WO2015075209A1 - Procede de detection d'une espece fluorescente reversiblement photoconvertible - Google Patents
Procede de detection d'une espece fluorescente reversiblement photoconvertible Download PDFInfo
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- WO2015075209A1 WO2015075209A1 PCT/EP2014/075336 EP2014075336W WO2015075209A1 WO 2015075209 A1 WO2015075209 A1 WO 2015075209A1 EP 2014075336 W EP2014075336 W EP 2014075336W WO 2015075209 A1 WO2015075209 A1 WO 2015075209A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/12—Circuits of general importance; Signal processing
Definitions
- the invention relates to a method for detecting a reversibly photoconvertable fluorescent species. Such a method has many applications, in particular in chemistry, biology and in the field of environmental measurements.
- “Species” means a chemical species such as a molecule or a complex, or a physical object such as a nanoparticle.
- the term "reversibly photoconversible species” (“photoswitchable” in the English literature) means a species having at least two distinct states, having different fluorescence properties, and being able to switch from one state to another reversibly by effect. light.
- Examples of reversibly photoconvertible fluorescent species are the "Dronpa” protein and the "Spinach - DFHBI” complex ("Spinach” being an aptamer of RNA and DFHBI a fluorogenic probe). These species can, in particular, be used as probes or markers.
- Imaging and more particularly fluorescence microscopy, has become an indispensable tool in biology, but also in other disciplines such as materials science. Its applications, however, are limited by the ability to observe a signal of interest in a background of fluorescence or noise. This problem is particularly acute in in vivo imaging applications, in which the fluorescent markers to be detected are dispersed in a complex auto-fluorescent and / or diffusing medium; the useful signal is then embedded in an intense background noise.
- fluorescence detection and imaging techniques Another limitation of conventional fluorescence detection and imaging techniques is that many fluorophores have broad emission bands; therefore, it is difficult to selectively detect several fluorescent markers in the same sample because their emission spectra tend to be superimposed.
- SAFIRe Synchronously Amplified Fluorescence Image Recovery
- the invention aims to overcome at least one of the aforementioned drawbacks of the prior art.
- An object of the invention making it possible to achieve this goal is a method of detecting at least one reversibly photoconvertable fluorescent species, comprising the following steps:
- step c) extracting the amplitude of the intensity component of said fluorescence radiation having the same periodicity as said periodically modulated lighting light and in phase quadrature with respect to she ;
- the average intensity of said illumination light and its modulation frequency are chosen so as to maximize said amplitude of the intensity component of said fluorescence radiation.
- At least one said reversibly photoconvertible fluorescent species (P) has a first and a second chemical state, at least one of said states being fluorescent, said or each said reversibly photoconvertable fluorescent species (P) being capable of being converted from said first state to said second state; state by a first reaction photo-induced, then return to said first state at a time by a thermo-induced reaction and a second photo-induced reaction and said illumination light may have a mean intensity of 1 ° and be modulated at a frequency f with:
- said sample may be illuminated by a substantially monochromatic illumination light.
- Said illuminating light comprises a first substantially monochromatic illumination light (FEX1) of wavelength
- FEX 2 substantially monochromatic illumination light
- ⁇ 2 substantially monochromatic illumination light
- At least one said reversibly photoconvertible fluorescent species (P) has a first and a second chemical state, at least one of said states being fluorescent, said or each said reversibly photoconvertable fluorescent species (P) being capable of being converted from said first state to said first state; second state by a first photoinduced reaction, then returning to said first state by a second photoinduced reaction, and wherein said first illumination light has a mean intensity of 7 ° and is modulated at a frequency f and said second light lighting has a substantially constant intensity 7 ° with: _ _ ⁇ 12.1 + ⁇ 21.1
- ⁇ 12.2 + ⁇ 21.2 ⁇ 2, ⁇ ⁇ and are respectively the kinetic constants of said first and said second reaction photo-induced by said first illumination light; and ⁇ 2,2 ⁇ 2 and ⁇ 2 ⁇ , 2 ⁇ 2 are . respectively, the kinetic constants of said first and said second reaction photo-induced by said second illumination light.
- Said sample may contain a plurality of said reversibly photoconvertible fluorescent species having different dynamic properties, said steps a) to c) being implemented successively for the detection of at least two so-called reversibly photoconvertible fluorescent species.
- Said steps b) and c) can be implemented by synchronous detection of said fluorescence radiation.
- Said sample may contain at least one other fluorescent species.
