Wavefront shaping for digital light sheet microscopy

Abstract

The present invention relates to a light sheet microscope, comprising one or more excitation laser beams (4); a volume defined by a coordinate system (x'(8), y'(12), z'(35)) containing an object plane (1); a component (38) for forming an excitation beam having a wavefront and power / amplitude density distribution from the excitation laser beam (4), the excitation laser beam (4) propagating in the object plane (1) as one or more continuous or modulated or discontinuous excitation beam waists (TWIs) extending along the x'(8) direction; and one or more components for translating the excitation beam waists (TWIs) in the object plane (1). The device comprises (6); at least one camera detector having at least one active region; a detection objective (7) for imaging an emission excited in an object plane (1) onto the camera detector, the emission including an emission excited by a telemetry beam waist (TWI); and a component for dynamically limiting at least one active region on the camera detector, characterized in that the telemetry beam waist (TWI) is coherently irradiated from one side with respect to the x' (8), z' (35) plane of the volume, and the image of the emission excited by the telemetry beam waist (TWI) is synchronized with at least one active region of at least one camera detector.

Description

Technical Field

[0001] This invention relates to a variant for beam shaping of light sheets generated by a scanning process of light sheet microscopy. Background Technology

[0002] Light sheet microscopy has evolved into a wide field with various implementations. It uses a thin sheet of light to excite only fluorophores located within the focal volume of the examination objective. This light sheet can be static, typically shaped using distorting optics, or dynamic, shaped by scanning. This is known as digital scanning light sheet microscopy. (Stelzer et al., [Light sheet fluorescence microscopy. Stelzer, EHK, Strobl, F., Chang, BJ.]) et al. An overview is given in the review [Nat Rev Methods Primers 1, 73 (2021)]. Light sheet microscopy is affected by the fact that the excitation light propagates within the image plane. An ideal light sheet would have to be uniformly constrained to be perpendicular to the image plane. In the case of digital scanning light sheet microscopy, uniformity in the scanning direction is generated over the time average of the scanning cycle. However, uniformity along the propagation direction is only possible in the case of so-called non-diffracting beams [Exact solutions for nondiffracting beams. I. The scalar theory. J. Durnin, Journal of the Optical Society of America Vol. 4, Issue 4, pp. 651-654 (1987)].

[0003] The most commonly used diffraction-free beam is the Bessel beam, which has a narrow straight line with a central intensity maximum surrounded by concentric cylindrical rings, where the integrated intensity in each ring is approximately equal to the integrated intensity of the central intensity. Strong sidelobes are detrimental to the requirements of light sheet microscopy. Another type of diffraction-free beam is achieved in lattice light-sheet microscopy, where a one-dimensional array of parallel Bessel beams is added to generate a structured diffraction-free intensity distribution. The distance between individual beams is chosen to partially suppress out-of-plane sidelobes. [Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution, E. Betzig] et. al.[SCIENCE, 24 Oct 2014, Vol 346, Issue 6208 and details of the theory in the supplementary material]. Lattice light sheets can be used in two main modes. One is as a structured illumination mode, where the 'lattice' is used to generate an illumination pattern in the sample, which can be moved in its transverse phase to generate a set of images that can be reconstructed into a single image with improved axial resolution. The other is the so-called 'jitter' mode, where the lattice is temporarily equalized by rapid transverse oscillations (jitter). Lattice light sheet illumination is a 'self-healing' beam of unlimited length. The supplementary material of the aforementioned paper states that 'neither an ideal Bessel beam nor an ideal 2D optical lattice can be directly used in light sheet microscopy'. The solution is a Bessel-Gaussian beam, which provides 'a hybrid of the properties of both: suppression of higher-order Bessel sidelobes due to the Gaussian envelope'… and a beam waist much longer than that of a conventional Gaussian beam due to the elimination of the F of the wave vector corresponding to the kp value within the inner diameter of the ring in Figure 12'. Layer cutting and field of view are inversely related variables in lattice light sheet microscopy. Bessel-Gaussian beams use finite ring illumination with a Gaussian radial intensity distribution.

[0004] Other implementations of the light sheet utilize Airy beams. Here, the accelerating phase generates a curved, continuous intensity distribution with a strong radial sidelobe pattern. Airy beams are not diffract-free along straight lines and are subject to strong sidelobe effects.

[0005] In digital scanning light sheet microscopy, layer cutting and stray light suppression can be achieved by synchronizing the slit-type rolling shutter of an sCMOS camera with the image of the scanning beam [DE 10 2010 013 223 B4]. This patent describes the use of focusing light and the use of a Bezier beam synchronized with the rolling shutter.

[0006] A variation of this method is outlined in the article [A shape-switch-block method for confocallight-sheet microscopy with sectioned Bessel beams and stimulated emission depletion, Köbele and Rohrbach, Communications Physics, 2020 3:201]. It uses a segmented Bessel beam combined with rolling shutter readings. Rolling shutter synchronization can also be applied to quasi-static light sheet systems as the focal region moves continuously along the propagation direction of the light sheet [US000011156822B2, DE102010 013 223 B4 - claim 11].

[0007] The use of conventional cylindrical focusing provides no stray light suppression because the entire area is simultaneously illuminated and detected. The Rayleigh length of the cylindrical focus must be adapted to the object to be imaged. Therefore, imaging a large field of view with a thin film is impossible. Both of these aspects are partially addressed by method [US000011156822B2], in which the waist position of the cylindrical focus of the film can vary along the propagation of the excitation light and is synchronized with the rolling shutter. Here, the width of the rolling shutter can be adapted to the Rayleigh length of the waist. In one dimension, the rolling shutter always collects light from the entire width of the field of view. The stray light suppression factor can be calculated as a fraction of the rolling shutter width relative to the full field of view of the sensor.

[0008] The use of a scanning focusing beam (Gaussian or flat-top illumination pupil) combined with a rolling shutter synchronized with the scan introduces the need to acquire a series of images, where the focal position must move along the propagation direction. This makes acquisition slow but provides ideal suppression of scattered light and can be used to introduce confocalization. The disadvantage here is that, due to variations in beam diameter, the confocalization varies with position within the beam.

