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1. WO2020156671 - APPAREIL ET PROCÉDÉ DE MANIPULATION D'UN FOYER DE LUMIÈRE D'EXCITATION SUR OU DANS UN ÉCHANTILLON ET MICROSCOPE

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

Claims

1 Apparatus for manipulating a focus of excitation light on or in a sample (10), particularly in a microscope (700; 800; 1000), comprising

a light source (L) for emitting excitation light (12),

an excitation beam path (20) for guiding the excitation light (12) onto or into the sample (10),

the excitation beam path (20) comprising an objective (21 ) for guiding the exci- tation light (12) onto or into the sample (10) and a wavefront modulator (40) for modulating the excitation light (12),

and a control device (50) for driving the wavefront modulator (40),

characterized in

that the control device (50) is designed for driving the wavefront modulator (40) to generate a number of shaped waves (61 , 62, 63) on or in the sample (10), that a focus (30) is generated at a specified location (31 , 32) on or in the sam- ple (10) by superposition of the shaped waves (61 , 62, 63) and

that, for manipulating the location (31 , 32) of the focus (30) on or in the sam- ple (10), a device (70) for imposing variably stepped phase shifts (f1 , f 2, f 3 ) upon the shaped waves (61 , 62, 63) is present, where the phase shifts (f1 , f 2,, f 3) imposed in each case on the shaped waves change stepwise between dif- ferent shaped waves (61 , 62, 63).

2. Apparatus according to claim 1 ,

characterized in

that each of the shaped waves (61 , 62, 63) is corrected for influences of the sample (10) such that the shaped waves resemble planar wavefronts in a focal plane on or in the sample (10).

3. Apparatus according to claim 1 or 2,

characterized in

that the device (70) for imposing variably stepped phase shifts upon the shaped waves or at least a component of this device (70) is arranged in a plane (1211 ) which is optically conjugate to a plane (1209) where the wavefront modula- tor (40) is arranged.

4. Apparatus according to one of the claims 1 to 3,

characterized in

that a lenslet array (1206) is present which is in particular arranged in a plane (1207) that is optically conjugate to a plane (1209) where the wavefront modulator (40) is arranged and/or to a plane (1211 ) where the device (70) for imposing variably stepped phase shifts are at least a component of this de- vice (70) is arranged.

5. Apparatus according to claim 4,

characterized in

that the lenslet array (1206) is arranged at a distance from a pupil plane which is equal to or at least approximately equal to a focal length (f1 ) of the individual lenslets (1208) of the lenslet array (1206).

6. Apparatus according to one of the claims 1 to 5,

characterized in

that the device (70) for imposing variable stepped phase shifts comprises at least one separate wavefront modulator which is, in particular, arranged in or near a plane which is optically conjugate to the plane where the wavefront mod- ulator (40) is arranged.

7. Apparatus according to one of the claims 1 to 6,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises

at least one electro-optic component (471 ), in particular at least one anisotropic crystal, with a stepped thickness, arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is ar- ranged, and

a driving device (475) for applying varying voltages (U(x), U(y)) to the at least one electro-optic component (471 ) to bring about varying magnitudes of the steps of the imposed phase shifts (f1 , f 2, f 3).

8. Apparatus according to claim 7,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises

a first stepped electro-optic component (471 ), arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modula- tor (40) is arranged, where the thickness of the first stepped electro-optic com- ponent (471 ) increases stepwise in a first direction (x) perpendicular to the di- rection (z) of the optical axis and

a second stepped electro-optic component (472), arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront mod- ulator (40) is arranged, where the thickness of the second stepped electro-optic component (472) increases stepwise in a second direction (y) perpendicular both to the direction (z) of the optical axis and the first direction (x)

9. Apparatus according to one of the claims 1 to 8,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises

at least one stacked gradient refractive-index glass component (G1 ), arranged in particular in or near a plane (573) which is optically conjugate to the plane where the wavefront modulator (40) is arranged, and

at least one x-y-scanner (S1 ),

wherein, for imposing variably stepped phase shifts upon the shaped waves (61 , 62, 63), the x-y-scanner (S1 ) guides the excitation light (12) onto different sec- tions of the at least one stacked gradient refractive-index glass component (G1 )

10. Apparatus according to claim 9,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises

a first stacked gradient refractive-index glass component (G1 ), arranged in par- ticular in or near a plane (573) which is optically conjugate to the plane where the wavefront modulator (40) is arranged, where the gradient of the refractive- index increases in a first direction (x) perpendicular to the direction (z) of the optical axis and

a second stacked gradient refractive-index glass component (G2), arranged in particular in or near a plane (574) which is optically conjugate to the plane where the wavefront modulator (40) is arranged, where the gradient of the refractive- index increases in a second direction (y) perpendicular to the direction (z) of the optical axis and perpendicular to the first direction (x).