- the method may also comprise the following step: d) determining the concentration of said or at least one said reversibly photoconvertible fluorescent species from the intensity component of said fluorescence radiation extracted during said step c).
- Said at least one said reversibly photoconvertable fluorescent species may be chosen from: a photochromic fluorescent protein; and a complex of a biomolecule, such as an aptamer or protein, with a fluorogenic probe.
- Said sample may comprise biological material.
- Another object of the invention is a fluorescence microscopy method implementing such a detection method.
- Another object of the invention is an optical remote sensing method implementing such a detection method.
- said sample may comprise a living organism, and at least one element selected from the presence and concentration of a said reversibly photoconvertible fluorescent species (P) may be measured from the intensity component of said fluorescence radiation extracted during said step c) without sampling on said living organism.
- P reversibly photoconvertible fluorescent species
- said illumination light (FEX) is emitted in one direction and said periodic modulation of said illumination light (FEX) is implemented by a modulation of said direction of emission of said illumination light (FEX).
- the invention is a method in which said lighting light (FEX) comprises a portion of the daylight and wherein said portion of the daylight contributes to the light intensity received by said daylight.
- fluorescent species reversibly photoconvertible (P) remaining less than or equal to said intensity 7 °.
- FIG. 1 illustrates the general principle of a method according to one embodiment of the invention
- FIGS. 2A and 2B are graphs illustrating how the in-phase and quadrature components of the fluorescence intensity depend on the dynamic parameters of a reversibly photoconvertable fluorescent species;
- FIGS. 3A, 3B, 3C, 3D, 3E and 3F are graphs illustrating the result of calculations of the normalized amplitude in quadrature of phase, as a function of different control parameters.
- FIG. 4 is a graph illustrating the application of a method according to one embodiment of the invention to the determination of the concentration of a reversibly photoconvertable fluorescent species
- FIGS. 5A and 5B are graphs illustrating the calculations performed to optimize quadrature detection in the case of excitation with two distinct light beams
- FIG. 6 illustrates the application of a method according to one embodiment of the invention to the detection of the reversibly photoconvertible fluorescent complex "Spinach";
- FIG. 7 illustrates the application of a method according to one embodiment of the invention to the selective imaging of a mixture comprising the reversibly photoconvertible fluorescent complex "Spinach" and a fluorophore interfering inside a device microfluidic;
- FIG. 8 illustrates the application of a method according to one embodiment of the invention to the selective imaging of a mixture comprising the reversibly photoconvertable fluorescent protein "Dronpa-2" and a fluorophore interfering within a microfluidic device;
- FIG. 9 illustrates the application of a method according to one embodiment of the invention to the selective imaging of biological material expressing the reversibly convertible fluorescent protein "Dronpa-3";
- FIG. 10 illustrates the application of a method according to one embodiment of the invention using two excitation beams to selective imaging of a mixture comprising the reversibly photoconvertable fluorescent protein "Dronpa-2" and a fluorophore interfere inside a microfluidic device;
- FIG 11 illustrates schematically the experimental apparatus used to obtain Figures 7;
- Figure 12 schematically illustrates an apparatus for use in remote sensing applications according to one embodiment of the invention
- Figure 13 schematically illustrates the experimental apparatus used to obtain Figure 10;
- Figure 14 schematically illustrates the experimental apparatus used to obtain Figure 8.
- a detection method comprises illuminating a sample E, containing a reversibly photoconvitable fluorescent species P, by a periodically modulated FEX excitation light beam.
- the modulation can also be obtained by periodically moving a lighting beam, for example in the context of observation by scanning microscopy; indeed, if we consider a localized region of a sample (for example a chamber or a duct of a microfluidic device), a periodic displacement of a lighting beam modifies the illumination in a manner analogous to a periodic modulation of the intensity of said beam.
- the excitation beam FEX is preferably substantially monochromatic, that is to say that its spectrum has a single maximum intensity, and / or a spectral width of no greater than 50 nm.
- the reversibly photoconvertable fluorescent species has two different states exchangeable under the action of light. It may be a fluorescent photochromic species, or any other system whose Dynamic behavior can be reduced to an exchange between two states under the action of light; these states may correspond to different stereochemical configurations of a molecule, to a bound / unbound state of a complex, etc.
- the first state - thermodynamically more stable - is indicated by 1 and represented by a solid square
- the second state - thermodynamically less stable - is indicated by 2 and represented by a hollow square.