[0009] US 2018 / 0292321 also describes extending the waist by translating it along the propagation axis. Furthermore, the modulator can be used to homogenize or synchronize the light source, or as a mask in the Fourier plane to generate a laser line focus. Also mentioned is the angular positioning and mechanical scanning of the coverslip relative to the objective lens.

[0010] The method of homogenizing the intensity distribution of the excitation beam using a digital micromirror device (DMD) by employing multiple elliptical double-arched DMD patterns is known in [Tommaso Galgani. Selective and volumetric fluorescence microscopy for the localization of single molecules in crowded environments. Physics [physics]. Université Paris sciences et lettres, 2021. French. ⟨NNT :2021UPSLS086> . ⟨tel-03847722>].

[0011] [JP2020502558A] describes light-sheet theta microscopy (LSTM) in which the excitation beam is tilted to the sample plane and the detection objective is kept perpendicular to the sample surface. The linear region is illuminated by the light sheet, where line readout from the camera and scanning along the transverse direction and the propagation direction of the light sheet are performed simultaneously by a holographic spatial light modulator.

[0012] The drawback of the aforementioned methods is that they require mechanical scanning and time averaging during the scanning process, which takes a considerable amount of time. They also do not offer any measures to improve the thickness of the film.

[0013] All the methods mentioned are affected by the fact that Rayleigh length is finite for thin light sheets. One distinction is the use of Bessel beams versus segmented Bessel beams. However, this has drawbacks, namely that strong sidelobes impair the shearing, and lateral structures require very precise synchronization of the rolling shutter [A shape-switch-block method for confocal light-sheet microscopy with sectioned Bessel beams and stimulate demission depletion, Köbele and Rohrbach, Communications Physics, 2020 3:201; Fig. 1b). Bessel beams utilize a conical wavefront, which excites each single point within the sample by light propagating along the surface of the cone. Bessel beams are said to be 'self-healing'. This is true if a local obstruction blocks or interferes with the beam. Larger-scale overall deformation of the beam will not 'self-heal' and will compromise the shearing capability of the Bessel beam. Here, cylindrical, Gaussian, or flat-top focusing are more robust options with the aforementioned limitations. Using segmented Bezier bundles reduces some of the problems associated with Bezier bundles, but layer cutting reduces the full-size utilization of the excitation objective pupil perpendicular to the light sheet. Summary of the Invention

[0014] The invention disclosed in this application relates to a new paradigm for illuminating samples within a digital scanning light sheet microscope, which overcomes the shortcomings of the aforementioned prior art light sheet microscopes. It is referred to as a tilted waist illumination light sheet (TWI).

[0015] It simplifies microscope setup and improves slice cutting by making full use of the aperture of the excitation objective perpendicular to the light plate.

[0016] The excitation light is formed in a way that reduces the risk of distortion during beam propagation through the sample.

[0017] It provides a high degree of suppression of scattered and out-of-focus light. It reduces the synchronization accuracy requirements between scanner movement and rolling shutter synchronization. The variant can also effectively suppress sidelobes at higher shear quality levels.

[0018] The object of the present invention is a light sheet microscope comprising one or more excitation laser beams (4); a volume defined by a coordinate system (x'(8), y'(12), z'(35)) containing an object plane (1); components (38) for forming the excitation laser beam (4), the excitation laser beam (4) having a wavefront and power / amplitude density distribution in one or more continuous or modulated or discontinuous excitation beam waists propagating into the object plane (1); one or more components (6) for translating the excitation beam waists in the object plane (1); at least one camera detector having at least one active area; and a detection objective (7) for... The emission excited in the object plane (1) is imaged onto a camera detector, the emission including emission excited by an excitation beam waist, and components for dynamically limiting at least one active region on the camera detector, characterized in that the one or more excitation laser beams (4) are arranged along the x' (8) axis and coherently irradiate the volume from one side of the z' (35) plane relative to the x' (8) plane as one or more tilted excitation beam waists (TWI), and the emission excited by the one or more excitation beam waists (TWI) along the x' axis is synchronized with the acquisition performed by at least one active region of at least one camera detector.

[0019] In a variant of the invention, component (38) generates an excitation beam having a power / amplitude density distribution having at least one 10-179° arc or elliptical arc shape in the Fourier transform of the wavefront and amplitude distribution relative to the volume in the y'(12), z'(35) plane and / or the x'(8), z'(35) plane. In other words, the excitation beam may have a power / amplitude density distribution in the shape of a Bessel beam fan-shaped with a central angle of 10-179°. Attached Figure Description

[0020] Figure 1 The general geometric layout of the optical system of the present invention is described. Figure 2 The geometry of the elongated excitation beam waist (8) is shown. Figure 3 An illumination plane with a series (25) of inclined illumination fine beams according to the present invention is shown.

[0021] Figure 4 (a) and (b) illustrate a Bessel bundle according to the prior art, while 4c illustrates an embodiment of the present invention.

[0022] Figure 5 (a) shows the geometry of the continuous TWI beams in the object region (10).

[0023] Figures 6 to 9 Simulated data of the yz cross section of the TWI beam and the vertical and horizontal center profiles are shown (a).

[0024] Figure 10 illustrates the robustness of the TWI bundle associated with rolling shutter readout.

[0025] Figure 11 An example is shown in which the present invention is used for robust STED de-excitation of out-of-plane excitation of TWI excitation beams.

[0026] Figure 12 shows the discontinuous variant of the TWI bundle.

[0027] Figure 13 (a) and (b) show a simple but functional version of the tilted waist bundle (TWI) in a quasi-continuous form.

[0028] Figure 14 An embodiment of the invention is shown, wherein the excitation light is imaged from the pivot point of the scanner (6) to the back focal plane of the excitation objective (26).

[0029] Figure 15 An embodiment of the invention is shown, wherein the collimation of the excitation light can be adapted on a scanner plane that introduces a variable propagation distance (21).

[0030] Figure 16 It shows the variation of the middle cone half angle A variant that does not change the position of the intermediate TWI bundle (17).

[0031] Figure 17 An embodiment of the invention is shown, wherein two light sheets are generated within the same sample.

[0032] Figure 18 A variant without a scanning eyepiece is shown.

[0033] Figure 19 Another embodiment of the invention is shown, in which a tilt angle of the object plane is introduced, which results in a tilt angle between the object plane and the image plane of the detection objective.

