EP2810050A1 - Module d'excitation multi-couleurs pour un systeme d'imagerie multi-photonique, systeme et procede associes - Google Patents
Module d'excitation multi-couleurs pour un systeme d'imagerie multi-photonique, systeme et procede associesInfo
- Publication number
- EP2810050A1 EP2810050A1 EP13705115.7A EP13705115A EP2810050A1 EP 2810050 A1 EP2810050 A1 EP 2810050A1 EP 13705115 A EP13705115 A EP 13705115A EP 2810050 A1 EP2810050 A1 EP 2810050A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- excitation
- excitation beam
- chromophores
- laser source
- module
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
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- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 5
- 229910052719 titanium Inorganic materials 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
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Classifications
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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
-
- 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
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B17/00—Systems with reflecting surfaces, with or without refracting elements
- G02B17/02—Catoptric systems, e.g. image erecting and reversing system
- G02B17/023—Catoptric systems, e.g. image erecting and reversing system for extending or folding an optical path, e.g. delay lines
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
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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
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6419—Excitation at two or more wavelengths
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
- G01N21/3151—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths using two sources of radiation of different wavelengths
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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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- 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
- G01N2201/06—Illumination; Optics
- G01N2201/061—Sources
- G01N2201/06113—Coherent sources; lasers
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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
- G01N2201/06—Illumination; Optics
- G01N2201/066—Modifiable path; multiple paths in one sample
- G01N2201/0668—Multiple paths; optimisable path length
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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
- G01N2201/06—Illumination; Optics
- G01N2201/069—Supply of sources
- G01N2201/0696—Pulsed
- G01N2201/0697—Pulsed lasers
Definitions
- Multi-color excitation module for a multi-photonic imaging system, associated system and method
- the present invention relates to a multi-color excitation module for a multi-photon imaging system.
- the field of the invention is more particularly that of multi-color excitation and multi-photon fluorescence imaging, that is to say the observation of so-called "fluorescence" return signals emitted by chromophores present in a sample, in response to the light absorption of a pulsed exciter laser beam.
- multi-photon imaging uses two- or three-photon excitation. It is particularly useful for deep and non-destructive observation of biological tissues.
- Chromophores can be naturally present in the sample. You can also plan to inject them. Fluorophores, that is to say chemical substances capable of emitting fluorescent light after excitation, are also more specifically referred to.
- the return signals are called "fluorescence", since they correspond to a light emission consecutive to the excitation of a molecule (here by absorption of at least two photons).
- the field of the invention is more particularly but not limited to that of two-photon microscopy.
- the present invention also relates to a multi-color multi-photon imaging system comprising an excitation module for exciting several different chromophores, as well as a method implemented in said module.
- a first solution consists in using at least one tunable laser source to excite the chromophores.
- An emission wavelength scan of the tunable laser source makes it possible to successively excite the at least three distinct chromophores.
- the change in the emission wavelength of said laser source takes at most several seconds for narrow-range femtosecond pulse laser sources generally used in multi-photon imaging systems.
- a disadvantage of this first solution is that it is impossible to simultaneously produce the return signals corresponding to each of said at least three chromophores.
- a second solution is to use a single laser source, to simultaneously excite at least three separate chromophores and having very close excitation frequencies.
- a disadvantage of this second solution is that the excitation frequency range is restricted, which reduces the range of chromophores capable of being excited effectively. Only spectrally close sets of chromophores (for example about 10 nm between two excitation peaks) can be imaged simultaneously. Another disadvantage of this second solution is that it does not independently control the excitation efficiency of the different chromophores.
- a third solution consists in using a pulsed laser source per chromophore that it is desired to excite simultaneously.
- a disadvantage of this third solution is of course its prohibitive cost since at least three pulsed laser sources are required.
- the objective of the present invention is to provide a multi-photon excitation module multi-color for a multi-photon imaging system, for simultaneously imaging at least three chromophores of a sample, and which does not present at least one of the disadvantages of the prior art.
