Wavefront shaping for digital light sheet microscopy

Abstract

The invention is directed to a light sheet microscope comprising one or more excitation laser light beams (4); a volume defined by the coordinate system (x'(8), y'(12), z'(35)) containing the object plane (1); a means (38) to form an excitation beam with a wavefront and a power / amplitude density distribution from the excitation laser light beams (4) propagating into one or more continuous or modulated or discontinuous excitation beam waists (TWI) along the x'(8) direction in the object plane (1); one or more means (6) to translate the excitation beam waists (TWI) in the object plane (1); at least one camera detector having at least one active area; a detection optics (7) to image the emission excited in the object plane (1) including the emission excited by excitation beam waists (TWI) onto the camera detector and a means to dynamically confine at least one active area on the camera detectors, characterized in that the excitation beam waists (TWI) are coherently illuminated from one side with respect to the x'(8),z'(35) plane of the volume and synchronizing the image of the emission excited by excitation beam waists (TWI) with at least active area of at least one camera detector.

Description

[0001] Wavefront shaping for digital light sheet microscopy The invention relates to variants of beam shaping for a light sheet generated by a scanning process for light sheet microscopy. BACKGROUND [1] Light sheet microscopy has evolved into a wide field with various different implementations. It uses a thin sheet of light to excite only the fluorophores located within the focal volume of a detection objective. This light sheet can be either static, typically shaped by means of anamorphotic optics, or dynamic, by means of scanning. This is referred to as digitally scanned light sheet microscopy. Overviews are given in a review by Stelzer et al. [Light sheet fluorescence microscopy. Stelzer, E.H.K., Strobl, F., Chang, BJ. et al. Nat Rev Methods Primers 1, 73 (2021)]. Light sheet microscopy suffers from the fact that the excitation light propagates within the image plane. An ideal light sheet would have to be confined homogenously perpendicular to the image plane. In case of digitally scanned light sheet microscopy the homogeneity in the scanning direction is generated over the time average over the scan period. However, homogeneity along the propagation direction is only possible with 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)]. [2] The most commonly used non-diffracting beam is the Bessel beam which has a narrow straight line of a central intensity maximum surrounded by concentric cylindrical rings, where the integrated intensity in each of the rings is approximately identical to the integrated intensity of the central intensity. The strong sidelobes compromise the requirement for light sheet microscopy. Another type of non-diffracting beams is realized in the lattice light sheet microscope, where an one-dimensional array of parallel Bessel beams are added to generate a structured non-diffracting intensity distribution. The distance of the individual beams are chosen to partly 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]. The lattice light sheet can be utilized in two main modes. One is the use as a structures illumination mode, where the ‘lattice’ is used to generate an illumination pattern in the sample, which could be shifted in its lateral phase to generate a set of images, which can be reconstructed into one image with improved axial resolution. The other is the so called ‘dither’-mode where the lattice is temporarily equalized by a fast lateral oscillation (dither). The lattice light sheet illumination is a ‘self-healing’ beam which is not limited in its length. The supplemental material of the above mentioned paper states that ‘Neither an ideal Bessel beam nor an ideal 2D optical lattice is directly useful for light sheet microscopy’. The solution is a Bessel-Gauss beam providing ‘a mix of the characteristics of each: a suppression of higher order Bessel sidelobes due to a Gaussian envelope’ … ‘and a much longer beam waist than a traditional Gaussian beam due to the elimination Fof wavevectors corresponding to k p values inside the figure 12inner diameter of the annulus’. Sectioning and the field of view are countercorrelated variables in Lattice light sheet microscopy. A Bessel-Gauss beam uses a finite annulus illumination with a Gaussian radial intensity distribution. [3] Other realizations of light sheets use Airy beams. Here an accelerating phase generates a curved continuous intensity distribution with a strong radial pattern of sidelobes. Airy beams are not non-diffracting along straight lines and also suffer from strong sidelobes. [4] Sectioning and the suppression of straylight can be suppressed in digitally scanned light sheet microscopy by means of a synchronization of the slit type rolling shutter of a sCMOS camera to the image of the scanned beam [DE 102010013223 B4]. The patent introduces the use of focused light and also the use of Bessel beams synchronized with the rolling shutter. [5] A variant of the approach is outlined in an article by Köbele and Rohrbach [A shape- switch-block method for confocal light-sheet microscopy with sectioned Bessel beams and stimulated emission depletion, Köbele and Rohrbach, Communications Physics, 20203:201]. It uses sectioned Bessel beams in combination with a rolling shutter readout. Rolling shutter synchronization can also be applied in quasi-static light sheet systems when the focal region is continuously moved along the propagation direction of the light sheet [US000011156822B2, DE 102010013223 B4 - Claim 11]. [6] The use of a classical cylinder-focus does not provide any stray-light rejection since the full area is illuminated and detected at the same time. The Rayleigh length of the cylindrical focus has to be adapted to the object to be imaged. Therefore imaging of large fields with a thin light sheet is not possible. Both aspects are partly addressed by the method [US000011156822B2] where the position of the waist of the cylindrical focus of the light sheet can be varied 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 beam waist. In one dimension the rolling shutter always collects light from the whole width of the field of view. The suppression factor of stray light can be calculated by the fraction of the rolling shutter width with respect to the width of the full field of the sensor. [7] The use of a scanned focused beam (Gaussian or Flat-Top illuminated pupil) in combination with a rolling shutter synchronized with the scanning introduces the need to acquire a series of images where the focus position is has to be moved along the propagation direction. This makes the acquisition slow but provides ideal suppression of scattered light and can be used to introduce confocalization. The disadvantage here is that confocalization varies with the position within the beam since the beam diameter varies. [8] US 2018 / 0292321 also describes extension of the waist by translating the waist along the propagation axis. In addition, a modulator may be used to homogenize or synchronize the light source, or be used as a mask in a Fourier plane to generate a laser line focus. Also mentioned is the positioning and mechanical scanning of a coverslip at an angle relative to the objectives. [9] The use of a digital micromirror device (DMD) to homogenize the intensity profile of the excitation beam by using multiple elliptical double arch 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.