[0034] Figure 20 An embodiment of the invention is shown, wherein a light sheet is generated and imaged using the same objective lens.

[0035] Figure 21 An embodiment of the invention is shown, wherein an additional tilt angle (39) is introduced between the TWI beam and the detection plane. Detailed Implementation

[0036] Variants of the invention are claimed, which achieve cost-effective means for generating excitation beams. These variants also address the need for flexibility in terms of acquisition speed, sensitivity, and tomography. It provides a simple method for axially confining the excitation beam and adapting it to object and objective lens sizes.

[0037] Other aspects of the invention implement a method for suppressing shadows in an image.

[0038] Further variations describe different technical implementations of the invention, using refractive lenses and / or aspherical elements, combinations of refractive and diffractive optical elements, such as reflective or transmissive DOEs, or programmable SLMs. DOEs or SLMs can also be used in two-way geometry to redistribute the intensity distribution of the incident beam and define the desired wavefront. Other variations of the invention relate to methods utilizing different coherent behaviors or different polarization modes. These methods always utilize relative motion between the TWI beam and the object, and synchronization with the sensor readout region. Relative motion (which itself is prior art) can be achieved by a galvanometer or MEMS mirror scanner, or also by sliding object or beam movement. In the case of beam scanning or movement, the restriction of the readout region can be achieved by so-called rolling shutter synchronization [DE 10 2010 013 223 B4] or by synchronized movement of a slit near or in the middle of the image plane. Synchronization is also possible if the sample is moved or if the image is scanned by means of a second relative movement implemented in the detection system. To collect light emitted from the waist of the TWI beam, a line camera or a time-delay integration (TDI) camera can be used.

[0039] To generate a light sheet, the excitation laser beam (4) is typically scanned using a scanner (6) and a scanning eyepiece (5), which is usually a telecentric F-θ objective or an achromatic objective, such that the propagation direction lies in the object plane (1), and the scanning motion moves the beam laterally in the object plane (see [reference]). Figure 1 ).

[0040] The axis of the scanning eyepiece and the normal to the object plane (1) (typically the axis of the detection optics) define a plane (3) that is perpendicular to the pupil plane (2) of the scanning eyepiece and also perpendicular to the object plane. This plane will be referred to below as the propagation detection plane (PD plane). For the definition of the coordinate system, please refer to [link to relevant documentation]. This invention relates to the design of an excitation beam asymmetric relative to the PD plane. This asymmetry may be related to the intensity and / or phase and / or polarization and / or coherence of the excitation laser. The excitation light generates a beam waist that extends along a straight line located in the object plane.

[0041] The waist of the beam can have a tilt angle relative to the excitation direction x. (9). According to the invention, the tilt angle can be in the range of 0 degrees and 45 degrees or even 60 degrees, ideally between 0 degrees and 30 degrees (see...). Figure 2 The region (10) imaged onto the detector (22) is oriented according to the elongation of the beam tail (8), and thus in Rotation relative to the normal of the object plane (1) when the value is not zero. Introducing a new coordinate system. It is advantageous (see Figures 1 to 4 In (8) (12) and (35) .exist In the case of, by The defined plane differs from the PD plane. A fundamental aspect of this invention is the excitation light contributing to the TWI beam from the... Planar coordinate system Half of the space radiation. This means that relative to... In the 2D Fourier space of a plane, only half of the planes exhibit non-zero values.

[0042] In one embodiment of the invention, the excitation beam generates a series of focal points (narrow beams) aligned along the beam waist, wherein the focal points may not be limited to Gaussian focal points, but may also be focal points with extended depth of focus (EDOF) or short Bezier-type focal points. (To be continued...) Figure 3 A more general description is given. The spacing between the focal points can be as large as needed, so that they do not exhibit significant mutual interference near the beam waist, thus forming a well-separated 'light chain' of beams. The intent of this embodiment of the invention is that the light from a single fine beam passes laterally across the beam waist line in the object plane. The fluorescence or scattered light generated by the excitation of the individual fine beams near the beam waist is then collected by the active area of ​​the synchronously rolling shutter. This corresponds to a well-defined sub-region in the acquired frame. This is as follows: Figure 3The sum of contributions from all the fine beams results in image data not limited by the Rayleigh length of a single fine beam. The fact that the fine beams pass laterally through the line connecting all the focal points disrupts the symmetry of the entire excitation beam containing all the fine beams. A series of fine beams are focused into a series of focal points. These focal points are arranged in a row, staggered laterally and axially. The invention includes this discrete but also continuous variant, as well as all intermediate variants. This will be explained in the next section.

[0043] In a more general description of the discrete embodiments of the present invention, the excitation beam electric field E Ex It can be described as: Equation 1 in It is the electric field of a single narrow beam in the sample. It is a position vector, and It is a unit vector along the waist direction, and and The list of real values ​​is thus generated and assigned to a single phase. Location A thin beam of light being translated. People will typically choose... and To generate equidistant positions and a constant phase difference, and It is independent of i. The excitation beam intensity distribution is then related to... Proportional. In incoherent cases, for example, if a broadband excitation source is used, or if the distance is large enough to reduce the interference. Therefore, simply adding up the intensities of each fine beam is sufficient.

[0044] If there is substantial interference between the fine beams in the sample region that is imaged onto the region (11) corresponding to the rolling shutter, the phase selection between the fine beams can be chosen based on the overall uniformity of the excitation.

[0045] In another embodiment of the invention, a continuous implementation of the excitation beam according to claim 1 is proposed. Equation 1 can be rewritten in a continuous form: Equation 2 here It is the wavenumber vector along the waist direction. If The center of the imaging area is taken at The imaging region then represents the effective elongation of the excitation beam. For ease of understanding rather than limitation, we assume that phase propagation is linear. A more simplified description can be obtained if the integral is extended to infinity. This reveals significant constraints: Equation 3 Normalization is not considered in the following equations. The field has the properties of a freely propagating wave. Given this, a coordinate transformation is meaningful, where the direction of the waist is along the coordinate axis x' of the new coordinate system. (direction). At the same time, one can write the equations for the other two coordinates (y', z') relative to the Fourier transform form: Equation 4 By using The propagation operator, as the wavenumber of the excited laser beam in a medium with refractive index n, can be used to redistribute the integral to a plane. The value of .