- an object of the present invention is to provide a module for a multi-photon imaging system, to effectively excite and simultaneously image at least three chromophores of a sample.
- Another object of the present invention is to provide a simple and inexpensive module for a multi-photon imaging system, to effectively excite at least three chromophores of a sample.
- Another objective of the present invention is to propose a module for a multi-photon imaging system, to effectively excite at least three chromophores of a sample, said three chromophores having excitation wavelengths distant from each other. others, for example at least 50 nm.
- the present invention aims to provide a multi-photonic imaging system comprising such a module, and a method implemented in said module.
- a module for a multi-photon imaging system for simultaneously exciting at least three chromophores of a sample, said module comprising:
- a first femtosecond laser source emitting a first excitation beam in the form of pulses having a repetition rate 1 / T and a (central) wavelength ⁇ able to excite the first one of the chromophores by multi-photon absorption said absorbed photons from the first excitation beam;
- a second femtosecond laser source emitting a second excitation beam in the form of pulses at a (central) wavelength ⁇ 2 able to excite a second of the chromophores by multi-photon absorption, said absorbed photons coming from the second excitation beam.
- the first excitation beam comprises a so-called “excitation” portion for exciting the sample, and a so-called “pumping” portion, this pumping portion serving as a pump beam for synchronously exciting the second femtosecond laser source.
- the second laser source is synchronous with the first laser source, that is to say the same repetition rate 1 / T.
- the module according to the invention further comprises an optical delay line arranged for spatially and temporally superimposing the second excitation beam and the excitation portion of the first excitation beam, so as to excite at least a third of the chromophores by multi-photon absorption (two-color), said absorbed photons from the first and second excitation beams.
- the second femtosecond laser source is thus formed synchronous with the first femtosecond laser source.
- the optical delay line (we will speak simply of
- Delay line in the following) spatially and temporally superimposes the second excitation beam and the remaining portion of the first excitation beam which is not used as a pump beam for the second laser source.
- the delay line may be arranged in particular to adjust the optical path traveled by the excitation portion of the first excitation beam, so as to superpose said excitation portion on the second excitation beam at the exit of the line. late.
- the delay line may be arranged to adjust the optical path traveled by the second excitation beam, so as to superimpose this beam on the excitation portion of the first excitation beam, at the output of the delay line.
- the first and second excitation beams at the output of the delay line, are superimposed spatially and temporally, at least three chromophores can be excited simultaneously by an excitation using simultaneous absorption of photons originating from:
- the module according to the invention is inexpensive.
- the additional delay line does not imply any real additional costs since it is a common optical element of the trade.
- the at least three respective chromophores can be excited by multi-photon absorption of several photons from:
- chromophores having very varied absorption wavelengths (for example at least 50 nm between two excitation wavelengths) according to the choice of the first and the second laser source.
- absorption wavelengths for example at least 50 nm between two excitation wavelengths
- tissues labeled with several fluorescent proteins whose emissions range from blue to red can be imaged rapidly and efficiently.
- the simultaneous excitation of the chromophores also allows their simultaneous detection. We can thus image the at least three chromophores belonging to a sample in which occur rapid movements, present at the scale of the second, without being affected by these movements during a comparison of the images or signals obtained for each chromophore.
- separation means are arranged upstream of the second femtosecond laser source to separate the first excitation beam into the excitation part and the pumping part.
- upstream and downstream refer to the direction of propagation of the first excitation beam.
- the separation means may comprise a dichroic mirror, to separate:
- the pumping part of the first excitation beam the pumping part of the first excitation beam
- the delay line may then in particular comprise a meeting element such as a dichroic mirror, arranged downstream of the second femtosecond laser source, to replace the delayed excitation beam on the same optical path as the non-excitation beam. delayed.
- a meeting element such as a dichroic mirror
- the delay line is preferably arranged outside the second femtosecond laser source. It can also be considered that the excitation portion and the pumping portion of the first excitation beam are at least partially merged.
