WO2018224503A1 - Dispositif et procede de microscopie multiphotonique - Google Patents
Dispositif et procede de microscopie multiphotonique Download PDFInfo
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- WO2018224503A1 WO2018224503A1 PCT/EP2018/064781 EP2018064781W WO2018224503A1 WO 2018224503 A1 WO2018224503 A1 WO 2018224503A1 EP 2018064781 W EP2018064781 W EP 2018064781W WO 2018224503 A1 WO2018224503 A1 WO 2018224503A1
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- optical parametric
- central wavelength
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- GQYHUHYESMUTHG-UHFFFAOYSA-N lithium niobate Chemical compound [Li+].[O-][Nb](=O)=O GQYHUHYESMUTHG-UHFFFAOYSA-N 0.000 claims description 2
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Classifications
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
- H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range
- H01S3/005—Optical devices external to the laser cavity, specially adapted for lasers, e.g. for homogenisation of the beam or for manipulating laser pulses, e.g. pulse shaping
- H01S3/0057—Temporal shaping, e.g. pulse compression, frequency chirping
-
- G—PHYSICS
- G02—OPTICS
- G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
- G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
- G02F1/35—Non-linear optics
- G02F1/3528—Non-linear optics for producing a supercontinuum
-
- G—PHYSICS
- G02—OPTICS
- G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
- G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
- G02F1/35—Non-linear optics
- G02F1/353—Frequency conversion, i.e. wherein a light beam is generated with frequency components different from those of the incident light beams
- G02F1/3532—Arrangements of plural nonlinear devices for generating multi-colour light beams, e.g. arrangements of SHG, SFG, OPO devices for generating RGB light beams
-
- G—PHYSICS
- G02—OPTICS
- G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
- G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
- G02F1/35—Non-linear optics
- G02F1/39—Non-linear optics for parametric generation or amplification of light, infrared or ultraviolet waves
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
- H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range
- H01S3/005—Optical devices external to the laser cavity, specially adapted for lasers, e.g. for homogenisation of the beam or for manipulating laser pulses, e.g. pulse shaping
- H01S3/0078—Frequency filtering
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
- H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range
- H01S3/005—Optical devices external to the laser cavity, specially adapted for lasers, e.g. for homogenisation of the beam or for manipulating laser pulses, e.g. pulse shaping
- H01S3/0092—Nonlinear frequency conversion, e.g. second harmonic generation [SHG] or sum- or difference-frequency generation outside the laser cavity
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G02—OPTICS
- G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
- G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
- G02F1/35—Non-linear optics
- G02F1/355—Non-linear optics characterised by the materials used
- G02F1/3551—Crystals
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- G—PHYSICS
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- G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
- G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
- G02F1/35—Non-linear optics
- G02F1/37—Non-linear optics for second-harmonic generation
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
- H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range
- H01S3/14—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range characterised by the material used as the active medium
- H01S3/16—Solid materials
- H01S3/1601—Solid materials characterised by an active (lasing) ion
- H01S3/1603—Solid materials characterised by an active (lasing) ion rare earth
- H01S3/1618—Solid materials characterised by an active (lasing) ion rare earth ytterbium
Definitions
- the present invention relates to multiphoton microscopy.
- Multiphoton microscopy allows biological tissue to be imaged to a certain depth non-invasively with micrometric three-dimensional resolution. It involves exciting fluorochromes at a given focal point with multiple photons simultaneously and scanning the surface to make a 2D or 3D image of the tissue response. Multiphoton microscopy is of particular interest in neuroscience.
- Deep imaging of a biological tissue by a multiphoton microscopy technique is mainly limited by three parameters: a) the absorption of light by the tissue and mainly by the water contained in the latter, b) the diffusion of photons in the tissue, and c) the power of the incident beam. These three parameters in particular limit the achievable imaging depth.
- one of the solutions for limiting absorption is to choose excitation wavelengths in the ranges of the absorption spectrum having a reduced absorption coefficient.