[0046] Equation 5 Integrals only display non-zero values. .therefore Equation 6 .

[0047] Because in Fourier space, for objects that are not in the coordinate system... The value of k on the circle, all values ​​disappear, where x' is the coordinate axis of the waist, and the wavefront of the wave generated by Equation 3 has a conical shape. If now through the waist Add propagation operators in the direction to propagate the field. For a wave at the center of , the following equation applies: Equation 7 Under the constraints of equation 6 Equation 8 .

[0048] This also applies to the non-Fourier transform version of the equation. Equation 8 shows the case with arbitrary waves. This general setup of the excitation beam always produces a diffraction-free beam. This means that the intensity along the propagation axis... The direction is constant. In the selection In the case of generating the waist, the intensity distribution of this waist is constant along the propagation axis. The parameter to be chosen is the angle of the wavefront cone. Equation 9, as well as Another parameter to select is Intensity distribution. The beam waist direction is typically within the imaging plane, and the intensity is relative to the object plane. The symmetry is advantageous. The advantage lies in the polar coordinates. (in and The generated beam is defined in the code as being related to the object plane. Then the symmetric beam will be generated by... generate.

[0049] Examples of such bundles are the Bessel bundles and segmented Bessel bundles described in [COMMUNICATIONS PHYSICS | https: / / doi.org / 10.1038 / s42005-020-00458-3 (Luise Köbele, Alexander Rohrbach) Fig. 1b)], where the propagation axis of the bundle waist is collinear with the optical axis of the excitation objective, and the wave is relative to the propagation detection plane (PD plane (3), see Figure 1 It has rotational / mirror symmetry. This means that in the prior art, it has relative to the propagation detection plane (PD plane (3), see Figure 1 The bindings of symmetry with respect to the object plane are subject to and One aspect of the invention reduces the TWI beam to The value or even a lower value: Equation 10 .

[0050] This has the effect that the lighting on the waist comes only from one side, and therefore within the waist... In direction and also Very limited or zero interference occurs in the direction.

[0051] if yes Figure 1 Given the unit vector along the waist in the coordinate system depicted, the conical phase in the pupil can be written as... Equation 11 in Equation 12 and ,and In the plane perpendicular to the axis of the scanning eyepiece ,in Equation 13 Equation 14 The aperture of the scanning eyepiece limits the usable z-value. This depends on the numerical aperture of the scanning eyepiece. and its focal length In paraxial approximation Below, among them , where n is the refractive index of the medium. If the wavefront at the apex of the pupil, i.e., at that point, points towards the center of the imaging field, then the maximum total numerical aperture can be utilized.

[0052] Equation 15 When the partial derivative at that point When this condition is met, the following equation and the partial derivatives of equation 13 are introduced. Equation 16 It can be deduced that maximizing the use of the pupil is important for... The following formula: Equation 17 Please note, The useful optimization values ​​can only be derived from Equation 17, if The pupil region that contributes to the waist is limited to the region of interest in the image plane, which has an elliptical shape in the pupil plane (13).

[0053] Use the lower portion of the provided numerical aperture This is also part of the present invention. In this case, it is still possible to use [the method] which has [the function] according to the insertion [of the device]. Modification of Equation 16 Calculate the value of equation 17 In this case, using a lower value is also acceptable. It also includes .

[0054] In this case, where And optimized The maximum value can be calculated based on equations 17 and 13. , from Equation 15 Inserting Equation 14, thus leading to Equation 18 Since the layering of the generated TWI beams mainly depends on It can be increased To reduce (See) Figure 7 and Figure 9 (Example in the text). The value is not related to .Increase Also increased This will be the angle between the TWI beam and the scanning direction. In cases where the object region has a high aspect ratio, a high value of up to 60° or higher may be beneficial.

[0055] One aspect of the invention is the breaking of the symmetry relative to the PD plane. This is always the case, if For example, as described in Equation 17. However, if And this is also true if the intensity is not symmetrical with respect to the PD plane, as described in Equation 10. A key aspect is shown together with the Bessel bundle (a) and the segmented Bessel bundle (b) in Figure 4 In (c), the wave distribution in the Fourier plane (13) is parallel to the excitation pupil plane (E plane (2), see Figure 1 The intensity distribution at the center of the image is shown together. It is evident that the choice of intensity distribution and local angular distribution in the pupil on the elliptical illumination line affects the waist length along x', the layering capability, and the intensity of the potential sidelobes with the maximum central intensity. Intensity modulation in the image plane can be suppressed, and the excitation scheme is robust to jitter errors of the scanner relative to the rolling shutter motion. There is no major limitation on the waist length. There is no limitation related to the Rayleigh length. If Equation 17 is taken into account, the full potential of layering can be provided in the direction perpendicular to the object plane (1) using the scanning eyepiece (5).

[0056] Figure 6 The calculated properties of the TWI beam are shown. Here, the excitation wavelength is 488 nm, and water is used as the immersion medium (n = 1.33), according to Equation 17. , and Used. According to Equation 18 (a) shows the intensity distribution of the TWI beam in the yz plane, with the central beam profile in both the z and y directions. (b) shows the range of the active region for imaging to the rolling shutter detector. The intensity distribution along the z-direction is obtained from the inner integral. A Gaussian profile fitted to this profile shows the FWHM at 430 nm along the z-direction. As an example, given a detection magnification of 20x and 4... The detector pixel size will make the number of active rows of the camera appear as (c) shows the intensity in Fourier space, and... Figure 4 (c) Equivalent. If as Figure 6 As shown in (d), by relative to the azimuth angle By smoothing the intensity using a truncated Gaussian intensity distribution, sidelobes can be suppressed without significant shear loss. All other parameters are related to those derived from... Figure 6 The same parameters are used in the calculations shown in (ac).

[0057] for Figure 6 The calculations shown are based on the selected conditions. , , The relationship between them is plotted in Figure 7 In China. All. , The excitation elliptic segment is generated on the Fourier plane, i.e., on a scanner utilizing the full vertical dimensions of the aperture. The elliptic segment connects the upper and lower vertices of the pupil. The layer cutting of the generated light sheet does not strongly depend on... The choice. On the other hand, if increasing The useful width of the rolling shutter must be adapted to a larger value. The TWI beam leaves no excitation notches above and below the beam waist. The full angle of the notch is... The cone of the detection light collected by one or more detection objectives should be smaller than the angle of the notch. Figure 7 It is shown that it is possible to select one of them. A reasonable small configuration without compromising layer cutting.

