppm; ¹³C, ¹³CD3SOCD3 in CD3SOCD3 39.5 ppm, ¹³CD3COCD3 in CD3COCD3 29.9 ppm), ¹⁹F, C7H5F3 in CHD2SOCD3 -63.7 ppm.

[0131] Mass spectra (chemical ionization and electron impact with NH3 or CH4) were obtained by the Mass Spectrometry Department of Chimie ParisTech, and high-resolution mass spectra by the Mass Spectrometry Department of the Institute of Organic and Analytical Chemistry (Orléans). Column chromatography was performed on silica gel 60 (0.040–0.063 nm; Merck). Analytical thin-layer chromatography (TLC) was performed on plates pre-coated with Merck silica gel 60 F254.

[0132] Compound 1: Trisodium 8-acetoxypyrene-L3,6-trisulfonic acid Trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, 2.28 g, 4.35 mmol) and sodium acetate (35.7 mg, 0.44 mmol) were suspended in acetic anhydride (25 mL) and refluxed for 35 h. The suspension was cooled to room temperature, diluted with tetrahydrofuran (THF), and filtered. The solid was washed with acetone and dried under vacuum, yielding a gray powder (2.26 g, 92%). 'H NMR (300 MHz, DMSO-d6): ô 9.23 (1H, d, J = 10 Hz), 9.15 (1H, d, J = 10 Hz), 9.10 (1H, d, J = 10 Hz), 9.07 (1H, s), 8.26 (1H, s), 8.11 (1H, d, J = 10 Hz), 2.56 (3H, s).

[0133] Compound 2: 8-Acetoxypyrene-L3,6-Trisulfonyl chloride Compound 1 (2.5 g, 4.5 mmol) was suspended in thionyl chloride (20 mL) and 10 drops of anhydrous dimethylformamide were added. The mixture The resulting mixture was stirred at 76°C for 5 h. The reaction mixture was then cooled to room temperature and poured into ice with vigorous stirring. A yellow / orange precipitate was obtained with effervescence of SO2 gas. The crude precipitate was filtered and dried under reduced pressure. Compound 2 was obtained as a yellow / orange powder and used without purification for the next step.

[0134] Compound Py-E: Tris(2,2,2-trifluoroethyl) 8-hydroxypyrene-L3,6-trisulfonate. To a solution of trifluoroethanol (1.3 mL, 18 mmol) in anhydrous dichloromethane (35 mL), anhydrous triethylamine (4.6 mL, 33 mmol) is added and cooled to 0°C in an ice bath. The solution of compound 2 (1.3 g, 2.3 mmol) in anhydrous dichloromethane (120 mL) is added dropwise at 0°C. The resulting mixture is then gently heated to room temperature and stirred under an inert atmosphere for 40 h. The organic phase is then separated and extracted with 1 M HCl (x3) and saturated with NaCl solution (x2). The crude residue is obtained by evaporating the dried organic phase over Na2SO4 under reduced pressure. It is purified by flash chromatography using n-pentane / ethyl acetate (70 / 30 v / v) as the eluent, which provides the Py-E product as a dark red powder (1.2 g, 74% yield). 1 H-NMR (300 MHz, acetone-d 6): ô 9.34 (1H, s), 9.23 (1H, d, J = 10 Hz), 9.16 (1H, d, J = 10 Hz), 8.98 (1H, d, J = 10 Hz), 8.92 (1H, d, J = 10 Hz), 8.56 (1H, s), 4.89 (6H, m). 13 C NMR (75 MHz, DMSO-d 6): ô 134.3, 132.8, 132.4, 131.9, 129.3, 128.9, 125.7, 125.4, 127.9, 124.3, 120.6, 116.9 (2C, q, JCF = 276 Hz) 64.6 (2C, q, JCF = 36 Hz), 66.0, 65.5, 65.0, 64.6 (q, JCF = 36 Hz). 19 F NMR (282 MHz, DMSO-d 6): ô -75.1 to -75.2 (m, 3CF3). MS (ESI): m / z cal, for C22Hi3F9OioS3: 702.94, found: 702.84 [MH]

[0135] Compound 3: 8-(4.5-Dimethoxy-2-nitrobenzyloxy)pyrene-L3.6-trisulfonyl chloride The NPy-S compound described in the publication M. Mandai, H. Sepasi. Tehrani, Q. Mai, E. Simon, M.-A. Plamont, C. Rampon, S. Vriz, I. Aujard, T. Le Saux, L. Jullien, Chem. Scz. 2023, 14, 13799-13811) (2.5 g, 3.5 mmol) is suspended in thionyl chloride (20 mL) and 10 drops of anhydrous dimethylformamide are added. The resulting mixture is stirred at 76°C for 5 h. The reaction mixture is then cooled to room temperature and poured into ice with vigorous stirring. A yellow / orange precipitate was obtained with effervescence of SO2 gas. The crude precipitate was filtered and dried under reduced pressure. Compound 3 is obtained in the form of a yellow / orange powder and used without purification for the next step.