- the first excitation beam passes through the second femtosecond laser source, then acting as a pump beam. At the output, it is at least partially reused as an excitation beam. In this case, it is possible to dispense with said separation means.
- the delay line can then be located directly inside the second femtosecond laser source.
- the delay line may be downstream of the second femtosecond laser source.
- means must be provided for separating the first excitation beam and the second excitation beam at the output of the second laser source. One of the two excitation beams is brought to the delay line. Then, the two excitation beams are recombined.
- the first femtosecond laser source is formed by an oscillator, for example a Titanium Sapphire laser (TiS) or a fiber laser.
- an oscillator for example a Titanium Sapphire laser (TiS) or a fiber laser.
- This type of femtosecond laser has a broad spectrum of emission in the near infrared, often centered around 800 nm.
- the second femtosecond laser source may be formed by an optical parametric oscillator (OPO).
- OPO optical parametric oscillator
- An optical parametric oscillator is a source implementing nonlinear optical interactions from a pump signal formed here by a portion of the first excitation beam emitted by the first femtosecond laser source.
- An advantage of OPOs is that they give access to wavelengths that are difficult to reach (wavelength greater than 1000 nm) with other types of laser sources.
- the OPO being pumped by the first femtosecond excitation beam, it can in turn emit a second excitation beam also femtosecond.
- the module according to the invention is adapted to excite chromophores emitting so-called "fluorescence" return signals spaced at least 50 nm apart from each other, where each return signal is expressed in units of wavelength.
- Each return signal has a peak centered on a wavelength which is that taken into account to measure said spacing of at least 50 nm.
- the module according to the invention may further comprise at least one telescope arranged to implement a spatial overlap, in the sample, of the second excitation beam and the excitation portion of the first excitation beam.
- the at least one telescope is preferably arranged in the optical path of one of the second excitation beam and the excitation portion of the first excitation beam.
- the telescope may be part of the delay line.
- a telescope can be provided on the optical path of the second excitation beam alone, and a telescope on the optical path of the excitation portion of the first excitation beam alone.
- the second and the first excitation beam have different wavelengths. In the absence of particular measurement and when these wavelengths are very far apart (for example more than 300 nm), they could be focused each to a different depth in the sample.
- the two excitation beams in the sample would not be spatially overlapped. It would therefore not be possible to obtain a return signal corresponding to the absorption of at least one photon of the first excitation signal and at least one photon of the second excitation signal.
- the at least one telescope makes it possible in particular to independently control the relative sizes and divergences of the second excitation beam and the excitation portion of the first excitation beam. It is thus possible to correct a possible offset of the second excitation beam and the excitation portion of the first excitation beam, in the axis of the depth of the sample.
- the first femtosecond laser source emits a first excitation beam at a wavelength (central) ⁇ able to excite one first of the two-photon absorption chromophores, said absorbed photons from the first excitation beam;
- the second laser source emits a second excitation beam at a (central) wavelength ⁇ 2 capable of exciting a second one of the two-photon absorption chromophores, said absorbed photons coming from the second excitation beam;
- the optical delay line is arranged to spatially and temporally superpose the second excitation beam and the excitation portion of the first excitation beam, so as to excite a third of the two-photon absorption chromophores, the two photons coming from one of the first excitation beam and the other of the second excitation beam.
- Two-photon absorption phenomena are thus used (also referred to as “2PEF” for "2-photon excited fluorescence”), which are most commonly used, in particular in the field of multi-photon microscopy.
- This embodiment is not limiting, and it may for example also consider the implementation of three-photon absorption phenomena (also referred to as “3PEF” for "3-photon excited fluorescence”).
- 3PEF three-photon absorption phenomena
- the invention also relates to a multi-photonic imaging system comprising a module according to the invention, and further comprising detection means with at least three channels, each channel being arranged to detect a respective return signal associated with a multi-absorption -photons corresponding.
- the number of channels depends on the number of chromophores detected (the term "number of chromophores” refers to a number of types of chromophores).
- the number of chromophores that can be detected depends on the number of photons used by the multi-photon absorption (it is considered that each chromophore can be excited by a distinct wavelength).