- FIG. 3 representing the absorption spectrum of water
- the higher the wavelength the higher the water absorption coefficient.
- the water absorbs little between 700 nm and 1000 nm and has windows around 1300 nm and 1700 nm in which the absorption is reduced.
- the scattering of light decreases as the wavelength increases.
- femtosecond lasers emitting ultrashort pulses of high energy are used.
- the photochromes accessible by these known techniques are those which are sensitive to excitation at a wavelength of half the wavelength of the incident beam, in the case of 2-photon microscopy, and / or to 1/3 of the length. of the incident beam, in the case of 3-photon microscopy. This limits the imaging possibilities to a limited choice of photochromics.
- the invention meets this need with the aid of a device comprising:
- a laser source emitting a first beam at a central wavelength of between 1010 nm and 1050 nm
- a spectral supercontinuum generator downstream of the laser source, generating a second beam at a central wavelength between 1670 nm and 1730 nm from a portion of the first beam
- an optical parametric amplification system downstream of the spectral supercontinuum generator, generating a third beam at a central wavelength between 2545 nm and 2690 nm from at least a portion of the second beam and a portion the first beam, and
- a second harmonic generator downstream of the optical parametric amplification system, the second harmonic generator generating a fourth beam at a central wavelength between 1270 nm and 1345 nm from at least a part of the third beam .
- Such a device makes it possible, from a single laser source emitting a beam having a central wavelength around 1030 nm, to obtain three beams respectively having central wavelengths around 1030 nm, 1300 nm and 1700 nm. nm.
- the fact that the three beams are obtained from a single beam ensures that they are synchronous.
- the wavelengths ⁇ ⁇ 5 ⁇ 2 and ⁇ 3 are linked by the following relation: 1 _ 1 1
- the obtained central wavelengths are located in the windows of less water absorption and are not very diffused by the tissues, in particular for the highest central wavelength beams, ie around 1300 nm and 1700 nm .
- the parametric amplification system allows, from a central wavelength pump around 1030 nm and a source of central wavelength around 1700 nm, which falls into a window of less absorption of light. water, to generate a complementary wavelength around 2600 nm, corresponding to double 1300 nm, which is also in a window of less water absorption.
- a single incident beam of central wavelength around 1030 nm allows, in a simple way, to generate two beams of different central wavelengths both in windows of lesser water absorption.
- the beams can be used in a dissociated or combined way. For example two or three of the beams are combined to imitate biological tissue in depth by multiphoton microscopy. Because of the wavelengths and beams that can be used, many possibilities for exciting photochromes are offered.
- the first beam has a central wavelength between 1020 nm and 1040 nm, more preferably equal to 1030 nm.
- the second beam has a central wavelength of between 1695 nm and 1710 nm, better still substantially equal to 1703 nm.
- the third beam has a central wavelength of between 2590 nm and 2620 nm, more preferably substantially equal to 2606 nm.
- the fourth beam has a central wavelength between 1295 nm and 1310 nm, more preferably equal to 1303 nm.
- the first beam is composed of pulse trains each of a duration less than or equal to 500 fs.
- the duration of the pulse is very small allows the spectral supercontinuum generator to easily generate the second beam. Indeed, the shorter the duration of the pulses of the first beam, the more the incident beam sees its spectrum expanded by the spectral supercontinuum generator.
- the laser source is a Ytterbium-doped fiber laser, the first beam having pulse trains of duration less than or equal to 400 fs and having an energy per pulse greater than 40 ⁇ .
- the device comprises a beam splitter upstream of the spectral supercontinuum generator for separating the first beam into two parts, the first part of the first beam making it possible to generate the second beam.
- This part of the first beam used for the generation of the second beam has a pulse energy of preferably between 2 ⁇ and 4 ⁇ and / or has a power that is between 5% and 10% of that of the first beam.
- the first part of the first beam represents 8% of the initial beam.
- the spectral supercontinuum generator is a transparent solid material, in particular glass, fibers or crystals, or better still a YAG (yttrium garnet aluminum) crystal.
