Multiphotonic microscopy method and device

Abstract

The invention relates to a device comprising: a laser source emitting a first beam with a central wavelength λ.sub.1 lying between 1010 nm and 1050 nm, a spectral supercontinuum generator downstream of the laser source, generating a second beam with a central wavelength λ.sub.2 lying between 1670 nm and 1730 nm from a part of the first beam, an optical parametric amplification system downstream of the spectral supercontinuum generator, generating a third beam with a central wavelength λ.sub.3 lying between 2540 nm and 2690 nm from at least a part of the second beam and a part of the first beam, and a second harmonic generator downstream of the optical parametric amplification system, the second harmonic generator generating a fourth beam with a central wavelength λ.sub.4 lying between 1270 nm and 1345 nm from at least a part of the third beam.

Claims

1. A device comprising: a laser source emitting a first beam with a central wavelength λ1 lying between 1010 nm and 1050 nm, a splitter downstream of the laser source to create a least a transmitted first beam and a reflected beam, a spectral supercontinuum generator downstream of the laser source and configured to receive the transmitted first beam and generate at least a spectrally broadened first beam from a part of the transmitted first beam, a spectral filter to filter said spectrally broadened first beam and form a second beam with a central wavelength λ2 lying between 1670 nm and 1730 nm, an optical parametric amplification system downstream of the spectral supercontinuum generator, said optical parametric amplification system comprising; a first optical parametric amplifier configured to amplify at least a part of said second beam and said reflected beam, and a second optical parametric amplifier configured to generate a third beam with a central wavelength λ.sub.3 lying between 2540 nm and 2690 nm, and a harmonic generator downstream of the optical parametric amplification system, the harmonic generator generating a fourth beam with a central wavelength λ.sub.4 lying between 1270 nm and 1345 nm from at least a part of the third beam.

2. The device as claimed in claim 1, the first beam having a central wavelength λ.sub.1 lying between 1020 nm and 1040 nm.

3. The device as claimed in claim 1, the second beam having a central wavelength λ.sub.2 lying between 1695 nm and 1710 nm.

4. The device as claimed in claim 1, the third beam having a central wavelength λ.sub.3 lying between 2590 nm and 2620 nm.

5. The device as claimed in claim 1, the fourth beam having a central wavelength λ.sub.4 lying between 1295 nm and 1310 nm.

6. The device as claimed in claim 1, the first beam being composed of pulse trains of a duration less than or equal to 500 fs.

7. The device as claimed in claim 1, the laser source being an ytterbium-doped fiber laser, the first beam presenting pulses of a duration less than or equal to 400 fs and presenting an energy per pulse greater than 40 μJ.

8. The device as claimed in claim 1, the spectral supercontinuum generator being a fiber with nonlinear effect.

9. The device as claimed in claim 1, the second optical parametric amplifier having an axial dimension greater than the first optical parametric amplifier, the beam at the input of the second optical parametric amplifier having a stronger power than that at the input of the first optical parametric amplifier.

10. The device as claimed in claim 1, comprising at least one stretcher downstream of the spectral supercontinuum generator and upstream of the optical parametric amplification system and at least two compressors downstream of the optical parametric amplification system, a first compressor for compressing the second beam and a second compressor for compressing the third beam.

11. The device as claimed in claim 1, comprising at least one stretcher downstream of the spectral supercontinuum generator and upstream of the optical parametric amplification system, a stretcher between the two optical parametric amplifiers, and at least two compressors downstream of the optical parametric amplification system, with one compressor for the second beam and one compressor for the third beam.

12. The device as claimed in claim 1, at least one of the optical parametric amplifiers further comprising a nonlinear crystal having a second order nonlinear successability and allowing a parametric phase tuning between the first and second beams.

13. The device as claimed in claim 1, the harmonic generator being a nonlinear crystal having a second order nonlinear successability and allowing a frequency-doubling phase tuning of the third beam.

14. The device of claim 1 which further comprises a method of using said device to generate at least one beam of central wavelength between 1010 nm and 1050 nm, between 1280 nm and 1345, and between 1670 nm and 1730 nm to excite one or more chromophores in a tissue.

15. The device of claim 1 which further comprises a method of using said device to generate a combination of two beams with a central wavelength lying, for one, between 1280 nm and 1345 nm and lying, for the other, between 1670 nm and 1730 nm.

16. The device of claim 1 which further comprises a method of using said device to generate a combination of two beams with a central wavelength lying, for the first, between 1010 nm and 1050 nm, lying, for the second, between 1270 nm and 1345 nm and lying, for the third, between 1670 nm and 1730 nm.