- For a two-photon absorption three combinations are possible (AA photons, BB photons, AB photons) so the system according to the invention comprises three channels.
- the signals received by each of the channels can be processed to separate for each channel a dedicated return signal and a parasite return signal corresponding to the return signal dedicated to a spectrally adjacent channel.
- the latter comprises in particular three-channel detection means
- the multi-photonic imaging system according to the invention forms one of:
- the invention is not limited to one application in particular, but can be applied to various multi-photon imaging geometries.
- the invention also relates to a method implemented in a module according to the invention. According to this method, the adjustment of the delay line is adjusted so as to spatially and temporally superimpose the second excitation beam and the excitation portion of the first excitation beam, by detecting the appearance of a corresponding return signal. at the excitation of at least a third of the chromophores by multi-photon absorption, said absorbed photons coming from the first and second excitation beams.
- the excitation of said at least one of the chromophores assumes simultaneous absorption by said chromophore of at least one photon coming from the first excitation beam and at least one photon coming from the excitation portion of the first beam of excitation. It is therefore necessary that the second excitation beam and the excitation portion of the first excitation beam are spatially and temporally superposed. The adjustment of the delay line is therefore particularly simple to implement.
- this method implements two-photon absorptions, and three return signals corresponding respectively to:
- the absorption probability simultaneous one photon of each of the two excitation beams is higher or lower. It follows that the intensity of the corresponding return signal is higher or lower. It is therefore possible to adjust the intensity of said return signal without modifying the intensities of the two other return signals respectively corresponding to the absorption of two photons of the first excitation signal or two photons of the second excitation signal.
- the relative intensity of the three return signals can be adjusted independently by adjusting respectively:
- the method according to the invention can be implemented to excite chromophores emitting so-called "fluorescence" return signals at least 50 nm apart from each other, where each return signal is expressed in units of wavelength.
- FIG. 1 illustrates an embodiment of a module and a system according to the invention
- FIG. 2 illustrates the first and second excitation beams in the absence of a delay line implementing their spatial and temporal superposition
- FIGS. 3A to 3D illustrate the multi-photon absorptions implemented in a module and system embodiment according to the invention
- FIG. 4 illustrates excitation spectra of different fluorescent proteins
- FIGS. 5A and 5B illustrate various acquisitions that can be obtained thanks to the module and system according to the invention
- FIG. 6 illustrates excitation spectra of different fluorescent proteins, and the spectral widths of detection of the associated detection channels
- FIG. 7 illustrates return signals acquired in a system according to the invention, by varying a time delay between the first and second excitation beams.
- FIGS. 8A to 8D illustrate an optimal spatial overlap test between the first and the second excitation beam.
- module 1 for a multi-photon imaging system 100.
- a module 1 for simultaneously imaging three chromophores of a sample through two-photon absorptions.
- this example is in no way limiting and it may be provided to implement the module and the imaging system according to the invention in the at least three photon absorption frame, in order to simultaneously image four chromophores at the same time. less than one sample.
- the module 1 comprises a titanium oscillator: sapphire 2 (Ti: S) emitting a first excitation beam 20 at a wavelength ⁇ equal to 820 nm.
- the titanium oscillator: sapphire 2 is a femtosecond laser, that is to say, emitting a pulsed signal whose pulses have a width of the order of ten or even a hundred femtoseconds.
- the emission wavelength ⁇ of this oscillator is chosen in particular to be able to excite by multi-photon absorption at least one chromophore of a sample to be studied.
- the titanium oscillator: sapphire 2 (Ti: S) must be excited by an excitation diode.
- the module 1 comprises only a single excitation diode.
- a portion of the first excitation beam 20 is fed to an optical parametric oscillator (OPO) 3 to serve as a pump beam.
- OPO optical parametric oscillator
- a dichroic mirror 41 arranged at 45 ° (angle in degrees where 180 ° is ⁇ radians) on the optical path of the first excitation beam 20 upstream of the OPO separates from a part this part called “pumping" of the first excitation beam, and other by a so-called "excitation" part of the first excitation beam.