- These two states have different luster. For the sake of simplicity, and by way of non-limiting example, it can be considered that only the state 1 is significantly fluorescent.
- the wavelength of the excitation beam, ⁇ allows both the excitation of the fluorescence emission of states 1 and 2, and also the conversion of state 1 to state 2 and vice versa .
- I a constant intensity
- state 1 is converted to state 2 with a kinetic constant ⁇ - ⁇ 2 ⁇ where ⁇ -12 is the photoconversion cross section of state 1 in state 2;
- state 2 is converted to state 1 with a kinetic constant ⁇ 2 ⁇ ⁇ + ⁇ ⁇ ⁇ 2 ⁇ , ⁇ 2 ⁇ being the effective photoconversion section of state 2 in state 1 and k A 2i being the kinetic constant of thermal relaxation from state 2 to state 1.
- the present inventors have realized that it is possible to choose the average intensity 1 ° of the illumination beam and its modulation frequency f so as to maximize the amplitude of the quadrature component of the fluorescence radiation.
- the optimal values of the parameters l ° and f which maximize this amplitude depend on the parameters ⁇ - ⁇ 2 , k A 2i and the fluorescent species reversibly photoconvertible considered.
- an excitation beam optimizing the amplitude of the quadrature component of the emission of a reversibly photoconvertable fluorescent species target will not optimize that of other fluorescent species (reversibly photoconvertible or not) that may be present in the sample. This gives a selective detection by dynamic contrast.
- an advantageous characteristic of the invention is that the optimum values of the parameters l ° and f can be calculated from the dynamic properties of the species to be detected, and more particularly from ⁇ - ⁇ 2, ⁇ 2 ⁇ and k A 2 i . It is therefore not necessary to resort to iterative optimization, by tests, as in certain techniques of the prior art. On the other hand, the optimum values of 1 ° and f can be determined analytically.
- Another advantage of the invention is that the selective detection can be quantitative.
- a calibration makes it possible to determine the concentration of the reversibly photoconvertable target fluorescent species from the magnitude of the quadrature component of the fluorescent intensity.
- the extraction of the amplitude of the fluorescent emission quadrature component does not pose any particular difficulty. It can be done, for example, by synchronous detection ("lock-in" in English) or by analysis of the Fourier transform of the fluorescence intensity.
- the modulation of the excitation beam can be obtained by known methods, for example the direct modulation of a radiation source or the use of a light, electro-optical or mechanical modulator.
- a single illumination beam - or a plurality of beams of the same wavelength and even modulation, or even diffuse monochromatic illumination - can be used.
- Qi and Q 2 are the molecular luster states 1 and 2 respectively, with Qi ⁇ Q 2 .
- the invention can be implemented with a modulation of "strong" amplitude ( ⁇ close to 1) while preserving the values of the parameters l ° and ⁇ provided below which then optimize the amplitude of the term of order 1 in quadrature of the modulation of the concentrations in 1 and 2.
- I F (i) Iy + ⁇ 8 ⁇ ( ⁇ ) + Ip ut cos (t) (1 -,)
- FIGS. 2A and 2B respectively illustrate the dependence of the normalized amplitude in phase 1 1 / Pf v t and the normalized amplitude in quadrature
- quadrature component of the fluorescent emission intensity - has a well-defined maximum, which is not the case for 1 1 SM . This maximum is centered on the point:
- condition (1 9) on 1 ° amounts to equalizing the kinetic constants of the thermal and photoinduced reactions, and the condition (20-21) on ù) or f to tune the modulation frequency to the o
- Equations 33 and 34 can be transformed to show the amplitudes i " m and i" or the in-phase or quadrature phase oscillating at the pulsation ⁇ .
- concentration of i can be written: i ° + ⁇ X i tl in sin (nu) + i " ot cos (nù) t)
- Equation 27 can be transformed into
- equations 4 and 29 are proportional to P tot (see equations 4 and 29). Since the derivation is linear, equation 36 implies that / (0 *) is proportional to P tot . The equation system giving access to a n and b n is linear. It can therefore be deduced that all the amplitudes a n and b n are individually proportional to P tot . Finally, we use equations 33 to 35 to deduce that the ⁇ °, i n, m and i n out are proportional to P tot . It has been shown previously that in the case of sinusoidal modulation of small amplitude light, 2 1 - out is optimal when the resonance conditions of equations 19 and 21 are fulfilled. In the absence of an analytical expression for 2 1> 011 ⁇ , such conclusions can not be drawn in the case of high amplitude periodic light modulation. Their relevance was assessed by numerical calculations.