Description

BRIEF DESCRIPTION OF SEVERAL VIEW(S) OF THE DRAWINGS

(1) The invention will be able to be better understood on reading the following detailed description, of nonlimiting exemplary implementations thereof, and on studying the attached drawing, in which:

(2) FIG. 1 schematically represents a device according to the invention,

(3) FIG. 2 illustrates the concept of central wavelength, and of a laser beam,

(4) FIG. 3 is an absorption spectrum of water as a function of wavelength,

(5) FIG. 4A is an image of a tissue obtained by third harmonic generation, and

(6) FIG. 4B is an image of the tissue of FIG. 4A obtained by 3-photon microscopy.

DETAILED DESCRIPTION

(7) Device

(8) An example of device 10 according to the invention is represented in FIG. 1.

(9) This device 10 comprises a pulsed laser source 12 emitting a beam 14 comprising ultrashort pulse trains with a repetition frequency of approximately 1.25 MHz. The pulses have a central wavelength λ.sub.1 around 1030 nm and a duration less than or equal to 400 fs, preferably lying between 250 fs and 400 fs, for example substantially equal to 350 fs.

(10) As is illustrated in FIG. 2, the central wavelength λ.sub.c is defined by the wavelength for which the maximum spectral intensity of the pulse is reached. The beam 14 is split into two beams 16 and 18 by a splitter 20, for example a semi-transparent mirror disposed at 45° to the incident beam.

(11) The transmitted beam 16 is sent into a spectral supercontinuum generator 22 such as a YAG crystal. The spectral supercontinuum generator 22 delivers as output a beam 24 presenting pulse trains of a duration less than or equal to 100 fs, for example around 70 fs, and a widened wavelength spectrum extending from at least 1030 nm to 1800 nm.

(12) This beam 24 passes through a stretcher 26 so that the duration of the pulse trains of the stretched beam 29 is of a duration lying between 200 fs and 400 fs, being for example substantially equal to 250 fs.

(13) The beam 18 reflected by the splitter 20 is once again split into two beams 28 and 30 by a splitter 32, composed for example of a semi-transparent mirror inclined at 45° to the incident beam.

(14) A delay line 33 is placed on the path of the reflected beam 18 upstream of the splitter 32, being set such that the beam 28 reflected by the splitter 32 and the stretched beam 29 are substantially synchronous.

(15) The beams 28 and 29 are sent to a dichroic mirror 34 oriented at 45° to the incident beam 29.

(16) The dichroic mirror 34 makes it possible to filter in transmission the stretched beam 29 at a central wavelength λ.sub.2 around 1700 nm into a transmitted beam 31 and to transmit in reflection the beam 28, so as to combine these two beams 28 and 31 into a beam 36 at the input of an optical parametric amplifier 38.

(17) Thus, this beam 36 has a pump component of central wavelength λ.sub.1 and a signal component of central wavelength λ.sub.2. The pulse trains of the two components in the beam 36 at the input are synchronous and the pulses have a similar duration.

(18) The optical parametric amplifier 38 generates at the output a beam 40 comprising three components: a pump component of central wavelength λ.sub.1, the residue of the beam 28, a signal component of central wavelength λ.sub.2, and an additional component of central wavelength λ.sub.3 around:

(19) $\frac{1}{\frac{1}{1030}-\frac{1}{1700}}\mathrm{nm}$
i.e. substantially equal to 2600 nm±10 nm.

(20) The beam 40 is stripped of its central wavelength components λ.sub.1 and λ.sub.3 by successive dichroic mirrors 42 and 44 to form a beam 46 having only a single component of central wavelength λ.sub.2 around 1700 nm.

(21) The mirror 42 rejects, by reflection at 45°, the component λ.sub.1 and transmits the components λ.sub.2 and λ.sub.3. The mirror 44 reflects the component λ.sub.2 and transmits the component λ.sub.3.

(22) The beam 46 reflected by the filter 44 passes through a stretcher 48 chosen so that the duration of the pulse trains at the output is between 200 fs and 400 fs, for example substantially equal to 250 fs.

(23) The stretched beam 49 at the output of the stretcher 48 is sent to a dichroic mirror 58 then into an optical parametric amplifier 54.

(24) The mirror 58 is oriented at 45° to the incident beam 49 and transmits it.

(25) The beam 30 transmitted by the splitter 32 is once again divided by a splitter 50 into two beams 52 and 53. Only the reflected beam 52 is retained to be sent to the dichroic mirror 58 then into an optical parametric amplifier 54. The splitter 50 makes it possible to reduce the power of the beam 52.

(26) As is illustrated, a delay line 56 is placed on the path of the beam 30 to the splitter 50, it being set so that the beams 52 and the beam 49 are substantially synchronous at the input of the optical parametric amplifier 54.