- the optical parametric oscillator (OPO) 3 then emits a second excitation beam 30 at a wavelength ⁇ 2 equal to 1180 nm.
- the emission wavelength ⁇ 2 of this oscillator is chosen in particular to be able to excite by multi-photon absorption at least one chromophore of the sample to be studied.
- the two emission wavelengths ⁇ and ⁇ 2 of these oscillators are chosen in particular to be able to excite by multi-photon absorption mixing these two wavelengths, at least one chromophore of the sample to be studied.
- the dichroic mirror 41 arranged at 45 ° on the optical path of the first excitation beam 20 directs the excitation portion of the first excitation beam to a delay line 4 of the module 1 according to the invention.
- the delay line 4 comprises at least two reflectors 42 defining an additional optical path for the excitation portion of the first excitation beam 20.
- the delay line further comprises a second dichroic mirror 43, arranged to replace the second excitation beam 30 and the excitation portion of the first excitation beam 20 on the same optical path.
- the dichroic mirror 43 is arranged at 45.degree. ° on the optical path of the second excitation beam and the excitation portion of the first excitation beam.
- the second excitation beam passes through the dichroic mirror 43 without being deflected.
- the excitation portion of the first excitation beam is deflected at 90 ° by the dichroic mirror 43.
- first excitation beam 20 the part of the first excitation beam 20 which has been brought towards the delay line 4 will simply be called "first excitation beam 20".
- first excitation beam 20 the part of the first excitation beam 20 which has been brought towards the delay line 4.
- second excitation beam 30 and the first excitation beam 20 are not superimposed. In fact, these two beams are of course spatially superimposed.
- Optical elements such as a telescope 90 within the delay line 4 may be provided to provide spatial overlap of the pulses by controlling the size and divergence of the exciting portion of the first beam of the beam. excitation, independently of the second excitation beam.
- a second telescope 90 ' is also arranged on the optical path of the second excitation beam alone, upstream of the second dichroic mirror 43.
- Each telescope 90, 90 makes it possible to independently adjust the divergence of each excitation beam, and preferably also independently of the size of these beams.
- telescopes with three or four lenses can be used.
- beam conditioning systems are based on active or adaptive optical elements.
- the invention consists in superimposing in space and in time at least two pulse trains of different wavelengths (first and second excitation beams), in a multiphotonic imaging system such as a multi-photon microscope.
- the first and second excitation beams 20, 30 are directed towards scanning means 5 in FIG. the plane (xOy), then scanning means 6 along the axis (Oz) corresponding to the axis of the depth of a sample 7.
- the scanning means 6 also comprise focusing optics for focusing the excitation signals at a desired location in the sample 7.
- Sample 7 comprises at least three chromophores emitting a fluorescence signal in response to absorption:
- a dichroic mirror 80 deflects them to a detection stage comprising a respective channel 81, 81 ', 81 "for each of the three fluorescence signals 82, 82', 82".
- three blue, yellow, red or blue, green, and red fluorescent protein signals can be imaged simultaneously.
- FIG. 2 illustrates the principle implemented according to the invention.
- FIG. 2 illustrates the pulse trains as a function of time, and in the absence of a delay line 4, the first excitation beam 20 (thick lines) and the second excitation beam 30 (dashed lines).
- the delay line makes it possible to cancel the time difference ⁇ t between the two pulse trains.
- a chromophore absorbing a photon of the first excitation beam 20 and a photon of the second excitation beam 30, producing a return signal at the wavelength, ie 484 nm (yellow)
- FIG. 3D illustrates the excitation signals used, in particular the virtual excitation signal artificially recreated by means of the invention.
- the abscissa axis corresponds to a wavelength in nm, the ordinate axis to an arbitrary unit intensity.
- FIG. 4 illustrates on an abscissa axis corresponding to a wavelength expressed in nm (nanometers):
- the excitation signals (ordinate axis in arbitrary unit, corresponding to a light intensity), in particular the virtual excitation signal artificially recreated by means of the invention.