11. Apparatus according to one of the claims 9 or 10,

characterized in

that optical means, comprising in particular at least one cylindrical lens, are pre- sent for forming a light sheet of excitation light (12) and for guiding the light sheet onto different sections of a stacked gradient refractive-index glass com- ponent (G1 , G2).

12. Apparatus according to one of the claims 1 to 11 ,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises at least one array (671 ) of corner mirrors with a one-dimensional structure, arranged in particular in or near a plane (672) plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged.

13 Apparatus according to claim 12,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3) comprises

a first array (671 ) of corner mirrors (671 ) with a one-dimensional structure, ar- ranged in particular in or near a plane (672) which is optically conjugate to the plane where the wavefront modulator (40) is arranged, where a direction of the one-dimensional structure is oriented in a first direction perpendicular to the di- rection of the optical axis and

a second array of corner mirrors with a one-dimensional structure, arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged, where a direction of the one-dimensional structure is oriented in a second direction perpendicular to the direction of the optical axis and perpendicular to the first direction

14 Apparatus according to one of the claims 1 to 13,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises an x-y-scanner (772), arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is ar- ranged, and a digital-mirror-device (771 ), positioned in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged.

15 Apparatus according to one of the claims 1 to 14,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises an x-y-scanner (872), arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is ar- ranged, a lenslet array (873), and a fixed mirror (874),

wherein the excitation light (12) is guided from the x-y-scanner (872) via the lenslet array (873) to the fixed mirror (874), is then reflected by the fixed mirror (874) back through the lenslet array (873) and is then guided via the x-y- scanner (872) in the direction of the objective (21 ).

16. Apparatus according to claim 15,

characterized in

that the fixed mirror (874) is tilted with respect to an optical axis,

that a beam splitter (890) is provided between the wavefront modulator (40) and the x-y-scanner (872) and

that the light reflected from the fixed mirror (874) is deflected, eg. by total reflec- tion, by the beam splitter (890) in the direction of the objective (21 ).

17. Apparatus according to one of the claims 1 to 16,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises

a first x-y-scanner (972), arranged in particular in or near a plane which is opti- cally conjugate to the plane where the wavefront modulator (40) is arranged, a lenslet array (973) and a lens (974) arranged downstream of the first x-y-scan- ner (972) and

a second x-y-scanner (975), arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged, downstream of the lenslet array (973) and the lens (974).

18. Apparatus according to one of the claims 1 to 17,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises

an x-y-scanner, arranged in particular in or near a plane which is optically con- jugate to the plane where the wavefront modulator (40) is arranged,

a spatial filter with a plurality of apertures downstream of the x-y-scanner and a lenslet array downstream of the spatial filter.

39. Apparatus according to claim 18,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises additionally a lenslet-device between the x-y-scanner for focusing excitation light (12) into apertures of the spatial filter

20. Apparatus according to one of the claims 1 to 19,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises at least

one staircase-shaped mirror (1074), arranged in particular in or near a plane (1075) which is optically conjugate to the plane where the wavefront mod- ulator (40) is arranged, where the heights (h(x)) of the stairs (1076) increase in one direction (x),

and an x-y-scanner (1072),

wherein, for imposing variably stepped phase shifts upon the shaped waves (61 , 62, 63), the x-y-scanner (1072) guides the excitation light (12) onto different sections of the staircase-shaped mirror (1074).

21. Apparatus according to claim 20,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises

a first staircase-shaped mirror (1074), arranged in particular in or near a plane (1075) which is optically conjugate to the plane where the wavefront mod- ulator (40) is arranged, where the heights (h(x)) of the stairs (1076) increase in a first direction (x) perpendicular to the direction (z) of an optical axis and a second staircase-shaped mirror, arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged, where the heights of the stairs increase in a second direction (y) per- pendicular to the direction (z) of the optical axis and perpendicular to the first direction (x).

22. Apparatus according to one of the claims 20 or 21 ,

characterized in

that at least one of the staircase-shaped mirrors consists of a plurality of stair- case-elements of approximately equal heigths.

23. Apparatus according to one of the claims 20 to 22,

characterized in

that optical means, comprising in particular at least one cylindrical lens, are pre- sent for forming a light sheet of excitation light (12) and for guiding the light sheet onto different sections of a staircase-shaped mirror (1074).

24. Apparatus according to one of the claims 1 to 23,

characterized in

that a beam splitter (890; 1090), preferably a polarizing beam splitter or a 50/50- beam splitter, is arranged downstream of the wavefront modulator (40)

25. Apparatus according to one of the claims 1 to 24,

characterized in

that the centers (64, 65, 66) of the intensity distributions corresponding to each of the shaped waves (61 , 62, 63) in the plane of the wavefront modulator (40) are localized on a non-uniform grid.