- a YAG crystal makes it possible in particular to have a path of the beam within it which is short, in particular of the order of one centimeter, which facilitates the production of a compact device.
- the crystal YAG supports better than a fiber a high power input beam, which is preferentially the case here.
- the optical parametric amplification system may include at least one optical parametric amplifier.
- the optical parametric amplification system comprises at least two successive optical parametric amplifiers, the first optical parametric amplifier amplifying said at least a portion of the second beam, from the second beam generated by the spectral supercontinuum generator and a part of the first beam, and the second optical parametric amplifier generating the third beam from at least a portion of the second beam amplified by the first optical parametric amplifier and a portion of the first beam.
- the second optical parametric amplifier then has at its input a portion of a first beam and a second beam of high power, which enables it to generate a second beam and a third beam of high power.
- the fact of using two optical parametric amplifiers makes it possible to have a good amplification efficiency, which makes it possible to have first, second and third output beams which each have a high power.
- the second optical parametric amplifier has an axial dimension greater than the first optical parametric amplifier, the portion of the first input beam of the second optical parametric amplifier being of a higher power than that at the input of the first parametric amplifier optical.
- the device comprises a beam splitter upstream of the optical parametric amplification system for separating the second part of the first beam into two parts, one of these parts constituting the pump of the first optical parametric amplifier and the other of these parts constituting the pump of the second optical parametric amplifier.
- the energies are distributed as follows, when the first beam has a pulse energy of 40 ⁇ , the pump of the first amplifier is 4 ⁇ , which represents about 10% of the first beam, and the pump of the second amplifier is 33 ⁇ , which is about 82% of the first beam.
- the device comprises 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.
- the compressors and stretchers are made of glass or crystals.
- a crystal or a glass makes it possible to have a short path of the beam within them, in particular of the order of a centimeter.
- stretcher is made of silicon.
- the first compressor compresses the second beam and is preferably of opposite sign dispersion to the stretcher.
- the first compressor may be silica.
- the second compressor compresses the third beam and is preferably a dispersion of the same sign as the stretcher.
- the second compressor is preferably upstream of the second harmonic generator.
- the second compressor may be silicon.
- the presence of the stretcher, the optical parametric amplification system and the compressors makes it possible to have a low frequency drift amplification, and to reduce the losses and the paths of the beams.
- the stretcher allows agreement of the duration of the pulses of the second beam constituting the signal of the optical parametric amplification system to that of the portion of the first beam constituting the pump of the amplification system.
- optical parametric and compressors allow after the optical parametric amplification system to find, for the second beam and the third beam, beams with trains of high energy pulses.
- the stretcher makes it possible to have a second beam having pulse trains with a duration of between 200 fs and 400 fs to be adapted to the duration of the first beam.
- the device comprises 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. , a compressor for the second beam and a compressor for the third beam, preferably upstream of the second harmonic generator.
- the compressors are configured to substantially compensate for the stretching or stretches and to obtain pulses for the second and third beams with a duration after compression of between 50 fs and 100 fs, preferably substantially equal to 70 fs.
- the optical parametric amplifier (s) may comprise a nonlinear crystal having a second order nonlinear susceptibility and allowing parametric phase matching between the first and second beams, preferably a Periodically Poled Lithium Niobate (PPLN) crystal, KTA (Potassium Titanyl Arsenate) or BBO (Beta Barium Borate),.
- PPLN Periodically Poled Lithium Niobate
- KTA Potassium Titanyl Arsenate
- BBO Beta Barium Borate
- the second harmonic generator may be a nonlinear crystal having a second order nonlinear susceptibility and allowing third beam frequency doubling phase tuning, preferably an AGS (Silver Thiogallate) crystal or PPLN crystal. .
- the device may include one or more delay lines. Such delay lines make it possible to adjust the phases of the different beams, and to obtain synchronous pulses in the optical parametric amplification and de facto output system.