[0058] Figure 8 shows a version of macroscopic imaging in a transparent solution (parameters: 488 nm, n = 1.55). , and The readout width is And according to Equation 18 Figure 8(d) shows a variant of the TWI sheet with Gaussian illumination that results in reduced side lobes. Figure 9 The conditions selected for the calculations shown in Figure 8 are illustrated. , , The relationship between them.

[0059] Figure 10 illustrates the robustness of the TWI bundle associated with rolling shutter readout. (a) shows a variant of the slice profile with relative movement of the fringes, which is integrated by the active region of the rolling shutter detector. Slice reduction occurs, but as shown in (b), the intensity stabilizes with relative movement. This will not occur with Bezier bundles or segmented Bezier bundles, making the TWI method very robust.

[0060] Figure 13 (a) and (b) show a simple but functional version of a tilted waist beam (TWI) in a quasi-continuous form. The incident collimated beam strikes the axial cone (14) at an off-axis position (22). The intermediate beam waist (17) is formed to have a length that depends on the width of the incident beam. At this position, two segmented apertures (24) can be inserted to confine the intermediate waist. The central beam propagates relative to the axis of the axial cone at an intermediate cone half-angle (19). The rear lens (15) is at a potential tilt angle. (18) Placement. The value of this angle may be between 0 and... Between. In the current example, the distance from the lens to the center of the intermediate beam waist (20) is the focal length of the lens. If the scanner is placed on the focal plane of the lens (15), the subsequent propagation distance (21) results in a circular or elliptical focal line and telecentric illumination on the scanner (6). The beam is then reflected toward the scanning eyepiece (5) and generates an image of the intermediate beam waist (TWI) within the object region (1). The tilt angle of the TWI beam relative to the optical axis of the scanning eyepiece is... (18) and the magnification introduced by lenses (15) and (5) are proportional. The beam is always asymmetrical with respect to the PD plane, and... The choice is irrelevant. The term Tilted Waist Illumination (TWI) refers to this asymmetry, which explains the fact that the waist is illuminated primarily from one side within the object plane (1).

[0061] In all embodiments of the invention, the use of an axial cone can be replaced by conical or spherical reflective surfaces, as well as toroidal, elliptical, and parabolic reflective surfaces and combinations thereof. An aspherical component of one or more such surfaces can be used to further modify the distribution of light along the TWI axis. This can be used to generate more uniform (flat-top) illumination along the TWI axis, thereby producing overall uniform sensitivity within the field of view.

[0062] Figure 14 One implementation of the TWI beam setup is shown, in which a microscope objective (26) is used to generate the TWI beam. Here, the position of the scanner (6) is imaged onto the back focal plane of the excitation objective (26) via intermediate imaging optics (5, 17, 5). The placement of the pivot point is important for aligning the TWI beam with the detector line. The constraint of the TWI beam can also be achieved by a segmented aperture (24) close to the intermediate image plane (17), as shown in Figure 12(a).

[0063] No other variations are shown here. A beam geometry can be achieved using two scanners with parallel axes, where a pivot point can be addressed at a distance from the scanner surface. Using this method, the use of intermediate imaging optics can be omitted.

[0064] Various variations of the scanning scheme can be employed by those skilled in the art: scanning can also be achieved through the relative movement of the excitation lens (26 or 5) along with part of the excitation optics relative to the object and part of the detection optics. In some cases, the image of the TWI beam waist will be moved relative to the detector. Descanning mechanisms and / or time-delay integration (TDI) cameras can also be used.

[0065] Further variations of the invention include a cone angle along the waist. Variations and components that dynamically change these variations. The variable cone angle must be inserted into Equation 11. Figure 15 One solution described is to leave the telecentric condition within the object space of the scanning eyepiece (5) or excitation objective (26). This allows the waist to be... The direction can be focused or expanded to fit the size of the sample or sub-region to be examined. It would also be advantageous to implement a magnification changer or a change in the inspection objective. Cone angle. It is related to the useful width of the activity detection area (the number of activity detector rows in rolling shutter mode).

[0066] Dynamically alter the characteristics of the TWI beam, especially towards smaller values. Change This is also part of the present invention. Figure 16 This demonstrates the possibility of radially moving the point where the excitation beam strikes the axial cone by synchronous movement of the axial cone (14) and a mirror (39) parallel to the axis of the axial cone. This parallel movement maintains the position of the intermediate TWI beam (17) but reduces the intermediate cone half-angle. This can be used to increase the width of the TWI beam. The width of the rolling shutter can also be increased. The useful width of the rolling shutter is related to... The correlation is approximately quadratic (squared) in nature.

[0067] The wave according to the invention can be generated by various means. Among them are single-pass or two-pass spatial light modulators (SLMs) and diffractive optical elements (DOEs). The conventional optical element to be used is also an axonocone. Details are described in the accompanying drawings.

[0068] In another embodiment of the invention, more than one excitation laser source is used. Wavefront shaping can be accomplished by using separate diffractive optical elements of different regions on the same diffractive optical element. A relatively small unilateral elliptical shape of the wavefront is advantageous. This makes it possible to shape several laser sources using relatively small diffractive optical elements. The combination of light sources can be accomplished by a dielectric beam combiner, where a prism with a geometric angle β is individually selected for each wavelength, its value being the same as γ. The elliptical beam then strikes the scanner plane at different locations but will achieve the same position for the TWI beam. Different wavelengths can also strike the scanner at different angles, resulting in different but parallel positions for the TWI beam at each wavelength. It is advantageous to use more than one camera and to split the light onto different cameras using a dielectric beam splitter. Multicolor fluorescence imaging can then be performed simultaneously by synchronizing the different cameras with the different TWI beams from different excitation lasers. The fact that the tilt angle of the TWI beam can be very small allows for almost complete suppression of crosstalk between channels.

[0069] In the present invention Figure 17In another embodiment shown, two light sheets are generated within the same sample, and imaging is performed on more than one camera using more than one objective lens (7). Imaging is typically performed from opposite sides. The two waists (8) of the excitation sheet can also be arranged parallel or collinear, provided that the two axes of the illumination objective lens (5) are not collinear.