(27) The dichroic mirror 58 makes it possible to combine the stretched beam 49 and the beam 52 within a beam 60 at the input of the amplifier 54. This beam 60 therefore has a pump of central wavelength λ.sub.1 and a signal component of central wavelength λ.sub.3. The pulse trains of the two components of wavelength λ.sub.1 and λ.sub.2 in the beam 60 are synchronous and the pulses have a similar duration.

(28) The optical parametric amplifier 54 generates at the output a beam 62 comprising three components: a pump of central wavelength λ.sub.1, the residue of the beam 28, a signal component of central wavelength λ.sub.2, and an additional component of central wavelength λ.sub.3.

(29) The optical parametric amplifiers 38 and 54 and the stretcher 48, as well as the dichroic mirrors 42, 44, 50 and 58 and the delay line 56 can form part of an integrated parametric amplification system 37.

(30) The three components of the beam 62 at the output of the optical parametric amplifier 54 are split by dichroic mirrors 64 and 66 into three beams 68, 70 and 72 having respective central wavelengths λ.sub.1, λ.sub.2 and λ.sub.3.

(31) The mirror 64 is placed at 45° to the incident beam 62, transmits the component λ.sub.3 and reflects the components λ.sub.1 and λ.sub.2. The mirror 66 is placed at 45° to the incident beam reflected by the mirror 64, transmits the component λ.sub.1 and reflects the component λ.sub.2.

(32) The reflected beam 70, of central wavelength λ.sub.2, is compressed by a compressor 74 so as to offset the effects on the beam 70 of the stretchings performed by the stretchers 26 and 48.

(33) The transmitted beam 72, of central wavelength λ.sub.3, is sent to a compressor 76 so as to offset the effects on the beam 72 of the stretchings performed by the stretchers 26 and 48 then to a second harmonic generator 78. The latter generates a beam 80 having two components, namely a component having a central wavelength λ.sub.3, the residue of the compressed beam 72, and a component having a central wavelength λ.sub.4 that is half of λ.sub.3, that is to say around 1300 nm.

(34) The beam 80 is filtered of its component of central wavelength λ.sub.3 by a dichroic mirror 82 placed at 45° to the incident beam 80 and the transmitted beam 86 has only the component of central wavelength λ.sub.4.

(35) Thus, the device 10 makes it possible to obtain, from a single beam 14 having a central wavelength λ.sub.1 around 1030 nm, three beams 68, 84 and 86 having central wavelengths λ.sub.1, λ.sub.2 and λ.sub.4 respectively around 1030 nm, 1700 nm and 1300 nm, the three beams obtained having synchronous pulses of repetition frequency of the order of 1.25 MHz.

(36) In an exemplary implementation, a prototype is produced by using as laser source 12 an ytterbium-doped fiber laser presenting pulse trains of an energy greater than or equal to 40 μJ composed of an industrial laser of Satsuma® type from the company Amplitudes System®.

(37) The spectral supercontinuum generator 22 is a YAG crystal.

(38) The stretchers 26 and 38 are silicon crystals of respective thicknesses of approximately 1 mm and 2 mm, or 2 mm and 1 mm.

(39) The optical parametric amplifiers 38 and 54 can each comprise a PPLN crystal. Preferably, the optical parametric amplifier 54 has a greater thickness than the optical parametric amplifier 38 to allow a better efficiency. For example, the optical parametric amplifier 38 is a PPLN crystal of a thickness substantially equal to 1 mm and the optical parametric amplifier 54 is a PPLN crystal of a thickness substantially equal to 3 mm.

(40) The compressor 74 is a sheet of glass of a thickness of approximately 75 mm. The compressor 78 is a silicon crystal of a thickness of approximately 5 mm. The second harmonic generator 78 is an AGS or PPLN crystal.

(41) The delay lines 33 and 56, the beam splitters 20, 32 and 50, and the dichroic mirrors 34, 42, 44, 58, 64, 66 and 82 are of conventional type and are known.

(42) In such a prototype, the power of the beam 16 corresponds to between 5% and 10% of that of the beam 14, that of the beam 28 corresponds to between 5% and 30% of that of the beam 14 and that of the beam 52 corresponds to between 60% and 90% of that of the beam 14.

(43) With such a device, the power of the beam 68 obtained corresponds to between 2% and 20% of that of the beam 14, the beam 84 presents pulse trains of a duration of approximately 65 fs and of an energy greater than 1 μJ and the beam 86 presents pulse trains of a duration of approximately 85 fs and of an energy equal to approximately 0.1 μJ.

(44) Multiphoton Microscopy Method

(45) The beams 68, 84 and/or 86 generated by the device described previously in relation to FIG. 1 can be combined to form a beam having components of central wavelengths λ.sub.1, λ.sub.2 and λ.sub.4.

(46) The beam formed can be used in a multiphoton microscope.