26. Apparatus according to one of the claims 1 to 25,

characterized in

that the magnitude of the imposed phase shifts (f1 , f 2, f 3) on each of the shaped waves is a linear function of the position of the center (64, 65, 66) of the intensity distribution of the respective shaped waves (61 , 62, 63) in the plane of the wavefront modulator (40).

27. Apparatus according to one of the claims 1 to 26,

characterized in

that the control device (50) is designed for driving the wavefront modulator (40) such that the shaped waves (61 , 62, 63) resemble apodized plane waves on or in the sample (10)

28. Method for manipulating a focus of excitation light on or in a sample (10),

particularly in a microscope (100),

comprising the steps of

guiding the excitation light (12) on an excitation beam path (20) to an objec- tive (21 ),

guiding, by means of the objective (21 ), the excitation light (12) onto or into the sample (10),

manipulating, by means of a wavefront modulator (40) in the excitation beam path (20), the excitation light (12),

characterized in

that the wavefront modulator (40) is driven to generate a number of shaped waves (61 , 62, 63) on or in the sample (12),

that stepped phase shifts (f1 , f 2, f 3) are imposed upon the shaped waves (61 , 62, 63), the phase shifts (f1 , f 2, f 3) changing stepwise between different shaped waves (61 , 62, 63), and

that, for manipulating the location (31 , 32) of the focus (30) on or in the sample (10), the magnitude of the steps of the phase shifts (f1 , f 2, f 3) between differ- ent shaped waves (61 , 62, 63) is varied

29. Method according to claim 28,

characterized in

that each of the shaped waves (61 , 62, 63) is corrected for influences of the sample (10) such that the shaped waves resemble, in each case, planar wave- fronts in a focal plane on or in the sample (10).

30. Method according to claim 28 or 29,

characterized in

that the location of the focus is manipulated in three dimensions.

31. Method according to one of the claims 28 to 30,

characterized in

that, for manipulating the location (31 , 32) of the focus (30) on or in the sam- ple (10), only the magnitude of the steps of the phase shifts (f1 , f 2, f 3) be- tween different shaped waves (61 , 62, 63) is varied.

32. Method according to one of the claims 28 to 31 ,

characterized in

that the wavefront modulator (40) is driven such that the shaped waves are ad- ditionally subjected to phase shifts which emulate the effect of a lenslet array.

33. Method according to one of the claims 28 to 32,

characterized in

that stepped phase shifts (f1 , f 2, f 3) are variably imposed on the shaped waves (61 , 62, 63) with the same wavefront modulator that generates the shaped waves (61 , 62, 63).

34. Method according to one of the claims 28 to 33,

characterized in

that a separate wavefront modulator, preferably a wavefront modulator that, compared with the wavefront modulator (40) generating the shaped waves (61 , 62, 63), is faster and can be operated in a lower resolution, is used to bring about varying magnitudes of the steps of the imposed phase shifts ( (f1 , f 2, f 3).

35. Method according to one of the claims 28 to 34,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3) comprises at least one electro-optic component (471 ), in particular at least one anisotropic crystal, with a stepped thickness, arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modula- tor (40) is arranged, and

that varying voltages are applied to the at least one electro-optic compo- nent (471 ) to bring about varying magnitudes of the steps of the imposed phase shifts (f1 , f 2, f 3)

36. Method according to one of the claims 28 to 35,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3) comprises at least one stacked gradient refractive-index glass component (G1 ), arranged in particular in or near a plane (573) which is optically conjugate to the plane where the wavefront modulator (40) is arranged, and

that, to bring about varying magnitudes of the steps of the imposed phase shifts (f1 , f 2, f 3) , the excitation light (12) is directed to varying sections of the at least one stacked gradient refractive-index glass component (G1 ).

37. Method according to one of the claims 28 to 36,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3) comprises at least one array (671 ) of corner mirrors with a one-dimensional structure, arranged in particular in or near a plane (672) which is optically con- jugate to the plane where the wavefront modulator (40) is arranged, and that, to bring about varying magnitudes of the steps of the imposed phase shifts (f1 , f 2, f 3), the at least one corner mirror (671 ) with a one-dimensional structure is variably tilted.

38. Method according to one of the claims 28 to 37,

characterized in

that, for imposing the variable stepped phase shifts upon the shaped waves (61 , 62, 63), the excitation light (12) is guided via an x-y-scanner (772), arranged in particular in or near a plane which is optically conjugate to the plane where the

wavefront modulator (40) is arranged, and a digital-mirror-device (771 ), posi- tioned in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged,

wherein each of the segments of the digital-mirror-device (771 ) is operated in coordination with the x-y-scanner (772) to cancel out phase ramps imposed by the x-y-scanner (772)

39. Method according to one of the claims 28 to 38,

characterized in

that, for imposing the variable stepped phase shifts upon the shaped waves (61 , 62, 63), the excitation light (12) is guided via an x-y-scanner (872), arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged, and via a lenslet array (873) to a fixed mirror (874),

that the excitation light (12) is then reflected by the fixed mirror (874) back through the lenslet array (873) and is then guided via the x-y-scanner (872) in the direction of the objective (21 ),

wherein, for varying the magnitude of the steps of the phase shifts (f1 , f 2, f 3) between different shaped waves (61 , 62, 63), the x y-scanner (872) is operated.