[0070] In this embodiment, at least two excitation laser beams (4) with the same wavelength are used to incoherently irradiate the excitation beam waist (TWI) from opposite directions. This can be achieved by separating one excitation laser beam (4).

[0071] TWI beam tilt relative to illumination axis Typically, this will be half the angle between the axes of the two illumination objectives (5). The advantage is that the small imaging chamber (29) inserted between the two detection objectives can move parallel to the lines of the TWI beam and the two detectors, and multiple detection regions (10) can be addressed. Wavefront preparation and intensity distribution of the incident beam (4) are not shown in the figure. This setup can be equipped with two scanners (6). Scanning of the two TWI beams can be performed simultaneously or sequentially. Alternatively, it is possible to use only one scanner and split the light exiting the scanner into two parts, but this is not shown in the figure. This separation can be achieved by a beam splitter or by separating a symmetrical wave into two parts, as shown in Equation 10. Contributed to one of the TWI beams, and This contributes to the second TWI beam. It can also be achieved by first separating the beams and then scanning both beams with two separate scanners. A basic setup without using the TWI beam is known from the system provided by VIVENTIS (Lausanne).

[0072] exist Figure 18 In another embodiment of the invention shown, the use of a scanning eyepiece is omitted. Here, a beam, generated by the axon cone (14) or other components, providing the desired wavefront and intensity distribution, is scanned by two reflections on the same scanner. A variation is shown here in which the beam strikes the scanner (8) from opposite positions. The intermediate reflection on the mirror (30) and the second reflection on the scanner result in compensation for the scanning angle. Thus, pure parallel movement of the TWI beam is achieved. Therefore, this setup is very simple and does not suffer from distortion from the scanning eyepiece. High TWI beam numerical aperture can be achieved for large fields of view. This setup may be ideal for macroscopic light sheet imaging. Alternatively, but not shown in the figure, the scanner motor can be equipped with two mirrors facing each other, such that the beam is first reflected by one mirror and then by the other mirror. Here, the scanner angle is also compensated, and parallel movement is achieved.

[0073] The invention can also be applied if the angle between the excitation and detection objectives is not 90°. Here, compensation for distortion caused by the tilted image plane must typically be utilized. This can lead to trapezoidal distortion in the image. The problem here is that the line simultaneously illuminated on the sensor by the TWI beam may rotate in the image space during scanning. One embodiment of the invention also addresses this limitation. By abandoning the telecentric condition in the image space of the scanning eyepiece, the TWI can be adapted to the trapezoidal distortion image on the detector, thus enabling synchronization with the rolling shutter (see...). Figure 19 ).

[0074] The present invention also includes the use of light-sheet microscopy to generate light sheets and to image them using the same objectives (Dunsby, C. Optically sectioned imaging by oblique plane microscopy. Opt. Express 16, 20306 (2008), see also). Figure 20 ).

[0075] In another embodiment of the invention, an STED TWI beam with wavelength 1 is used to excite the sample, while a second modified STED TWI beam with wavelength 2 is used to deplete fluorescence by de-excitation of the excited molecules. The modification of the second beam is... In phase A step transition results in destructive interference in the TWI beam plane at wavelength 1. This is similar to the implementation by Luise Köbele and Alexander Rohrbach (cited above). The difference is that, for this invention using both excited TWI and STED-TWI beams, no lateral structure is introduced. This makes the setup robust and facilitates the use of STED-TWI beam saturation to confine fluorescence emission to a thin layer within the TWI beam. The cone angle can be chosen differently for the excited TWI and STED-TWI beams. And azimuth intensity distribution. This can be used to optimize the effect of the STED-TWI beam. For example, a Gaussian focus can be generated using the Gaussian envelope of the excitation TWI beam, which is then compressed by the two symmetric maxima of the STED TWI beam, resulting in high-resolution tomography without sidelobes. Figure 11 The STED-TWI beam cross-section (a) and z-profile (b) considering rolling shutter readout are shown. The parameters used for calculation are... , , , and , In addition, a Gaussian envelope was applied. (c) shows a diagram with the same parameters but... The excited TWI beam, along with the STED TWI beam (b) and an exemplary depleted TWI profile. There are no major limitations on the potential length of the TWI beam. Specifically, adding to the STED-TWI beam... The truncated Gaussian envelope in the direction helps generate robust de-excitation because the unwanted minimum between sidelobes is reduced. In another embodiment of the invention, it can be used to generate a primary dual-color activation photoinitiator layer, as used in the XOLO printing system. The scanned tilted sheet can then be synchronized with the vertical illumination of the structured activation light, for example, using a DLP projector or light structured by a fast-scanning acousto-optic deflector. The cross-sections of the TWI activation beam and the projected polymer beam will be identical along the entire length of the TWI beam. The intensity of the TWI beam can be adapted along the TWI beam without illuminating volumes closer to the light source, and thus absorbing less. Completely uniform printing is possible. TWI sheet activation and imaging can be combined to guarantee long-term uniform activation and high-resolution quality control of the print volume.

[0076] Description of the accompanying drawings / executives Figure 1 The general geometric layout of the optical system is described, wherein the object plane XY (1) (referred to as the I plane in the figure), the excitation pupil plane YZ (2) is referred to as the E plane, the propagation-detection plane (XZ) (3) is referred to as the PD plane, the excitation laser beam (4), the scanning eyepiece (SO) (5), the scanner (6), and the detection objective (DO) (7) are used to describe the prior art and the present invention.

[0077] Figure 2 The geometry of the elongated excitation beam waist (8) is described, which may have an angle of inclination relative to the excitation direction X. (9). The image region (10) is aligned with the waist direction (8). In the telecentric case, the region (11) corresponding to the camera's rolling shutter propagates along the Y' (12) direction perpendicular to the waist direction (8) along the sensor's row. The excitation direction may be tilted relative to the optical axis of the excitation objective.

[0078] Figure 3 An illumination plane with a series of tilted illumination beams (25) according to the invention is shown. The waists of the beams are arranged in a straight line. The figure also depicts the area corresponding to the rolling shutter of the camera (11).