- the excitation signals obtained artificially or not according to the invention correspond to absorption peaks of several fluorescent proteins which will thus be able to be imaged thanks to a module 1 and a multi-photon imaging system 100 according to the invention. invention.
- Curve 401 corresponds to an eCFP protein (for "Enhanced Cyan
- Fluorescent Protein that is to say a protein emitting a return signal
- Curve 402 corresponds to an eYFP protein (for "Enhanced Yellow
- Fluorescent Protein that is to say a protein emitting a return signal
- Curve 403 corresponds to a tdTomato protein (a protein emitting a return signal called "fluorescence" bright red).
- Curve 404 corresponds to a mCherry protein (a protein emitting a return signal called "fluorescence" red cherry).
- mouse brain tissue labeled with these different proteins could be imaged.
- Each excitation wavelength (in particular the so-called “virtual" wavelength and corresponding to said central excitation signal) can excite several different types of chromophores. This appears here for the signal at 1100 nm which corresponds to a maximum of absorption for both mCherry and tdTomato proteins.
- Chromophore names are indicated in parentheses, separated by commas when multiple chromophores can be used.
- FIGS. 5A and 5B A method for adjusting the delay line 4 according to the invention will now be described with reference to FIGS. 5A and 5B. It can be seen in FIG. 1 that the reflectors 42 of the delay line 4 can be mounted on a motorized translation, so as to be able to adjust the adjustment of said delay line in order to superimpose the two excitation beams 20, 30 as closely as possible.
- the delay control induced by the delay line 4 on the first excitation beam 20 also provides a means of adjusting the luminous intensity of the return signal obtained by frequency mixing (absorption of two photons, one coming from the first excitation beam and the other from the second excitation beam), independently of the return signals depending only on a single excitation beam.
- the existence of a third feedback signal obtained by frequency mixing confirms that the first and second excitation signals are spatially and temporally superimposed with a precision equal to the resolution of the multi-photon imaging system 100. We therefore see that the invention offers an "alignment test" particularly simple to implement.
- the control of the average power of the two excitation beams also makes it possible to control the relative intensities of the three return signals.
- the intensities of the so-called fluorescent feedback signals are respectively, proportional to:
- Pi 2 where Pi is the average power of the excitation portion of the first excitation beam 20 (absorption of two photons of the first excitation beam);
- P 2 2 is the average power of the second excitation beam 30 (absorption of two photons of the second excitation beam);
- ⁇ is the delay between the two pulse trains respectively of the first and the second excitation beam, downstream of the delay line (absorption of two photons, one of the part d excitation of the first excitation beam and the other of the second excitation beam), and g is the temporal inter-correlation function of the two excitation beams, proportional to exp (-x 2 ) in the case of excitation beams with Gaussian temporal profiles.
- FIGS. 5A and 5B which each have from right to left: the image obtained by means of the return signal corresponding to a two-photon absorption of the first excitation beam;
- the imaged sample is a Drosophila with a triple fluorescent markings blue, green and red.
- the central image shows the presence of a strong return signal corresponding to an absorption of two photons, one of the first excitation beam and the other of the second excitation beam.
- the central image shows the presence of a very weak return signal corresponding to an absorption of two photons, one of the first excitation beam and the other of the second excitation beam.
- This result can also be used to compensate for so-called "cross talk" interference effects between the first and the second excitation beam, and which tend to reduce the relative intensity of the return signal corresponding to a two-photon absorption, one of the first excitation beam and the other of the second excitation beam.
- This principle can also be used to maintain constant over time the absolute intensities of the three return signals as detected, during a depth scan of a sample (thus varying a beam attenuation coefficient). excitation and feedback signals).
- the acquisitions are obtained with pixels of dimensions 0.8 * 0.8 * 3 pm 3 .
- three-dimensional images are produced by scanning in the direction of the depth of the sample, for example a three-dimensional image every 45 seconds.