[0010] [JP2020502558A] describes light sheet theta microscopy (LSTM) in which the excitation beam is oblique to the sample plane which the detection objective remains perpendicular to the sample surface. Linear regions are illuminated by light sheets, with simultaneous line readout of the camera and scanning in the lateral direction as well as in the direction of propagation of the light sheet by means of a holographic spatial light modulator.

[0011] The disadvantages of the foregoing approaches are the need for mechanical scanning and time-averaging during scanning, which require much time. They offer no measures to improvement of the thickness of the light sheet.

[0012] All mentioned approaches suffer from the fact that the Rayleigh length is limited for thin light sheets. One difference is the use of Bessel beams and sectioned Bessel beams. But this has the disadvantage that strong sidelobes compromise the sectioning and that the lateral structure requires a very precise synchronization of the rolling shutter [A shape-switch-block method for confocal light-sheet microscopy with sectioned Bessel beams and stimulated emission depletion, Köbele and Rohrbach, Communications Physics, 20203:201; Fig.1b)]. Bessel beams make use of a conical wavefront which excites each single point within the sample by light traveling along the surface of a cone. Bessel beams a known to be ‘self- healing’. This is true if local obstacles block or disturb the beam. A more global distortion of the beam will not be ‘self-healed’ and destroy the sectioning capabilities of the Bessel beam. Here cylindrical or gaussian or flat-top focuses are the more robust alternative with the limitations mentioned above. The use of a sectioned Bessel beam reduces some of the problems which occur with Bessel beams, but the sectioning reduces the use of the full dimension of the pupil of the excitation objective perpendicular to the sheet. OBJECT OF THE INVENTION

[0013] The aim of the invention disclosed in this application is related to a new paradigm to illuminate a sample within a digitally scanned light sheet microscope which overcomes the disadvantages of state of the art light sheet microscopes mentioned above. It is referred to as Tilted Waist Illumination light sheet (TWI).

[0014] It simplifies the setup of the microscope, improves sectioning through a potentially full use of the aperture of the excitation objective perpendicular to the light sheet.

[0015] The excitation light is formed in a way which reduces the risk of beam distortion during the propagation through the sample,

[0016] It provides a high degree of suppression of scattered and out-of-focus light,

[0017] It reduces the requirements of precision of the synchronization between scanner movement and rolling shutter synchronization. Variants also suppress sidelobes effectively at high sectioning quality.

[0018] Object of the invention is a light sheet microscope comprising one or more excitation laser light beams (4); a volume defined by the coordinate system (x’(8), y’(12), z’(35)) containing the object plane (1); a means (38) to form the excitation laser light beams (4) with a wavefront and a 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 means (6) to translate the excitation beam waists in the object plane (1); at least one camera detector having at least one active area; a detection optics (7) to image the emission excited in the object plane (1) including the emission excited by excitation beam waists onto the camera detector and a means to dynamically confine at least one active area on the camera detectors characterized in that the said one or more excitation laser beams (4) are disposed along the x’(8) axis and coherently illuminate the volume from one side with respect to the x’(8),z’(35) plane as one or more tilted excitation beam waists (TWI) and that the emission excited by the one or more excitation beam waists (TWI) along the x’ axis is synchronized with the acquisition by at least one active area of at least one camera detector.