[0136] NPy-E compound: Tris(2,2,2-trifluoroethyl) 8-((4,5-dimethoxy-2-nitrobenzyl)oxy)p yrene-1,3.6-trisulfonate To a solution of trifluoroethanol (0.8 mL, 11 mmol) in anhydrous dichloromethane (25 mL), anhydrous triethylamine (2.8 mL, 20 mmol) is added and cooled to 0°C in an ice bath. The solution of compound 3 (1.0 g, 1.4 mmol) in anhydrous dichloromethane (120 mL) is then added dropwise at 0°C. The resulting mixture is then gently heated to room temperature and stirred under an inert atmosphere for 40 h. The crude residue is obtained by adding n-pentane to the reaction mixture. The crude residue is then purified by washing with n-pentane / DCM (60 / 40 v / v; 3 x 30 mL), yielding NPy-E as a bright yellow powder (1.05 g, 83% yield). 1 H-NMR (300 MHz, DMSO-d 6): ô 9.27 (1H, s), 9.21 (1H, d, J = 10 Hz), 9.11 (1H, d, J = 10 Hz), 9.01 (1H, d, J = 10 Hz), 8.94 (1H, d, J = 10 Hz), 8.68 (1H, s), 7.82 (1H, s), 7.63 (1H, s), 6.10 (2H, s), 5.07 (6H, m), 4.01 (3H, s), 3.93 (3H, s). 13 C NMR (75 MHz, DMSO-d 6): ô 154.6, 153.3, 148.31, 140.0, 133.3, 132.4, 131.8, 131.7, 129.2, 127.9, 124.2, 120.5, 116.8 (2C, q, JCF = 276 Hz), 127.8, 124.2, 120.5, 116.8 (IC, q, JCF = 276 Hz), 127.7, 125.9, 125.7, 125.5, 125.0, 124.9, 124.3, 123.0, 122.9, 120.5, 114.6, 111.8, 108.5, 68.8, 66.2, 65.7, 65.2, 64.7 (3C, q, JCF = 36 Hz), 56.4, 56.2. 19 F NMR (282 MHz, DMSO-d 6): ô -75.2 to -75.4 (m, 3CF3). MS (CI, NH 3): m / z cal, for [M+NH4]+: 917.04, found: 917.18 [M+NH4]+. Instruments

[0137] Fluorescence and absorption spectrometer for TUV / Vis. UV / Vis absorption spectra were recorded on a UV / Vis spectrophotometer (Cary 300 UV-Vis, Agilent Technologies, Santa Clara, CA) at 293 K equipped with a 1x1 Peltier thermostatic cell holder (Agilent Technologies). Samples were contained in either 1 cm x 1 cm (3 mL; cuvette contents were agitated), 0.3 cm x 0.3 cm (54 qL; cuvette contents were not agitated), or 0.15 x 0.15 cm (23 qL; cuvette contents were not agitated) quartz cuvettes (Hellma Optics, Jena, Germany).

[0138] NMR Spectrometer NMR measurements were performed using a Bruker Ascend 500WB probe. Chemical shifts of 19F were measured using the instruments' internal calibration. Spectra were processed using MestReNova software.

[0139] Light source: A Thorlabs M405L4 LED and a Lumileds Luxeon Rubix L1RX-BLU 1000000000 LED are mounted on a star-shaped printed circuit board supported by a heat sink and are used as light sources at 405 and 470 nm, respectively. Multimode fiber optics with a pitch index of 0-1000 µm (NA=0.50), available from Thorlabs, are used.

[0140] A Thorlabs PM100A power sensor with S130VC photodiode detector is used to measure light power, with a NE06A OD=0.6 neutral density filter when the light intensity exceeds the instrument limits.