- Figure 6 has the additional advantage of simultaneous multi-channel detection.
- the abscissa axis corresponds to a wavelength in nm.
- the ordinate axis corresponds to a return signal intensity, in arbitrary unit.
- Curves 601, 602 and 603 correspond to return signal spectra.
- Curve 601 corresponds to an endo protein emitting a return signal called "fluorescence" blue).
- Curve 602 corresponds to a GFP (for "green fluorescent protein” protein, that is to say a protein emitting a green fluorescence return signal.
- Curve 603 corresponds to an RFP protein (for "red fluorescent protein", that is to say a protein emitting a red fluorescent "return” signal.
- the dotted slots 611, 612, 613 correspond respectively to:
- the spectral width detected by the detection channel dedicated to the red return signal is the spectral width detected by the detection channel dedicated to the red return signal.
- the emission being simultaneous, the detection is also simultaneous. For each channel (here more particularly for the channel corresponding to the slot 612), it is therefore possible to separate the contributions of the different return signals.
- RAI is the normalized (known) contribution of the chromophore A in the C1 and C2 channels
- RB1 is the normalized (known) contribution of the chromophore A in the C1 or C2 channel
- Cl and C2 are measured intensities.
- - A and B are intensities to be determined.
- FIG. 7 illustrates the return signals 404, 401 and 402 of FIG. 4.
- the abscissa axis corresponds to the femtosecond delay ⁇ between the second excitation beam and the excitation portion of the first excitation beam, at the output of the delay line.
- the y-axis corresponds to the intensity in arbitrary units.
- an additional return signal (corresponding to a multi-photon absorption of photons coming from both the first and the second excitation beam) indicates an optimal setting of the temporal overlap between the first and second excitation beams.
- FIGS. 8A to 8D more particularly illustrate an optimal spatial overlap test between the second excitation beam, and the excitation portion of the first excitation beam, downstream of the delay line.
- FIG. 8A illustrates the focusing zones 81 and 82, respectively, of the first and second excitation beams, when the spatial overlap is not achieved between these two beams.
- Figure 8B shows the image obtained in this case. We obtain two distinct signals, one red and the other blue.
- FIG. 8C illustrates the focusing zones 181 and 182, respectively, of the first and second excitation beams, when the spatial overlap is made between these two beams.
- Figure 8D shows the image obtained in this case. A single white signal is obtained, corresponding to the superposition of three distinct signals, red, blue and green.
- the blue return signal and the red return signal are successively acquired, then the images corresponding to these signals are superimposed on the same image.
- the images respectively corresponding to the red signal and to the blue signal can be deformed and deformed differently, in particular at the edge of the field of view (these deformations come from the impact, on the corresponding excitation signals, chromatic aberrations displayed by the focusing optics 6).
- an offset is made between the focusing distance of the first excitation signal and that of the second excitation signal. For this, we seek to detect a blue signal and a red signal from the same focusing plane (the two signals each corresponding to a multi-photon absorption of several photons from the same excitation signal).
- the time offset between the two excitation signals is canceled, thanks to the delay line.
- the appearance of a third signal corresponding to a multi-photon absorption of at least one photon of the first excitation signal and at least one photon of the second excitation signal indicates when this offset is canceled.
- the return signals are always observed in the center of the field of view, where they are not deformed by chromatic aberrations.
- FIGS. 8A to 8D it is possible to verify at the edge of the field of vision from which angle the chromatic aberrations of the focusing optics become troublesome for a good overlap of the two excitation signals, by using the test illustrated by FIGS. 8A to 8D. More precisely, we gradually move away from the center of the field of view and we spot the disappearance of the third return signal. Thanks to the invention, a final image is obtained grouping the various return signals much closer to reality. Indeed, the absence of the third feedback signal shows that we have reached an area of the field of view corresponding to distorted red and blue images. We can therefore remove this part of the return signal and keep only the return signal unaffected by chromatic aberrations.