- FIG. 3 shows the dependence of the normalized amplitude I 1 ""
- - I / not ⁇ j on the luminous flux 7 ° (in ein.s .m) and the adimensionned pulsation ⁇ ° 2 when a 1. numerical calculation is performed on the truncation of the Fourier series / (0 *) at the order 1, 2,3,4 and 5 respectively for the panels A, B, C, D and E.
- the panel F presents the dependence normalized amplitude on the same variables observed in a low-amplitude modulation regime
- the markers correspond to the isodensity curves (0.01 for the dash-points, 0.03 for the dots and 0.05 for the dashes)
- I itiotm I presents an optimum in space (7 ⁇ , ⁇ ), for which the position and the amplitude are very close to those observed in the case of a low amplitude sinusoidal modulation.
- the mistake made while taking the analytical expression, valid only for low amplitude modulation, is less than 20%, whatever the amplitude of use.
- Such an error is of the order of the experimental error by setting the mean luminous intensity and the pulsation to their resonance values, / ° ' R and ⁇ ⁇ .
- the quadrature intensity will present a plurality of local maxima, one for each said species.
- FIG. 4 makes it possible to highlight the selectivity of the detection according to the invention, as well as its quantitative character.
- the interfering species form an edge cube "n", with the target species in the center.
- n edge cube
- rfraiioîi is
- the concentration is overestimated by a factor greater than 8.
- titrabon is
- l lcos and l cos are the amplitudes of the quadrature components of the concentrations of the states 1 of the target and the interfering species X.
- two distinct excitation beams can be used, at different wavelengths: each of the two beams, of electromagnetic spectrum and of different light intensity, can impose different rate constants on the reactions linking the two states of the species P.
- One of the disadvantages of the use of two beams is to complicate the implementation of the detection method.
- it advantageously makes it possible to overcome the limitation of the acquisition speed related to the thermal relaxation from state 2 to state 1 in the case of the use of a single light beam.
- the species P is illuminated by a light of intensity / (t), comprising components ⁇ (t) and / 2 (t), respectively of wavelengths ⁇ 1 and ⁇ 2 .
- a light of intensity / (t) comprising components ⁇ (t) and / 2 (t), respectively of wavelengths ⁇ 1 and ⁇ 2 .
- this system is used with illumination at the wavelength of sinusoidal periodic intensity oscillating around the average value 7 ° at a pulse o ⁇ and with a small amplitude of oscillation ⁇ 7 ° ( ⁇ "1), on which is superimposed an illumination of wavelength ⁇ 2 , with ⁇ 1 ⁇ ⁇ 2 , of constant intensity 7 °.
- I (t) if [1 + ⁇ sin (ut) ⁇ + if. (41 ⁇
- fc 12 (i) k ° l2 1 [1 + ⁇ sin ( ⁇ )] + fcf : (46) i) - 3 ⁇ 4.i [i + e siii ⁇ + fcf + fcâ (47) and the system of equations Differences governing the temporal evolution of concentrations 1 and 2 are solved during a first order development for this light disturbance.
- the * 12.2 * -2, i + ⁇ 21.2 + ⁇ 21 (51 ) denotes the reaction rate in the steady state and the contributions of photochemical processes by the modulated light at the transition from 1 to 2 and respectively from 2 to 1 by illuminating at 7 °.
- the oscillating fluorescent emission is then:
- the embodiment of the invention using two distinct excitation beams differs from the embodiment of the invention using a single excitation beam in the limit where the exchanges between the states 1 and 2 are essentially due to the photochemical contributions during the use of two beams.
- the fluorescent species P used is Dronpa-2. It is characterized by the quintuplet of parameters ( ⁇ 12 1 , ⁇ 21 1 , ⁇ 12 2 , ⁇ 21 2 , ⁇ 2 ) such that ⁇ 12 1 ⁇ 12 2 and ⁇ 21 2 ⁇ 21 1 . Its amplitude
- Equation 54 P ⁇ -z ⁇ measure the composition difference ⁇ 2 0 Standing 2 speed after a jump intensity luminous amplitude ⁇ - e . This component is maximized when the photochemical reactions induced by the two light sources take place at the same speed.
- ⁇ "1 / ⁇ ° 2 the exchange is slow compared to the variations of light, and the pair ⁇ 1, 2 ⁇ does not have enough time to answer.