[0141] Measurement of transmittance and emitted fluorescence An optical fiber is used as a sensor with its end immersed in the liquid light guide, approximately 0.5 cm above the end of the illumination fiber. The other end of the "sensor" fiber is connected to an Fl 10-SMA-532 lens. The light extracted from the fiber is directed onto an FF-506-Di03 dichroic mirror. The resulting transmission and fluorescence beams are filtered with 535 / 20 nm and 405 / 20 nm filters, respectively, before being focused with a 16 mm lens (available as ACL25416U-A, Thorlabs) onto the sensor of the photodetector modules (AFBR-S4KTIA3315B, Broadcom).

[0142] Imaging Fluorescence imaging was performed using a uEye UL3080CP IDS camera equipped with a 535 / 20 nm filter and a 50 mm f / 1.8 AF Nikkor lens. Exposure times ranged from 1 to 200 ms depending on the fluorescence intensity and photoactivation kinetics. Images were recorded at a resolution of 2456 x 2054 and then reduced by a factor of 2 during processing. For light intensity extraction, the image area corresponding to the sample was selected.

[0143] Data processing:

[0144] The evolution over time of the recorded signal S(t) (fluorescence level, HPLC peak integral, and NMR peak integral) are treated: either with a mono-exponential function according to the following equation: (SI) S(t)=S(oo)+Axexp(-t / r) or with a bi-exponential function according to the following equation: (S2) S(t)=S(oo)+AiXexp(-t / Ti)+A2Xexp(-t / T2) in order to recover the characteristic time r which is then used either to recover the photoactivation cross-section o with the following equation: (S3) a= (|-kA) / l| either to retrieve the desired light intensity I to be used in the equation: (S4) I=0-kA) / o when the cross section o associated with the photochemical process considered and the rate constant associated with the thermal return of the photoactivated state kA (here non-zero only in the case of reversibly photoactivatable species Dronpa-2) are known.

[0145] Photochemical experiments

[0146] Implementation of fluorescence imaging to characterize the first configuration of the first embodiment of the invention. Protocol: 230 pL of lOpM Dronpa-2 in 10 mM Tris / 140 mM NaCl buffer at pH 7.4 are introduced into the receiving compartment 11 located between the coaxial tubes 12a and 13a. The temporal evolution of its fluorescence emission at 535 nm was recorded using a camera during the sequential application: (i) of a constant 470 nm light injected into the liquid tube 15, followed by a reset to the initial state from a constant 405 nm light with another LED and (ii) a constant 405 nm light from the liquid tube feed, followed by the external application of a constant 470 nm light with another LED. The film images were processed to extract the relaxation time of the fluorescence change at each pixel of the sample area by monoexponential fitting with the SI equation. The resulting map was then converted into a light intensity map. The photon efficiency of the first configuration can be evaluated experimentally from the ratio of the integral of the lateral photon flux delivered to the rough area Z2 of the 3 mm inner tube 12a (as measured by the Dronpa-2 actinometer) to the photon flux supplied to the end of the optical fiber (measured with a power sensor). This measurement was performed by injecting 470 nm of light at 0.111 W of power, which is equivalent to a photon flux of 4.2 × 10⁶ words*. Under the final illumination conditions, a photon flux density of 1.2 × 10² mol m² s⁻¹ was measured for the laterally scattered light, assuming uniform delivery of scattered photons onto the rough outer surface Z₂ of the 3 mm inner tube 12a (r = 1.5 mm, h = 3.5 cm). Therefore, over 90% of the photon efficiency of the setup was recovered.