40. Method according to one of the claims 28 to 39,

characterized in

that, for imposing the variable stepped phase shifts upon the shaped waves (61 , 62, 63), the excitation light (12) is guided via a first x-y-scanner (972), arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged, a lenslet array (973) and a lens (974) arranged downstream of the first x-y-scanner (972) and a second x-y-scan- ner (975), arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged, downstream of the lenslet array (973) and the lens (974), and

wherein, for varying the magnitude of the steps of the phase shifts (f1 , f 2, f 3) between different shaped waves (61 , 62, 63), the first x-y-scanner (972) and the second x-y-scanner (975) are operated, and

wherein the second x-y-scanner (975) is operated in coordination with the first x-y-scanner (972) to cancel out phase-ramps imposed onto the shaped waves (61 , 62, 63) by the first x-y-scanner (972)

41. Method according to one of the claims 28 to 40,

characterized in

that, for imposing the variable stepped phase shifts (f1 , f 2, f 3) upon the shaped waves (61 , 62, 63), the excitation light (12) is guided via an x-y-scanner, arranged in particular in or near a plane which is optically conjugate to the plane where the wavefront modulator (40) is arranged, onto a spatial filter with a plu- rality of apertures,

that the excitation light transmitted through the plurality of apertures is colli- mated with a lens and guided further in the direction of the microscope objec- tive (21 ) and

that, for varying the magnitude of the steps of the phase shifts (f1 , f 2, f 3 ) between different shaped waves (61 , 62, 63), the x y-scanner is operated.

42. Method according to claim 41 ,

characterized in

that the excitation light (12) is focused with a lenslet array into the apertures of the spatial filter

43. Method according to one of the claims 28 to 42,

characterized in

that the device (70) for imposing variable stepped phase shifts (f1 , f 2, f 3 ) comprises at least one staircase-shaped mirror (1074), arranged in particular in or near a plane (1075) which is optically conjugate to the plane where the wave- front modulator (40) is arranged, where the heights (h(x)) of the stairs (1076) increase in one direction (x), and

that, to bring about varying magnitudes of the steps of the imposed phase shifts (cp1 , f2, cp3), the excitation light (12) is guided onto different sections of the staircase-shaped mirror (1074)

44 Method according to one of the claims 36 to 43,

characterized in

that a light sheet of excitation light (12) is formed and that the light sheet is guided onto different sections of the staircase-shaped mirror or the stacked gra- dient refractive-index glass component, respectively.

45 Method according to one of the claims 28 to 44,

characterized in

that the centers (64, 65, 66) of the intensity distributions corresponding to each of the shaped waves (61 , 62, 63) in the plane of the wavefront modulator (40) are localized on a non-uniform grid

46 Method according to one of the claims 28 to 45,

characterized in

that the magnitude of the imposed phase shift (f1 , f 2, f 3) on each of the shaped waves (61 , 62, 63) is a linear function of the position of the center (64, 65, 66) of the intensity distribution of the respective shaped waves (61 , 62, 63) in the plane of the wavefront modulator (40)

47 Method according to one of the claims 28 to 46,

characterized in

that at least some of the shaped waves (61 , 62, 63) resemble apodized plane waves in a focal plane on or in the sample (10).

48 Microscope, particularly a nonlinear microscope, comprising

an apparatus (201 ; 202; 203) for focusing excitation light (12) onto or into a sample (10) according to one of the claims 1 to 27, where the objective is a microscope objective (21 ),

a detection beam path for guiding detection light (17), in particular fluorescence light, in the direction of a detector (220),

the detector (220) for detecting the detection light (17), and

a control unit (18) for controlling the apparatus (201 ; 202; 203) for focusing ex- citation light (12) and the detector (220) and for evaluating the detection data received from the detector (220).

49. Microscope according to claim 48,

characterized in

that the detection beam path includes a main beam splitter (210), particularly a dichroic beam-splitter, for the separation of excitation light (12) and detection light (17).

50. Microscope according to one of the claims 48 or 49,

characterized in

that the detector (220) is arranged in a non-descanned portion of the detection beam path.

51. Microscope according to one of the claims 48 to 50,

which is a multi-photon-fluorescence microscope, in particular a 2-photon-fluo- rescence microscope or a 3-photon-fluorescence microscope, a SHG-micro- scope, a THG-microscope, or a CARS-microscope.