- the terms i n and V- out then disappear.
- the panel A of FIG. 5 presents the normalized amplitude of the quadrature oscillations of phase 1 ⁇ norm II ot ⁇ in the case where 1 ⁇ 2, ⁇ + ⁇ 21 , ⁇
- the panel B of FIG. 5 presents the normalized amplitude of the oscillations in quadrature phase ⁇ ⁇ 1 ⁇ " ⁇ ⁇ ⁇ - ⁇ ⁇ h A
- the invention has also been experimentally validated.
- a first validation uses, as a reversibly photoconvertable fluorescent species, the "Spinach - DFHBI" system, illustrated in FIG.
- DFHBI fluorogen that can be complexed with "Spinach” and exists as two isomers, cis and trans. The system therefore exists in four states:
- the bound (complexed) states are fluorescent, unlike free states, and in particular the I bound state is both more stable and brighter than the state 2 b0U nd-
- the trans-cis (2 ⁇ 1) isomerization reaction occurs both thermally (symbol “ ⁇ ” in Figure 6) and under the effect of illumination (symbol “hv”), while the reaction cis-trans (1 -> 2) is exclusively photoinduced (symbol "hv").
- a microfluidic device comprising four channels (depth 200 ⁇ , width 50 ⁇ ) was used as a sample; channels 1 to 4 of the device were filled with the following solutions:
- the system (Spinach - DFHBI) is the target species and Fluorescein a fluorophore interferes.
- the panel “a” of FIG. 7 shows a fluorescence image of this microfluidic device acquired, by means of a microscope, in the presence of a constant illumination; panel “b” shows the corresponding normalized fluorescence intensity, 1 ° F, norm, integrated over the length of the imaged channels.
- the panels “c” and “d” correspond to the case of a modulated lighting and a quadrature detection, under optimal conditions for the detection of the target species.
- a second validation, illustrated in Figure 8 uses, as a reversibly photoconvertable fluorescent species, the protein "Dronpa- 2".
- the Dronpa-2 system is the target species and Fluorescein a fluorophore interferes.
- the panel “a” of FIG. 8 shows a fluorescence image of this microfluidic device acquired, by means of a microscope, in the presence of constant illumination.
- the panel “b” corresponds to the case of a modulated lighting and a quadrature detection, under optimal conditions for the detection of the target species.
- the fluorescence intensities I or V, norm measured in accordance with the invention, are substantially proportional to Dronpa-2 concentrations: a ratio of 4 and 2 for chambers 1 and 3 compared to the chamber 4. It is also observed that the fluorescence intensity I or V, norm is close to zero in the square containing only fluorescein.
- Figure 9 illustrates the application of a method according to one embodiment of the invention to the selective imaging of biological material expressing the reversibly convertible fluorescent protein "Dronpa-3".
- the panels "a” and “b” of Figure 9 illustrate the selective imaging of "Dronpa-3" expressed in mammalian cells.
- Each panel has two images, one corresponding to a photograph taken in epifluorescence and the other corresponding to a photograph taken in selective imaging of HEK293 cells expressing both "Dronpa-3", which is a reversibly photoconvertable fluorescent species, in the nucleus, and "EGFP", which is not a reversibly photoconvertible fluorescent species, on their membrane.
- the panel “a” has a fixed cell while the panel “b” has a living cell whose image is taken after a period of modulation of the light signal.
- the scale bar represents 50 ⁇ .
- each panel presents two images, one corresponding to a photograph taken in epifluorescence and the other corresponding to a photograph taken in selective imaging.
- the panel “c” presents epifluorescence images, while the panel “d” presents images acquired by microscopy by single-plane illumination microscopy (SPIM).
- SPIM single-plane illumination microscopy
- the dotted white rectangle indicates the thinnest part of the excitation light plane. Selective imaging makes it possible to observe the actin network more specifically than in epifluorescence.
- the images of panels “a”, “b” and “c” in Figure 9 are taken at 37 ° C and the panel image "d” is taken at 20 ° C.
- the scale bar represents 50 ⁇ .
- Another validation uses, as a reversibly photoconvertible fluorescent species, the "Dronpa- 2" protein that is excited by two distinct light beams, at the substantially monochromatic wavelengths of 480 nm and 405 nm. .
- the Dronpa-2 system is the target species and Fluorescein a fluorophore interferes.