- a first delay line may be located on the path downstream of the first optical parametric amplifier and may match the wavelength of the second amplified beam in said amplifier. This delay line may be located on the path of the first beam constituting the pump or the second beam constituting the signal, preferably on the first beam constituting the pump. The tunability between the pump and the signal is favored by the use of the stretcher which temporally spreads the wavelengths of the second beam.
- a second delay line may be located on the path downstream of the second optical parametric amplifier, and can optimize the efficiency of said amplifier.
- the portion of the first beam sent at the output of the device has a pulse energy greater than 0.5 ⁇ , for example substantially equal to 1.5 ⁇ .
- the second beam amplified by the optical parametric amplification system has pulse trains of duration between 50 fs and 100 fs and a pulse energy greater than 1 ⁇ , for example substantially equal to 2.5 ⁇ .
- the fourth beam has pulse trains of duration between 50 fs and 100 fs and a pulse energy greater than 0.2 ⁇ , for example substantially equal to 0.4 ⁇ .
- the device may include one or more filters for separating unwanted wavelengths from desired wavelengths at the input or output of certain components of the device.
- These filters are advantageously dichroic mirrors, which allow light to pass at certain wavelengths and reflect light at other wavelengths. These dichroic mirrors can be oriented at 45 ° of the incident beam to be filtered.
- the device may notably comprise such filters at the output of the first and second parametric amplifiers.
- the subject of the invention is also a multiphoton microscopy imaging method comprising the step of exciting one or more chromophores in a tissue from at least one beam, preferably a combination of wavelength beams. central between 1010 nm and 1050 nm, between 1270 nm and 1345 and / or between 1670 nm and 1730 nm, obtained at the output of a device according to the invention, as defined above.
- the pulse trains of the different beams are transmitted in the fabric synchronously.
- a photochromic can be excited by a photon at 1300 nm and a photon at 1700 nm simultaneously, equivalent to an excitation by a photon at 737 nm, or by a photon at 1300 nm and two photons at 1700 nm simultaneously, equivalent to excitation by a photon at 514 nm, or by three photons at 1700 nm, equivalent to excitation by a photon at 567 nm.
- the excitation of the photochromic (s) is made from a combination of two beams at a central wavelength comprised for one between
- the excitation of the photochromic (s) is made from a combination of three beams with a central wavelength comprised for the first between 1010 nm and 1050 nm, included for the second between 1280 nm and 1320 and included for the third between 1650 nm and 1750 nm.
- FIG. 1 schematically represents a device according to the invention
- FIG. 2 illustrates the notion of central wavelength, and of a laser beam
- FIG. 3 is an absorption spectrum of water as a function of the wavelength
- FIG. 4A is an image of a tissue obtained by third harmonic generation
- FIG. 4B is an image of the tissue of FIG. 4A obtained by 3-photon microscopy.
- FIG. 10 An example of a device 10 according to the invention is shown in FIG.
- This device 10 comprises a pulsed laser source 12 emitting a beam 14 comprising ultrashort pulse trains at a repetition frequency of about 1.25 MHz.
- the pulses have a central wavelength ⁇ around 1030 nm and a duration less than or equal to 400 fs, preferably between 250 fs and 400 fs, for example substantially equal to 350 fs.
- the central wavelength c is defined by the wavelength for which the maximum of the spectral intensity of the impulse is reached.
- the beam 14 is separated into two beams 16 and 18 by a separator 20, for example a semi-transparent mirror disposed at 45 ° of the incident beam.
- the transmitted beam 16 is sent into a spectral supercontinuum generator 22 such as a YAG crystal.
- the spectral supercontinuum generator 22 outputs a beam 24 having pulse trains of a duration less than or equal to 100 fs, for example around 70 fs, and an extended wavelength spectrum extending at least from 1030 nm to 1800 nm.
- 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 of between 200 fs and 400 fs, for example being substantially equal to 250 fs.