[0019] In a variant of the invention, means (38) generates an excitation beam with a power / amplitude density distribution having the shape of at least one circular or elliptical arc of 10 - 179° in the Fourier transformation of the wavefront and amplitude distribution with respect to the y’(12),z’(35) plane and / or the x’(8),z’(35) plane of the volume. In other words, the excitation beam may have a power / amplitude density distribution with the shape of a sector of a Bessel beam with a central angle of 10 - 179°. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 describes the general geometrical layout of the optical system of the invention

[0021] Figure 2 shows the geometry of the elongated excitation beam waist (8)

[0022] Figure 3 shows the illumination plane with a tilted series of illumination beamlets (25) according to the invention.

[0023] Figure 4 (a and b) shows Bessel beams according to the prior art, whereas 4c shows an embodiment of the invention.

[0024] Figure 5 (a) shows the geometry of a continuous TWI beam in the object area (10).

[0025] Figure 6 to Figure 9 show simulated data for the y-z cross section of the TWI beam together with vertical and horizontal central profiles (a).

[0026] Figure 10 shows the robustness of the TWI beam in connection with a rolling-shutter readout.

[0027] Figure 11 shows an example where the invention is used to produce a robust STED deexcitation of out of plane excitation of a TWI excitation beam.

[0028] Figure 12 shows a discontinuous variant of a TWI beam.

[0029] Figure 13 (a) and (b) shows a simple but functional version of the tilted waist beam (TWI) in a quasi-continuous form.

[0030] Figure 14 shows an embodiment of the invention where the excitation light is imaged from the pivot point of the scanner (6) to the back-focal plane of an excitation objective (26).

[0031] Figure 15 shows an embodiment of the invention where the collimation of the excitation light can be adapted at the plane of the scanner introducing a variable propagation distance (21).

[0032] Figure 16 shows a variant to change the intermediate cone half angle ^^ᇱwithout changing the position of the intermeditate TWI beam (17).

[0033] Figure 17 shows an embodiment of the invention where two light sheets are generated within the same sample.

[0034] Figure 18 shows a variant that does not use a scanocular.

[0035] Figure 19 shows another embodiment of the invention, where 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.

[0036] Figure 20 shows an embodiment of the invention where the light sheet is generated and imaging is performed through the same objective.

[0037] Figure 21 shows an embodiment of the invention where an additional tilt angle (39) between the TWI beam and the detection plane is introduced. DETAILED DESCRIPTION

[0038] Variants of implementation of the invention are claimed which realize cost effective means for generating the excitation beam. Variants of the invention also address the need for flexibility in terms of acquisition speed, sensitivity and sectioning. It provides easy methods to axially confine the excitation beam, the size of the object and of detection objective.

[0039] Other aspects of the invention realize methods to suppress shadows in the images.

[0040] Further variants describe different technical implementations of the invention using refractive lenses and / or aspheric elements, combinations of refractive and diffractive optical elements such as DOEs in reflection or transmission, or programmable SLMs. DOEs or SLMs could also be used in a double-pass geometry to redistribute the intensity profile of the incoming beam and define the required wavefront. Other variants of the invention relate to methods for exploiting different coherence behaviors or different polarization patterns. The method always utilizes a relative movement between the TWI beam and the object and a synchronization to a readout region of a sensor. The relative movement is state of the art can be realized by means of galvanometric or MEMS mirror scanners or also by a sliding type of object or beam movement. In case of a beam scanning or movement the confinement to the readout region can be realized by a so-called rolling shutter synchronization [DE 102010013 223 B4] or by a synchronous movement of a slit near the image plane or in an intermediate 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 the light emitted from the waist of the TWI beam, line cameras or time-delayed integration cameras can be used.

[0041] To generate a light sheet, an excitation laser beam (4) is typically scanned by means of a scanner (6) and a scanning ocular (5) in the form of a generally telecentric F-theta objective or an aplanatic objective such that the direction of propagation lies in the image plane (1) and the scanning movement displaces the beam laterally in the image plane (see Figure 1).

[0042] The axis of the scanning eyepiece and the normal of the image plane (1) (usually the axis of the detection optics) define a plane (3) that is perpendicular to the pupil plane (2) of the scanning ocular and perpendicular to the image plane. This plane will hereinafter be referred to as the propagation-detection plane (PD-plane). For the definitions of the coordinate system see

[0043] The invention relates to the design of an excitation beam which is asymmetric with respect to the PD plane. This asymmetry can relate to the intensity and or phase and or to the polarization and or to the coherence of the excitation laser. The excitation light generates a beam waist that is extended along a straight line that is located in the image plane.