- Panel “A” of FIG. 10 shows a fluorescence image of this microfluidic device acquired, by means of a microscope, in the presence of constant illumination.
- the panel “B” corresponds to the case of a quadrature detection, in optimal conditions for the detection of the target species.
- the fluorescence intensities l out F, norm measured in accordance with the invention, are substantially proportional to the concentrations of Dronpa-2: a ratio 4 and 2 for the chambers 1 and 3 compared to the chamber 4. It is also observed that the fluorescence intensity I or V, norm is close to zero in the square containing only fluorescein.
- FIG. 11 illustrates an apparatus for carrying out a method according to one embodiment of the invention, of the type used for carrying out some of the experimental validations described above.
- Such an apparatus illustrated by way of non-limiting example, comprises an SLM light source consisting of a bar of light-emitting diodes, powered by an AM power source.
- the modulation of the excitation light beam FEX generated by said source is obtained by modulation of the electric power supply. Since the emission of the light-emitting diodes is broadband, the beam FEX is filtered by a first optical filter F1, before being directed onto a sample, constituted in this case by a microfluidic device DMF.
- the sample thus illuminated is observed, by its rear face, by an objective OBJ which takes the fluorescence radiation and focuses it into a beam FLU.
- the latter is filtered (filters F2, F3) and directed, via an M mirror and an LF lens, on a CAM camera.
- a PR processor (in fact, a conveniently programmed computer) drives the AM power source and the CAM camera to perform quadrature detection as described above.
- the CAM camera can be replaced by a point sensor of light.
- FIG. 12 illustrates an apparatus for implementing a method according to another embodiment of the invention, making it possible to remotely detect reversibly photoconvertible fluorescent probes in the environment.
- the high selectivity made possible by the quadrature detection is necessary to make the useful signal emerge from the very intense background formed by the ambient light.
- the light source SLM ' is a continuous mode laser oscillator.
- the excitation beam FEX is expanded and collimated by two lenses L1, L2, then modulated by an electro-optical modulator MEO.
- Two orientable mirrors M1 and M2 are used to scan a target to observe CO (a surface of the ground or of a body of water), containing at least one fluorescent species reversibly photoconvertible P (Dronpa, for example).
- FLU fluorescence radiation is collected by a CAM camera lens, which acquires an image of the target.
- a processor PR drives the modulator MEO and CAM 'camera so as to perform a quadrature detection as described above.
- FIG. 13 illustrates an apparatus for carrying out a method according to one embodiment of the invention, of the type used for carrying out some of the experimental validations described above.
- Such an apparatus illustrated by way of non-limiting example, comprises two light sources SLM1 and SLM2 constituted by two light-emitting diode bars.
- the light source SLM1 is powered by an AM1 power source and the light source SLM2 is powered by an AM2 power source.
- the modulation of the excitation light beam FEX1 generated by said source is obtained by modulation of the electric power supply.
- the beams FEX1 and FEX2 are filtered by two optical filters F1 and F1, before being directed onto a sample, constituted in this case by a microfluidic device DMF.
- the sample thus illuminated is observed, by its rear face, by an objective OBJ which takes the fluorescence radiation and focuses it into a beam FLU.
- the latter is filtered (filter F2) and directed, via a mirror M and a lens LF, on a CAM camera.
- a PR processor (in fact, a conveniently programmed computer) drives the power sources AM1, AM2 and the CAM camera to perform quadrature detection as described above.
- the CAM camera can be replaced by a point sensor of light.
- FIG. 14 illustrates an apparatus for carrying out a method according to one embodiment of the invention, of the type used for carrying out some of the experimental validations described above.
- Such an apparatus illustrated by way of non-limiting example, comprises an SLM light source constituted by a bar of light-emitting diodes, powered by an AM power source.
- the modulation of the excitation light beam FEX generated by said source is obtained by modulation of the electric power supply. Since the emission of the light-emitting diodes is broadband, the beam FEX is filtered by a first optical filter F1, before being directed onto a sample, constituted in this case by a microfluidic device DMF.
- the sample thus illuminated is observed by an objective OBJ which takes the fluorescence radiation and focuses it into an FLU beam.
- a PR processor (in fact, a conveniently programmed computer) drives the AM power source and the CAM camera to perform quadrature detection as described above.
- the CAM camera can be replaced by a point sensor of light.
- the quadrature detection method that has just been described can also exploit any other spectroscopic observable that makes it possible to distinguish the two states of a reversibly photoconvertable species.