- the beam 18 reflected by the separator 20 is again separated into two beams 28 and 30 by a separator 32, consisting for example of a semi-transparent mirror inclined at 45 ° of the incident beam.
- a delay line 33 is placed in the path of the reflected beam 18 upstream of the separator 32, being set so that the beam 28 reflected by the separator 32 and the drawn beam 29 are substantially synchronous.
- the beams 28 and 29 are sent on a dichroic mirror 34 oriented at 45 ° to the incident beam 29.
- the dichroic mirror 34 makes it possible to filter in transmission the stretched beam 29 at a central wavelength ⁇ 2 around 1700 nm in a transmitted beam 31 and to transmit in reflection the beam 28, so as to combine these two beams 28 and 31 in a beam 36 at the input of an optical parametric amplifier 38.
- this beam 36 has a pump component of central wavelength ⁇ and a signal component of central wavelength ⁇ 2 .
- the pulse trains of the two components in the input beam 36 are synchronous and the pulses have a similar duration.
- the optical parametric amplifier 38 generates at the output a beam 40 comprising three components:
- the beam 40 is freed from its central wavelength components ⁇ and ⁇ 3 by successive dichroic mirrors 42 and 44 to form a beam 46 having only one component of central wavelength ⁇ 2 around 1700 nm .
- the mirror 42 rejects the component ⁇ 1 by reflection at 45 ° and transmits the components ⁇ 2 and ⁇ 3 .
- the mirror 44 reflects the component ⁇ 2 and transmits the component ⁇ 3
- the beam 46 reflected by the filter 44 passes through a stretcher 48 chosen so that the duration of the pulse trains output is between 200 fs and 400 fs, for example substantially equal to 250 fs.
- the drawn beam 49 at the output of the stretcher 48 is sent on a dichroic mirror 58 and then in an optical parametric amplifier 54.
- the mirror 58 is oriented at 45 ° of the incident beam 49 and transmits it.
- the beam 30 transmitted by the separator 32 is again divided by a separator 50 into two beams 52 and 53. Only the reflected beam 52 is retained to be sent on the dichroic mirror 58 and then in an optical parametric amplifier 54.
- the separator 50 allows to reduce the power of the beam 52.
- a delay line 56 is placed in the path of the beam 30 to the splitter 50, being set so that the beams 52 and the beam 49 are substantially synchronous to the input of the optical parametric amplifier 54 .
- the dichroic mirror 58 makes it possible to combine the stretched beam 49 and the beam
- This beam 60 thus having a pump of central wavelength ⁇ ⁇ and a signal component of central wavelength ⁇ 3 .
- the pulse trains of the two wavelength components ⁇ and ⁇ 2 in the beam 60 are synchronous and the pulses have a similar duration.
- the optical parametric amplifier 54 generates at the output a beam 62 comprising three components:
- 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 may be part of an integrated parametric amplification system 37.
- the three components of the beam 62 at the output of the optical parametric amplifier 54 are separated by dichroic mirrors 64 and 66 into three beams 68, 70 and 72 having central wavelengths ⁇ 1; ⁇ 2 and ⁇ 3 respectively.
- the mirror 64 is placed at 45 ° of the incident beam 62, transmits the component ⁇ 3 and reflects the components ⁇ 1 and ⁇ 2.
- the mirror 66 is placed at 45 ° of the incident beam reflected by the mirror 64, transmits the component ⁇ 1 and reflects the component ⁇ 2.
- the reflected beam 70, of central wavelength ⁇ 2 is compressed by a compressor 74 so as to compensate for the effects on the beam 70 of the stretches made by the stretchers 26 and 48.
- the transmitted beam 72 of central wavelength ⁇ 3 , is sent on a compressor 76 so as to compensate for the effects on the beam 72 of the stretches performed by the stretchers 26 and 48 and then on a second harmonic generator 78. generates a beam 80 having two components, namely a component having a central wavelength ⁇ 3 , the residue of the compressed beam 72, and a component having a central wavelength ⁇ 4 half of ⁇ 3 , that is to say say around 1300 nm.