(47) Before the combination of the different beams 68, 84 and/or 86, the latter can each pass or not pass through a delay line making it possible to finely adjust the synchronization of the beams in the case where they might not be totally synchronous at the level of the sample. This can make it possible to compensate the different optical paths of each of these beams between the source and the microscope.

(48) As is illustrated in FIG. 3, the wavelengths λ.sub.1, λ.sub.2 and λ.sub.4 are situated in the windows of lesser absorption of water, and offer a good penetration into the tissues, which makes it possible to excite fluorochromes at depth.

(49) This excitation can be done with one or more simultaneous photons of the same wavelength or of different wavelengths.

(50) For example, in the case where the excitation is performed by two simultaneous photons of wavelengths λ.sub.i and λ.sub.j, the excitation is equivalent to that of two photons of the same wavelength:

(51) $\frac{2}{\frac{1}{\lambda i}+\frac{1}{\lambda j}}\mathrm{nm}$

(52) For example, in the case where the excitation is performed by three simultaneous photons of wavelengths λ.sub.i and λj, that is to say of which at least two photons are of the same wavelength, the excitation is equivalent to that of three photons of the same wavelength:

(53) $\frac{3}{\frac{2}{\lambda i}+\frac{1}{\lambda j}}\mathrm{nm}\mathrm{or}\frac{3}{\frac{1}{\lambda i}+\frac{2}{\lambda j}}\mathrm{nm}$

(54) By way of example, different combinations of excitation with two photons of wavelengths λ.sub.1, λ.sub.2 or λ.sub.4 or three photons of wavelength λ.sub.1, λ.sub.4 or λ.sub.2 are realized in Tables 1 and 2 below. The equivalent wavelength is indicated each time.

(55) TABLE-US-00001 TABLE 1 Two-photon excitation with one or two colors out of λ.sub.1 = 1030, λ.sub.4 = 1300, λ.sub.2 = 1700 nm. Photon 1, photon 2 (nm) 1030, 1030, 1300, 1300, 1300, 1700, 1030 1300 1700 1300 1700 1700 Equivalent photon for a 1030 1149 1283 1300 1473 1700 2-photon excitation (nm) Equivalent photon for a 515 574 641 650 736 850 1-photon excitation (nm)

(56) TABLE-US-00002 TABLE 2 Three-photon excitation with two colors (λ.sub.1 = 1030 nm, λ.sub.4 = 1700 nm). Photon 1, photon 2, photon 3 (nm) 1030, 1030, 1030, 1700, 1030, 1030, 1700, 1700, 1030 1700 1700 1700 Equivalent photon for a 3-photon excitation 1030 1186 1397 1700 (nm) Equivalent photon for a 1-photon excitation 343 395 466 567 (nm)

(57) TABLE-US-00003 TABLE 3 Three-photon excitation with two colors (λ.sub.4 = 1300 nm, λ.sub.2 = 1700 nm). Photon 1, photon 2, photon 3 (nm) 1300, 1300, 1300, 1700, 1300, 1300, 1700, 1700, 1300 1700 1700 1700 Equivalent photon for a 3-photon excitation 1300 1411 1542 1700 (nm) Equivalent photon for a 1-photon excitation 433 470 514 567 (nm)

(58) Similar tables can be effected for an excitation with four photons, and so on.

(59) The number of equivalent wavelengths achievable is much greater in the context of the invention than in the context of an excitation with several simultaneous photons of the same wavelength.

(60) These equivalent wavelengths range in the invention from the visible to the infrared.

(61) The multiphoton microscopy as described above therefore makes it possible to excite a greater diversity of photochromes emitting from the visible to the infrared and to obtain images of tissues with multiple contrasts as a function of excited photochromes.

(62) An example of use of the beams obtained to perform multicolor microscopy is described below.

Example

(63) Beams of central wavelengths λ.sub.2, substantially equal to 1700 nm, and λ.sub.4, substantially equal to 1300 nm, are obtained simultaneously using the device as described in relation to FIG. 1.

(64) These beams are sent simultaneously to a sample of fluorescence-marked chicken embryo spinal cord.

(65) Several images are obtained simultaneously.

(66) The photons of wavelength λ.sub.4 make it possible to generate a first image, illustrated in FIG. 4A, obtained by third harmonic generation allowing visualization of the morphology of the tissue without fluorescence.

(67) The photons of wavelength λ.sub.4 make it possible also to generate a second image obtained by 3-photon microscopy, in which the excited fluorochromes re-emit in the green.

(68) The photons of wavelength λ.sub.2 make it possible to generate a third image obtained by 3-photon microscopy, in which the excited fluorochromes re-emit in the red.

(69) The second and third images are combined in FIG. 4B to form a single image in two colors.

(70) The device according to the invention therefore makes it possible to generate beams of different wavelengths that can be used together to produce the multiphoton imaging. It is then possible to simultaneously obtain images of different colors that can be superposed.