[0147] Characterization of the photochemical properties of NP y -E HPLC. 3 mL of 2 µM Npy-E in 2 mM pH 8.4 Trizma buffer / acetonitrile 1 / 9 (v / v) in a 1 x 1 cm² quartz cuvette under stirring are subjected to constant illumination at 405 nm, calibrated to 6.8 x 10-1O4E·m2·s-1 with the Npy-Ad actinometer. 30 µL aliquots are removed at regular intervals and then analyzed by high-performance liquid chromatography using an Agilent 1260 Infinity LC system equipped with an autosampler and a diode array detector ([Fig. 13]). Chromatographic separations are performed using an Atlantis T3 column (Waters Corp.), percolated at 0.7 mL / min and temperature-controlled at 25 °C. The detection of caged or decaged pyranines is carried out at 400 and 530 nm respectively. 15 qL are injected into the chromatographic system. The compounds are eluted with a mobile phase composed of two solvents A (methanol) and B (ammonium acetate buffer, pH 5).5) Using a gradient to optimize analyte separation. Initially, the column is equilibrated with a mobile phase consisting of 70% A and 30% B. Six minutes after injection, the proportion of A is linearly increased to 95% and two minutes later to 98%. After this step, the mobile phase composition is fixed at the initial condition in 0.5 min, and the column is equilibrated for 7.5 min before the next injection. The retention times for caged pyranine (NPy-E) and free pyranine (Py-E) are 7.4 and 4.3 min, respectively, under these experimental conditions. We measured 5592 and 4417 s for the characteristic times recovered from the time evolution of the peak integrals of NPy-E and Py-E in the HPLC chromatograms, respectively. Using equation S3 with kA = 0, we subsequently extracted 0.29 and 0.03 m²·mol⁻¹ for the cross-section of NPy-E with photon excitation at 405 nm.

Claims

1.

2. Demands System (1) for receiving and illuminating a photoreactive medium, said system comprising: at least one light source (2), at least one receiving compartment (11) having at least one zone (Zl) in which the photo-reactive medium is located, means for guiding the light emitted by the light source (2) and means for diffusing said light, characterized in that the at least one receiving compartment (11,110) is delimited between two walls (12, 13; 103, 104), the system (1) comprising at least one compartment adjacent (14; 111) to the receiving compartment (11,110) of the reactive medium, separated from it by one of the walls (13; 104) delimiting the receiving compartment (11, 110), the adjacent compartment (14;111) to the receiving compartment (11, 110) containing a fluid medium as means for guiding the light emitted by the light source (2), to guide the light towards at least one area of ​​the compartment (Z2) adjacent to the area (Z1) of the receiving compartment (11, 110) in which the photo-reactive medium is located, area (Z2) of the adjacent compartment (14, 111) in which are further light diffusion means arranged to diffuse the light through the wall (12; 104) separating the two compartments towards the area (Z1) of the receiving compartment (11; 110) in which the photo-reactive medium is located.; A system according to claim 1, characterized in that it comprises an additional adjacent compartment (18; 112) to the receiving compartment (11; 110) of the photoreactive medium, separated from it by the other wall (13; 103) delimiting the receiving compartment (11, 110) of the photoreactive medium, the additional adjacent compartment (18; 112) to the receiving compartment (11, 110) of the photoreactive medium containing a fluid medium as means for guiding the light emitted by a light source towards at least one zone (Z3) of the additional adjacent compartment (18; 112) opposite the zone (Z1) of the receiving compartment (11, 110) in which the photoreactive medium is located, zone (Z3) of the additional adjacent compartment (18; 112) in which are light-diffusion means arranged to diffuse light through the wall (13; 103) separating the two compartments towards the area of ​​the receiving compartment in which the photo-reactive medium is located, the receiving compartment (11; 110) of the photo-reactive medium being intercalated between the two compartments adjacent to it, each comprising a fluid as guiding means and means for diffusing light.

3. System according to any one of claims 1 or 2, characterized in that the receiving compartment (11) of the photo-reactive medium is defined between two coaxial cylindrical walls (12; 13), one (12) inner of smaller cross-section than the other called outer (13), the inner wall (12) defining the adjacent compartment (14) containing the fluid medium as means of guiding the light emitted by the light source and means of diffusing the light.

4. System according to claim 3, characterized in that it comprises a third coaxial cylindrical wall (17) of larger cross-section forming the additional adjacent compartment (18).

5. System according to any one of claims 3 and 4, characterized in that the system comprises at least two tubes (12a, 13a) having a closed end, one of which has a diameter smaller than the diameter of the other, the tube (12a) of smaller diameter, referred to as the inner tube, being housed concentrically in the tube (13a) of larger diameter, referred to as the outer tube, the receiving compartment (11) of the photo-reactive medium being formed by the space between the walls of the tubes (12a, 13a), the adjacent compartment (14) being delimited by the inner tube (13a) and comprising a fluid medium as means for guiding the light emitted by the light source and means for diffusing the light.

6. A system according to claim 5, characterized in that it comprises an additional tube (17a) in which the two inner (12a) and outer (13a) tubes are mounted, said additional tube (17a) defining with the outer tube (13a) an additional adjacent compartment (18) to the receiving compartment (11) of the photoreactive medium, said additional adjacent compartment (18) comprising a fluid medium as means for guiding the light emitted by a light source towards at least one zone (Z3) of the additional adjacent compartment (18) opposite

7.