- the beam 80 is filtered from its central wavelength component ⁇ 3 by a dichroic mirror 82 placed at 45 ° of the incident beam 80 and the transmitted beam 86 only has the central wavelength component ⁇ 4 .
- the device 10 makes it possible to obtain, from a single beam 14 having a central wavelength ⁇ around 1030 nm, three beams 68, 84 and 86 having central wavelengths ⁇ , ⁇ 2 and ⁇ . 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.
- a prototype is made using as a laser source 12 a fiber laser source doped with Ytterbium having pulse energy trains greater than or equal to 40 ⁇ consisting of an industrial laser Satsuma® type from Amplitudes System®.
- Spectral supercontinuum generator 22 is a YAG crystal.
- the stretchers 26 and 38 are silicon crystals of respective thicknesses of about 1 mm and 2 mm, or 2 mm and 1 mm.
- the optical parametric amplifiers 38 and 54 may each comprise a PPLN crystal.
- the optical parametric amplifier 54 is of greater thickness than the optical parametric amplifier 38 to allow better performance.
- the optical parametric amplifier 38 is a PPLN crystal with a thickness substantially equal to 1 mm and the optical parametric amplifier 54 is a PPLN crystal with a thickness substantially equal to 3 mm.
- the compressor 74 is a glass plate with a thickness of about 75 mm.
- the compressor 78 is a silicon crystal with a thickness of about 5 mm.
- the second harmonic generator 78 is an AGS or PPLN crystal.
- 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 known.
- the power of the beam 16 corresponds to between 5% and 10% that of the beam 14, that of the beam 28 corresponds to between 5% and 30% that of the beam 14 and that of the beam 52 corresponds to between 60% and 90% that of beam 14.
- the power of the beam 68 obtained is between 2% and 20% that of the beam 14, the beam 84 has pulse trains with a duration of about 65 fs and energy greater than 1 ⁇ and the beam 86 has pulse trains with a duration of about 85 fs and energy equal to about 0.1 ⁇ .
- the beams 68, 84 and / or 86 generated by the device described above in connection with FIG. 1 can be combined to form a beam having central wavelength components ⁇ ⁇ 5 ⁇ 2 and ⁇ 4 .
- the formed beam can be used in a multiphoton microscope.
- the latter may each pass or not by a delay line making it possible to finely adjust the synchronization of the beams in the event that they are not totally synchronous at the level of the beam. sample. This can compensate the different optical paths of each of these beams between the source and the microscope. As illustrated in FIG. 3, the wavelengths ⁇ , ⁇ 2 and are located in windows of less water absorption, and have good penetration into the tissues, which makes it possible to excite the fluorochromes by depth.
- This excitation can be done with one or more simultaneous photons of the same wavelength or different wavelengths.
- the excitation is equivalent to that of two photons of the same wavelength:
- the excitation is equivalent to that of three photons of the same wavelength wavelength: nm
- Multiphoton microscopy as described above thus makes it possible to excite a greater diversity of photochromes emitting infrared visible light and to obtain tissue images with multiple contrasts as a function of excited photochromes.
- Beams of central wavelength ⁇ 2 substantially equal to 1700 nm, and ⁇ 4 , substantially equal to 1300 nm, are obtained simultaneously with the aid of the device as described in relation with FIG.
- the photons of wavelength ⁇ 4 make it possible to generate a first image, illustrated in FIG. 4A, obtained by third harmonic generation allowing a visualization of the morphology of the tissue without fluorescence.
- the photons of wavelength ⁇ 4 also make it possible to generate a second image obtained by 3-photon microscopy, in which the excited fluorochromes re-emit in green.
- the photons of wavelength ⁇ 2 make it possible to generate a third image obtained by 3-photon microscopy, in which the excited fluorochromes re-emit in the red.
- the second and third images are combined in Figure 4B to form a single two-color image.
- the device according to the invention thus makes it possible to generate beams of different wavelengths that can be used together to perform multiphoton imaging. It is then possible to simultaneously obtain images of different colors that can be superimposed.