- the absorbance can be considered (in particular in the low absorption regime, where the Beer-Lambert law can be linearized); in this case, instead of detecting a fluorescence radiation, a transmitted light intensity is detected which is related to an incident light intensity, and then the amplitude of its phase quadrature component is determined.
- the reflectance Equations 19 - 21 and 58 - 59, which define the optimal lighting conditions, apply regardless of the spectroscopic observable considered.
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- Optics & Photonics (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2931185A CA2931185A1 (fr) | 2013-11-21 | 2014-11-21 | Procede de detection d'une espece fluorescente reversiblement photoconvertible |
AU2014351811A AU2014351811A1 (en) | 2013-11-21 | 2014-11-21 | Method for detection of a reversibly photo-convertible fluorescent species |
JP2016554908A JP6417422B2 (ja) | 2013-11-21 | 2014-11-21 | 可逆的に光切り替え可能な蛍光種の検出方法 |
EP14802429.2A EP3071948B1 (fr) | 2013-11-21 | 2014-11-21 | Procede de detection d'une espece fluorescente reversiblement photoconvertible utilisant l'imagerie hors phase apres modulation optique |
US15/034,402 US10267732B2 (en) | 2013-11-21 | 2014-11-21 | Method for detection of a reversibly photo-convertible fluorescent species |
Applications Claiming Priority (2)
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FR1361476 | 2013-11-21 | ||
FR1361476A FR3013452B1 (fr) | 2013-11-21 | 2013-11-21 | Procede de detection d'une espece fluorescente reversiblement photoconvertible |
Publications (1)
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WO2015075209A1 true WO2015075209A1 (fr) | 2015-05-28 |
Family
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Family Applications (1)
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PCT/EP2014/075336 WO2015075209A1 (fr) | 2013-11-21 | 2014-11-21 | Procede de detection d'une espece fluorescente reversiblement photoconvertible |
Country Status (7)
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US (1) | US10267732B2 (fr) |
EP (1) | EP3071948B1 (fr) |
JP (1) | JP6417422B2 (fr) |
AU (1) | AU2014351811A1 (fr) |
CA (1) | CA2931185A1 (fr) |
FR (1) | FR3013452B1 (fr) |
WO (1) | WO2015075209A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018041588A1 (fr) | 2016-09-02 | 2018-03-08 | Centre National De La Recherche Scientifique | Procede de detection d'especes fluorescentes reversiblement photocommutables a haute frequence |
EP3795982A1 (fr) | 2019-09-18 | 2021-03-24 | Centre National de la Recherche Scientifique | Procédé et appareil pour détecter une espèce chimique photochimiquement active dans un échantillon |
EP3812744A1 (fr) | 2019-10-24 | 2021-04-28 | Centre National de la Recherche Scientifique | Appareil permettant d'effectuer des mesures photochimiques sur un liquide ou sur un échantillon contenant un liquide |
EP3839484A1 (fr) | 2019-12-17 | 2021-06-23 | Centre National de la Recherche Scientifique | Procede de detection d'un marqueur fluorescente reversiblement photoconvertible dans un echantillon |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170065182A1 (en) * | 2015-09-09 | 2017-03-09 | Washington University | Reversibly switchable photoacoustic imaging systems and methods |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2089029A (en) * | 1980-11-28 | 1982-06-16 | Scintrex Ltd | Method and Apparatus for the Remote Detection of Certain Minerals of Uranium, Zinc, Lead and Other Metals |
WO2001027883A1 (fr) * | 1999-10-13 | 2001-04-19 | Spectra Science Corporation | Codage et authentification par reponse a la modulation de mesure de phase et emission spectrale |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9000740D0 (en) * | 1990-01-12 | 1990-03-14 | Univ Salford | Measurement of luminescence |
WO2007040459A1 (fr) * | 2005-10-06 | 2007-04-12 | Nanyang Technological University | Elimination d'un bruit de fond en fluorescence |
AU2013225655A1 (en) * | 2012-03-02 | 2014-09-18 | The Regents Of The University Of California | System and method for time-resolved fluorescence imaging and pulse shaping |