[0079] Figure 4 (a) shows a Bezier bundle with a rotationally symmetric intensity distribution and a strong central maximum but relatively strong secondary maximum (lower left sketch). The upper sketch shows a schematic diagram of the Fourier transform of the pupil function. The illumination in the pupil is concentrated on a circle. The arrows depict the area suitable for rolling shutter selection. It must be limited to the central maximum. Figure 4(b) shows a segmented Bezier bundle where a portion of the illumination circle is blocked. This results in a less prominent submaximum, expanding the suitable area for rolling shutter selection, but also shows a less defined layer cut because the height of the aperture is not fully utilized. Figure 4 (c) shows an embodiment of the bundle according to the invention, wherein the tilt axis of the bundle waist is related to the cone angle of illumination in the pupil. Together they are selected, and the illumination is a segment of an ellipse reaching the upper and lower vertices of the pupil.

[0080] Figure 5 (a) The geometry of the continuous TWI beams in the object region (10) is shown in the schematic diagram. At the endpoint (28) of the TWI region, light originates from a beam with a segment angle. One segment of the circle extends through the waist (8) to the other side of the waist, and forms a mirror segment of the same size again at the end of the object region. At the center of the object region (27), the profile is symmetrical, with the incident light on one side and the outgoing light on the other side. This longitudinal and transverse asymmetry is a key aspect of the invention.

[0081] Figures 6 to 9 Simulated data of the yz cross section of the TWI beam and the vertical and horizontal center profiles (a) are shown. The slice profiles take into account the rolling shutter readings but do not include the effects of the detection point spread function.

[0082] Figure 10 illustrates the robustness of the TWI bundle associated with rolling shutter readout. (a) shows a variant of the slice profile with relative movement of the fringes, which is integrated by the active region of the rolling shutter detector. The slice is reduced, but as shown in (b), the intensity stabilizes with relative movement.

[0083] Figure 11 An example is shown in which the present invention is used for robust STED de-excitation of out-of-plane excitation of TWI excitation beams.

[0084] Figure 12 shows a discontinuous variant of the TWI beam, in which a series of 11 beams (here with flat-top illumination of the pupil) are superimposed according to Equation 1, where the tilt angle is... , , , The axial distance between the focal points is 43 μm. The width of the readout region is set to 18 μm. (a) shows a schematic diagram (left) of the yz cross section and Fourier space, which shows that the discrete TWI beam also presents a set of discrete elliptical regions (extended radial structure) in Fourier space. This version produces softer shadows because a larger portion of the pupil is used. (b) shows a top view of the intensity of the central plane of the TWI beam. (c) shows the uniformity of illumination, taking into account scanning and rolling shutter readings. (d) shows the tomography obtained under certain conditions. The different profiles represent different positions between the central focal point and the intermediate position of the next focal point, showing the overall stability of the TWI excitation, as in the discrete case. There is no principle limit to the number of focal points applied, and the tomography is not compromised if the TWI beam is extended. If the multiplexing of the beam is generated by diffractive optics or SLM, individual aberrations can be corrected, especially those that may occur due to the fact that individual beams propagate different distances within the medium. Other aberrations, such as field curvature, tilt, and all effects related to the field coordinates, can also be corrected. The length of the TWI beam can also be dynamically adjusted via SLM and by segmented apertures at the edges in the intermediate image space (see example (24) in Figure 12). This dynamic adaptation can help reduce the total dose of light illuminating the sample and illuminate only where needed. This adaptation can be linked to the individual planes being scanned or to the scanner motion itself.

[0085] Figure 13 (a) and (b) show a simple but functional version of the tilted waist beam (TWI) in a quasi-continuous form. The incident collimated beam strikes the axial cone (14) at an off-axis position.

[0086] Figure 14 An embodiment of the invention is shown, wherein the excitation light is imaged from the pivot point of the scanner (6) to the back focal plane of the excitation objective (26).

[0087] Figure 15 An embodiment of the invention is shown, wherein the collimation of the excitation light can be adapted on the scanner plane by introducing a variable propagation distance (21). This allows the TWI beam waist to... The direction can be focused or expanded to fit the size of the sample or sub-region to be examined.

[0088] Figure 16 It shows the variation of the middle cone half angle A solution that does not change the position of the intermediate TWI bundle (17).

[0089] Figure 17An embodiment of the invention is shown, wherein two light sheets are generated within the same sample, and imaging is performed on more than one camera using more than one objective lens (7). In this example, two excitation beams propagate from opposite sides but at an angle relative to each other. The TWI directions of the two beams are aligned.

[0090] Figure 18 A variant without a scanning eyepiece is shown. Parallel movement of the TWI beam is achieved by double reflection of the incident beam on the scanner (8). A similar result can also be achieved by subsequently using two synchronized scanners with parallel scanning axes.

[0091] Figure 15 An example is shown, where relative to Figure 13 In the telecentric setting shown, the telecentric condition in the object space of the scanning eyepiece (5) has been abandoned due to the increase in propagation distance (21). This angle The beam waist varies along the TWI beam in the sample. Here, the light is concentrated along the waist and illuminates the shorter portions of the sample. The opposite can be achieved by extending the propagation distance (21).

[0092] Figure 19 Another embodiment of the invention is illustrated, in which a tilt angle of the object plane is introduced, resulting in a tilt angle between the object plane and the image plane of the detection objective. Here, the tilt angle is corrected according to the prior art via an externally tilted intermediate image plane. This results in a trapezoidal distortion image on the detector. Parallel lines in the object will not be imaged as parallel lines on the detector. This can be compensated for by moving the pivot point of the scanner along the optical axis of the excitation lens, thereby abandoning the telecentric condition.

[0093] Figure 20 An embodiment of the invention is shown, wherein a light sheet is generated and imaged using the same objective lens. Potential correction for trapezoidal distortion is addressed in a manner similar to that described above.