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KR1020207000106A KR20200013039A (ko) | 2017-06-09 | 2018-06-05 | 다광자 마이크로스코피 방법 및 디바이스 |
JP2019567612A JP2020522757A (ja) | 2017-06-09 | 2018-06-05 | 多光子顕微鏡測定方法及び装置 |
EP18729654.6A EP3635824A1 (fr) | 2017-06-09 | 2018-06-05 | Dispositif et procede de microscopie multiphotonique |
US16/620,422 US11256157B2 (en) | 2017-06-09 | 2018-06-05 | Multiphotonic microscopy method and device |
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US20140367591A1 (en) * | 2012-02-02 | 2014-12-18 | Ecole Polytechnique | Multicolour excitation module for a multiphoton imaging system and associated method and system |
FR3013857A1 (fr) * | 2013-11-28 | 2015-05-29 | Fastlite | Systeme pour generer des impulsions optiques courtes de duree inferieure a la periode de la porteuse optique utilisant le principe de l'amplification parametrique. |
US20160064891A1 (en) * | 2004-03-25 | 2016-03-03 | Imra America, Inc. | Optical parametric amplification, optical parametric generation, and optical pumping in optical fibers systems |
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WO2015130651A1 (fr) * | 2014-02-28 | 2015-09-03 | Imra America, Inc. | Génération et distribution d'impulsions ultracourtes à longueurs d'ondes multiples, avec des applications en microscopie |
FR3023423B1 (fr) * | 2014-07-03 | 2016-07-08 | Amplitude Systemes | Systeme laser uv-visible a impulsions ultra-courtes de forte puissance et/ou de forte energie |
US9244332B1 (en) * | 2014-12-22 | 2016-01-26 | Deutsches Elektronen-Synchrotron Desy | Pulse light source device and method for creating fs pulses |
DE102016202098A1 (de) * | 2016-02-11 | 2017-08-17 | Ape Angewandte Physik Und Elektronik Gmbh | Optische Verstärker-Anordnung, Laser-Verstärker-System und Verfahren zur Erzeugung eines breiten sichtbar bis infraroten, jedenfalls nah-infraroten, Spektrums von kohärenten ultra-kurzen Lichtpulsen mit einer optischen Verstärker-Anordnung |
EP3469667B1 (fr) * | 2016-06-10 | 2020-12-16 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Dispositif source de lumière pulsée et procédé pour créer des impulsions laser fs à cep stable |
US10281331B2 (en) * | 2017-02-23 | 2019-05-07 | Robert Alfano | Resonant stimulated Raman scattering microscope |
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- 2018-06-05 JP JP2019567612A patent/JP2020522757A/ja active Pending
- 2018-06-05 WO PCT/EP2018/064781 patent/WO2018224503A1/fr active Application Filing
- 2018-06-05 EP EP18729654.6A patent/EP3635824A1/fr not_active Withdrawn
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US20160064891A1 (en) * | 2004-03-25 | 2016-03-03 | Imra America, Inc. | Optical parametric amplification, optical parametric generation, and optical pumping in optical fibers systems |
US20140367591A1 (en) * | 2012-02-02 | 2014-12-18 | Ecole Polytechnique | Multicolour excitation module for a multiphoton imaging system and associated method and system |
FR3013857A1 (fr) * | 2013-11-28 | 2015-05-29 | Fastlite | Systeme pour generer des impulsions optiques courtes de duree inferieure a la periode de la porteuse optique utilisant le principe de l'amplification parametrique. |
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FR3067524B1 (fr) | 2019-07-26 |
EP3635824A1 (fr) | 2020-04-15 |
US11256157B2 (en) | 2022-02-22 |
KR20200013039A (ko) | 2020-02-05 |
JP2020522757A (ja) | 2020-07-30 |
FR3067524A1 (fr) | 2018-12-14 |
US20200088642A1 (en) | 2020-03-19 |
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