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FR1250990A FR2986668B1 (fr) | 2012-02-02 | 2012-02-02 | Module d'excitation multi-couleurs pour un systeme d'imagerie multi-photonique, systeme et procede associes. |
PCT/EP2013/052002 WO2013113861A1 (fr) | 2012-02-02 | 2013-02-01 | Module d'excitation multi-couleurs pour un systeme d'imagerie multi-photonique, systeme et procede associes |
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JP6307903B2 (ja) * | 2014-02-04 | 2018-04-11 | 株式会社Ihi | 物質特定方法および物質特定システム |
JP6740131B2 (ja) | 2014-02-21 | 2020-08-12 | スリーディインテグレイテッド アーペーエス3Dintegrated Aps | 手術器具を備えたセット、手術システム、及びトレーニング方法 |
JP6776327B2 (ja) | 2015-07-21 | 2020-10-28 | スリーディインテグレイテッド アーペーエス3Dintegrated Aps | カニューレアセンブリキット、套管針アセンブリキット、スリーブアセンブリ、低侵襲性手術システム及び方法 |
US11020144B2 (en) | 2015-07-21 | 2021-06-01 | 3Dintegrated Aps | Minimally invasive surgery system |
DK178899B1 (en) | 2015-10-09 | 2017-05-08 | 3Dintegrated Aps | A depiction system |
JP6729878B2 (ja) | 2016-04-05 | 2020-07-29 | ウシオ電機株式会社 | 多光子励起用スーパーコンティニウム光生成光源、多光子励起用スーパーコンティニウム光生成方法、多光子励起蛍光顕微鏡及び多光子励起方法 |
FR3067524B1 (fr) | 2017-06-09 | 2019-07-26 | Centre National De La Recherche Scientifique | Dispositif et procede de microscopie multiphotonique |
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US6369928B1 (en) * | 2000-11-01 | 2002-04-09 | Optical Biopsy Technologies, Inc. | Fiber-coupled, angled-dual-illumination-axis confocal scanning microscopes for performing reflective and two-photon fluorescence imaging |
DE10120425C2 (de) * | 2001-04-26 | 2003-12-18 | Leica Microsystems | Scanmikroskop |
DE10151217B4 (de) * | 2001-10-16 | 2012-05-16 | Carl Zeiss Microlmaging Gmbh | Verfahren zum Betrieb eines Laser-Scanning-Mikroskops |
JP2009150649A (ja) * | 2005-03-29 | 2009-07-09 | Osaka Univ | 空間情報検出装置 |
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JP2008076361A (ja) * | 2006-09-25 | 2008-04-03 | Toshiba Corp | 顕微鏡装置及び分析装置 |
US8554035B2 (en) * | 2006-10-26 | 2013-10-08 | Cornell Research Foundation, Inc. | Production of optical pulses at a desired wavelength using soliton self-frequency shift in higher-order-mode fiber |
US8994932B2 (en) * | 2008-12-20 | 2015-03-31 | Purdue Research Foundation | Multimodal platform for nonlinear optical microscopy and microspectroscopy |
JP2011141311A (ja) * | 2010-01-05 | 2011-07-21 | Nikon Corp | 多光子顕微鏡 |
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- 2013-02-01 EP EP13705115.7A patent/EP2810050A1/fr not_active Ceased
- 2013-02-01 WO PCT/EP2013/052002 patent/WO2013113861A1/fr active Application Filing
- 2013-02-01 US US14/376,040 patent/US20140367591A1/en not_active Abandoned
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CAMPAGNOLA P J ET AL: "High-resolution nonlinear optical imaging of live cells by second harmonic generation", BIOPHYSICAL JOURNAL, ELSEVIER, AMSTERDAM, NL, vol. 77, no. 6, 1 December 1999 (1999-12-01), pages 3341 - 3349, XP002248209, ISSN: 0006-3495 * |
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JP6254096B2 (ja) | 2017-12-27 |
FR2986668B1 (fr) | 2014-02-21 |
WO2013113861A1 (fr) | 2013-08-08 |
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