8. the zone (Zl) of the receiving compartment (11) in which the photo-reactive medium is located, zone (Z3) of the additional adjacent compartment (18) in which the light diffusion means are arranged to diffuse the light through the wall (12a) separating the two compartments towards the zone (Zl) of the receiving compartment (11) in which the photo-reactive medium is located, the receiving compartment (11) of the photo-reactive medium being intercalated between the two adjacent compartments (14, 18) comprising a liquid medium as guiding means and the light diffusion means. System according to claim 1 or 2, characterized in that it comprises at least three parallel flat walls (103, 104, 105), a receiving compartment (110) for a photoreactive medium being defined between two successive walls (103, 104) and an adjacent compartment (111) being defined between one (104) of the walls of the receiving compartment (110) for a photoreactive medium and the third wall (105), said adjacent compartment (111) comprising a fluid medium as means for guiding the light emitted by a light source (2) towards at least one area of ​​the adjacent compartment (111) opposite the area of ​​the receiving compartment (110) in which the photoreactive medium is located,area of ​​the adjacent compartment (111) in which are located the light diffusion means arranged to diffuse the light through the wall (104) separating the two compartments towards the area of ​​the receiving compartment (110) in which is located the photoreactive medium. A system according to claim 7, characterized in that it comprises at least one additional adjacent compartment (112) to the receiving compartment (110) of the photoreactive medium, separated from it by the other wall (103) delimiting the receiving compartment (110) of the photoreactive medium, and defined by a fourth parallel flat wall (106), the additional adjacent compartment (112) to the receiving compartment (110) of the photoreactive medium containing a fluid medium as a means of guiding the light emitted by a light source, towards at least one area of ​​the additional adjacent compartment (112) opposite the area of ​​the receiving compartment (110) in which the photoreactive medium is located, area of ​​the additional adjacent compartment (112) in which are the light diffusion means arranged to diffuse the light through the wall separating the two compartments towards the area of ​​the receiving compartment (110) in which is the photo-reactive medium, the receiving compartment (110) of the photo-reactive medium being intercalated between the two adjacent compartments (111, 112) comprising a fluid medium as guiding means and the light diffusion means.

9. System according to any one of claims 1 to 8, characterized in that the fluid medium is a liquid medium such as dimethyl sulfoxide (DMSO).

10. System according to claim 1 to 9, characterized in that the refractive index of the fluid medium and the refractive index of the material constituting the wall between the receiving compartment (11, 110) and said at least one adjacent compartment (14, 18; 111, 112) are such that the difference between these refractive indices is less than 20%, preferably less than 10% and even more preferably the refractive indices are identical.

11. System according to claim 1 to 10, characterized in that the refractive index of the material constituting the wall (12, 13, 103, 104) between the receiving compartment (11, 111) and said at least one adjacent compartment (14, 18; 111, 112) is greater than that of the receiving compartment (11, 110), i.e. the refractive index of the photo-reactive medium.

12. System according to any one of claims 1 to 11, characterized in that the light diffusion means are made up of micro and / or nano particles suspended in the fluid medium forming the light guidance means.

13. System according to any one of claims 1 to 12, characterized in that the diffusion means are formed by a rough surface of at least one face of the wall (12, 13; 103, 104) separating the receiving compartment (11; 111) of the photo-reactive medium and an adjacent compartment (14, 18; 111; 112) comprising the light guiding means.

14. A system according to any one of claims 1 to 13, characterized in that the light source (2) consists of at least one light-emitting diode or a laser source positioned upstream of the light-guiding means, the light from which is:

15. either directly emitted to a compartment comprising a fluid medium as means of guiding the light, or delivered to said compartment comprising a fluid medium as means of guiding the light, by an optical fiber (21) connected to the light source (2) at one end and the other end of which is immersed in the fluid medium to allow the propagation of light in the fluid medium. System according to any one of claims 1 to 14, characterized in that the fluid medium as light guiding means is suitable for transmitting the absorbance and / or luminescence of the photo-reactive medium, the system comprising means configured to detect said absorbance and / or fluorescence, such as at least one additional optical fiber (21a), connectable to absorbance and luminescence measuring devices.

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