-
2013
- 2013-11-21 FR FR1361476A patent/FR3013452B1/fr not_active Expired - Fee Related
-
2014
- 2014-11-21 AU AU2014351811A patent/AU2014351811A1/en not_active Abandoned
- 2014-11-21 EP EP14802429.2A patent/EP3071948B1/fr active Active
- 2014-11-21 US US15/034,402 patent/US10267732B2/en active Active
- 2014-11-21 CA CA2931185A patent/CA2931185A1/fr not_active Abandoned
- 2014-11-21 WO PCT/EP2014/075336 patent/WO2015075209A1/fr active Application Filing
- 2014-11-21 JP JP2016554908A patent/JP6417422B2/ja active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2089029A (en) * | 1980-11-28 | 1982-06-16 | Scintrex Ltd | Method and Apparatus for the Remote Detection of Certain Minerals of Uranium, Zinc, Lead and Other Metals |
WO2001027883A1 (fr) * | 1999-10-13 | 2001-04-19 | Spectra Science Corporation | Codage et authentification par reponse a la modulation de mesure de phase et emission spectrale |
Non-Patent Citations (4)
Title |
---|
CH. . RICHARDS ET AL.: "Synchronously Amplified Fluorescence Image Recovery (SAFIRe", J. PHYS. CHEM. B, vol. 114, 2010, pages 660 - 665 |
MARRIOTT ET AL.: "Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cell", PNAS, vol. 105, no. 46, 18 November 2008 (2008-11-18), pages 17789 - 17794 |
PENGCHENG WANG ET AL: "Photochemical properties of Spinach and its use in selective imaging", CHEMICAL SCIENCE, vol. 4, no. 7, 1 January 2013 (2013-01-01), pages 2865, XP055131010, ISSN: 2041-6520, DOI: 10.1039/c3sc50729g * |
Q. WEI; A. WEI: "Optical Imaging with Dynamic Contrast Agents", CHEM. EUR. J., vol. 17, pages 1080 - 1091 |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018041588A1 (fr) | 2016-09-02 | 2018-03-08 | Centre National De La Recherche Scientifique | Procede de detection d'especes fluorescentes reversiblement photocommutables a haute frequence |
FR3055704A1 (fr) * | 2016-09-02 | 2018-03-09 | Centre National De La Recherche Scientifique | Procede de detection d'especes fluorescentes reversiblement photocommutables a haute frequence |
JP2019532280A (ja) * | 2016-09-02 | 2019-11-07 | サントル ナシオナル ドゥ ラ ルシェルシェ シアンティフィクCentre National De Larecherche Scientifique | 高周波数で可逆的に光スイッチング可能な蛍光種を検出する方法 |
US10718712B2 (en) | 2016-09-02 | 2020-07-21 | Centre National De La Recherche Scientifique | Method for detecting fluorescent species that are reversibly photoswitchable at a high frequency |
EP3795982A1 (fr) | 2019-09-18 | 2021-03-24 | Centre National de la Recherche Scientifique | Procédé et appareil pour détecter une espèce chimique photochimiquement active dans un échantillon |
WO2021052668A1 (fr) | 2019-09-18 | 2021-03-25 | Centre National De La Recherche Scientifique | Procédé et appareil permettant la détection d'une espèce chimique photochimiquement active dans un échantillon |
EP3812744A1 (fr) | 2019-10-24 | 2021-04-28 | Centre National de la Recherche Scientifique | Appareil permettant d'effectuer des mesures photochimiques sur un liquide ou sur un échantillon contenant un liquide |
WO2021078419A1 (fr) | 2019-10-24 | 2021-04-29 | Centre National De La Recherche Scientifique | Appareil pour réaliser des mesures photochimiques sur un échantillon liquide ou contenant un liquide |
EP3839484A1 (fr) | 2019-12-17 | 2021-06-23 | Centre National de la Recherche Scientifique | Procede de detection d'un marqueur fluorescente reversiblement photoconvertible dans un echantillon |
WO2021121898A1 (fr) | 2019-12-17 | 2021-06-24 | Centre National De La Recherche Scientifique | Procédé de détection d'une espèce chimique réversiblement photocommutable dans un échantillon |
Also Published As
Publication number | Publication date |
---|---|
EP3071948B1 (fr) | 2017-10-18 |
JP6417422B2 (ja) | 2018-11-07 |
US20160356716A1 (en) | 2016-12-08 |
FR3013452A1 (fr) | 2015-05-22 |
AU2014351811A1 (en) | 2016-06-09 |
US10267732B2 (en) | 2019-04-23 |
JP2016537658A (ja) | 2016-12-01 |
EP3071948A1 (fr) | 2016-09-28 |
FR3013452B1 (fr) | 2016-02-05 |
CA2931185A1 (fr) | 2015-05-28 |
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