[0094] Figure 21 An embodiment of the invention is shown, wherein an additional tilt angle (39) is introduced between the TWI beam and the detection plane. For example, This can be achieved by selecting an asymmetric value interval relative to the image plane. Here, the TWI beam is tilted relative to the image plane, which would impair slice cutting if the extended range of the rolling shutter readout line is activated. An advantage is that the sample can be illuminated from a hemisphere. This allows light sheet readout within the microtiter plate without tilting the detection objective or image plane relative to the coverslip of the microtiter plate. A light sheet parallel to the coverslip is generated. Excitation can be performed using a high-NA objective or by utilizing additional optics from the side. The only limitation is the total internal reflection angle on the coverslip. Low-refractive-index materials like FEP can be used instead of the coverslip, allowing for the selection of small tilt angles (39). This setup can be considered a scanning halo microscope, except that the rolling shutter can be used to suppress scattered light. Less than 1 Layer cutting is feasible. This setup can also be used for detection objectives with lower magnification but larger working distances, which have independent excitation optics positioned at an angle relative to the image plane. This angle can also be zero if the axis of the excitation objective lies within the object plane. The lower half of the excitation objective will still be used to illuminate the object plane from below.

[0095] In a similar embodiment not shown in the figure, the z' direction (35) may also be additionally tilted relative to the optical axis z of the detection objective, such that z' ≠ z. The tilt may be relative to x', such that the TWI will still be perpendicular to z, but the scan translation will be in the x'y' plane tilted relative to the axis of the detection objective.

[0096] Glossary 1. Object plane (XY) 2. Excitation Pupil Plane (YZ) 3. Propagation-Detection Plane (XZ) (PD Plane) 4. Excite the laser beam 5. Scanning eyepiece (SO) 6 Scanners 7. Inspection objective (DO) 8 x', the direction of the sloping waist 9. Inclination angle 10. Imaged object region 11. The area illuminated by the waist in the object plane 12 y', perpendicular to X' and Z = Z', with the direction corresponding to the row of the image sensor.

[0097] 13. The pupil of the scanning eyepiece (5) 14-axis cone 15 Lenses 16. Middle waist 17. Mid-waist length 18. Inclination angle from the intermediate waist to the optical axis 19. Half angle of the middle cone 20 First transmission distance 21 Second propagation distance 22 Radial beam displacement 23 Detectors 24 segmented aperture 25 A series of individual bundles 26 Excitation Objective 27 TWI slices / center of object region 28 TWI slices / beginning / end of object region 29 Sample Room 30 Reflectors 31. Detection cone 32 Regions on optics that contribute to the TWI beam 33. X-axis of the scanning eyepiece 34. The y-axis perpendicular to X and Z 35 Z = Z' axis 36. Sloping waist length 37. Focal points of individual fine beams contributing to the TWI beam 38. Components that form the excitation wavefront and power / amplitude density distribution of a laser beam. 39 out-of-plane TWI tilt angle 40 Extended radial structure.

Claims

1. A light sheet microscope comprising one or more excitation laser beams (4); a volume defined by a coordinate system (x'(8), y'(12), z'(35)) containing an object plane (1); a component (38) for forming the excitation laser beam (4), the excitation laser beam (4) having a wavefront and power / amplitude density distribution propagating into one or more continuous or modulated or discontinuous excitation beam waists in the object plane (1); one or more components (6) for translating the excitation beam waist in the object plane (1); at least one camera detector having at least one active region; a detection objective (7) for imaging an emission excited in the object plane (1) onto the camera detector, the emission including an emission excited by the excitation beam waist; and a component for dynamically limiting at least one active region on the camera detector. Its features are, The one or more excitation laser beams (4) are positioned along the x' (8) axis and coherently irradiate the volume from one side of the z' (35) plane relative to the x' (8) to serve as one or more tilted excitation beam waists (TWIs), and the emission excited by the one or more excitation beam waists (TWIs) along the x' axis is synchronized with the acquisition performed by at least one active area of ​​at least one camera detector.

2. The light-sheet microscope according to claim 1, characterized in that, The component (38) generates an excitation beam with a power / amplitude density distribution, wherein the excitation beam has a shape of at least 10-179° of circular or elliptical arc in the Fourier transform of the wavefront and amplitude distribution.

3. The light sheet microscope according to claim 1 or 2, characterized in that, The elliptical or circular power / amplitude distribution has an extended radial structure (40) that forms a distribution of a primary conical wave, the interference of which forms an axial confinement and / or modulation or discontinuous excitation beam waist (TWI) in the object plane (1).

4. The light sheet microscope according to any one of claims 1 to 3, characterized in that, At least two excitation laser beams (4) with the same wavelength are used to generate the excitation beam waist (TWI) from opposite directions.

5. The light sheet microscope according to any one of claims 1 to 4, characterized in that, The component (38) is selected from the group consisting of diffractive optical elements, mirrors, lenses, SLMs, aspherical elements, axonospindles, or combinations thereof.

6. The light sheet microscope according to any one of claims 1 to 5, characterized in that, The component (38) includes an aperture and / or a segmented aperture and / or a variable density aperture.

7. The light sheet microscope according to any one of claims 1 to 6, characterized in that, The at least one camera detector is a rolling shutter camera.

8. The light sheet microscope according to any one of claims 1 to 7, characterized in that, The component used to dynamically limit at least one active area on the camera detector is at least one scanning slit.

9. The light sheet microscope according to any one of claims 1 to 7, characterized in that, The component used to dynamically limit at least one active area on the camera detector is a scanner combined with a time delay integration (TDI) camera.

10. The light sheet microscope according to any one of claims 1 to 9, characterized in that, The excitation beam is propagated in the object plane (1) in the form of a light sheet using one or more galvanometer-type (galvanometer) scanning mirrors, which reflect the excitation beam once or twice.

11. The light sheet microscope according to any one of claims 1 to 10, characterized in that, At least two excitation laser beams (4) with different wavelengths are used to incoherently irradiate the respective excitation beam waists (TWI) in the separated portions of the object plane (1).

12. The light sheet microscope according to any one of claims 1 to 11, characterized in that, Using at least two excitation laser beams (4), at least two images of the emission excited by the excitation beam waist (TWI) are created on at least two active regions of at least one camera detector.

13. The light sheet microscope according to any one of claims 1 to 12, characterized in that, The component (38) forms a first excitation waist (TWI) to excite the chromophore, and a second excitation waist (TWI) has a minimum intensity in the object plane (1) to excite the chromophore, thereby generating an STED light sheet mode.

14. The light sheet microscope according to any one of claims 1 to 12, characterized in that, The two components (38) form two light plates in the object plane (1), and the two light plates are arranged to excite the sample from different directions.

15. The light sheet microscope according to any one of claims 1 to 14, characterized in that, The excitation beam TWI is generated by the detection objective (7).

Images