[0044] The waist of the beam might have a tilt angle ^^ (9) with respect to the excitation direction X. The tilt angle can have angles ranging between 0 and 45 or even 60 degrees ideally between 0 and 30 degrees according to the invention (see Figure 2). The area (10) which is imaged onto the detector (22) is oriented according to the elongation of the beam tail (8) and therefor is rotated with respect the normal of the image plane (1) in case of ^^ (9) beingnot zero. It is advantageous to introduce a new coordinate system ^^ᇱ, ^^ᇱ, ^^′ (see (8) for ^^′,(12)for ^^′and (35) ^^ᇱ ൌ ^^ in Figure 1 to Figure 4. The plane defined by ^^ᇱ, ^^′ differ from the PD-plane in case of ^^ ് 0. The fundamental aspect of the invention is that the excitation lightcontributing to the TWI beam is radiating from one half-space of the coordinate system^^ᇱ, ^^ᇱ, ^^′ separated by the ^^ᇱ, ^^′ plane. This means that in the 2D-Fourierspace with respect tothe ^^ᇱ, ^^′-plane only half of the plane shows non-zero.

[0045] In one embodiment of the invention the excitation beam generates a series of foci (beamlets) aligned along the beam waist where the foci might not be limited to gaussian foci but could be also foci with an extended depth of focus (EDOF) or short Bessel-like foci. A more general description is given later in conjunction with Figure 3. The separation of the foci might be as large as necessary that they do not show substantial interference with each other in the vicinity of the beam waist forming a ‘light chain’ of well separated beams. The intention of this embodiment of the invention is that the light of an individual beamlet transversally passes the line of the beam waist within the object plane. The fluorescent or scattered light generated by the excitation of the individual beamlets in the vicinity of the beam waist is than collected by the active area of the synchronized rolling shutter. It contributes to a well-defined subarea of the acquired frame. This is depicted in Figure 3. The sum of the contributions from all beamlets leads to image data which are not limited by the Rayleigh length of the individual beamlet. The fact that the beamlets transversally pass the line with connects all foci breaks the symmetry of the overall excitation beam which is the subsummation of all beamlets. The series of beamlets are focused in a series of foci. These are transversally and axially displaced in a row. The invention includes this discrete but also a continuous variant and all intermediate variants. This is elucidated in the following section.

[0046] In a more general description of the discrete embodiment of the invention is the excitation beam electrical field EEx can be described as:

[0047] where ^^^,^is the electrical field of an individual beamlet in the sample are, ^⃗^ is the position vector and ^̂^ is a unit vector along the direction of the waist and the lists of realvalues of ^^^ and ^^^ thus generate translated beamles assigned to the positions ^⃗^ െ ^^^^̂^ withindividual phases ^^^. One would typically chose of ^^^and ^^^to generate equidistant positions and constant phase differences and ^^^,^independent of i. The excitation beam intensity distribution is then proportional to|^^^௫^^⃗^^|ଶ. In an incoherent case, e.g., if a broad band excitation source is used or if the distance|^^^^̂^|is large enough to render interferences small, it would be also sufficient to just add the intensities of the individual beamlets.

[0048] The choice of the phase between the beamlets can be chosen according the overall homogeneity of the excitation in case that there is substantial interference of the beamlets with each other within the area of the sample which is imaged onto the area (11) which corresponds to the rolling shutter.

[0049] In another embodiment of the invention a continuous realization of the excitation beam according to claim 1 is proposed. Eq. 1 can be rewritten in a continuous form:

[0050] Here ^^^⃗ ௪ is the wave number vector along the waist direction ^̂^. In case ^⃗^ ൌ 0^⃗ would beassigned to the center of the imaging area ^^^ିൗ 2 ,^^ାൗ 2 ^ address the useful elongation of theexcitation beam. For the ease of understanding but not limiting the phase propagation is assumed to be linear. A more simplified description can be elucidated if the integral is extended to infinity. This shows prominent restrictions:

[0051] Normalizations are not taken into account also in the following equations. The property of the field is that of a freely propagating wave. To take this into account, it makes sense to carry out a coordinate transformation in which the direction of the beam waist is along acoordinate axis x' of the new coordinate system (the direction of ^^^⃗ ௪). At the same time, one can write down the equation with respect to the other two coordinates (y',z') of the Fourier transformed form:Eq. 4 ^^^ ᇱା^ᇱ ^^ ௫ᇱ^௫൫^^ ൌ 0, ^^௬ᇱ, ^^௭ᇱ൯ ൌ ^ ି^ ^^^^൫^^ , ^^௬ᇱ, ^^௭ᇱ൯^^ ^ᇲ^^^^′

[0052] With the use of the propagation operator with ^^ ൌ as the wavenumber ofexcitation laser beam in the medium with refraction index ^^ one can reassign the integral tothe values of ^^^^ in one plane.

[0053] The integral shows only non-zero values if ^^ଶ^ െ ^^௬ᇲଶ െ ^^௭ᇲଶ ൌ ^^ଶ௫ᇱ. Thus Eq. 6 ^^ଶ െ ^ ᇲଶᇲଶᇲଶ^ ^௫ ൌ ^^௬ ^ ^^௭ ൌ ^^^^^^^^^^.

[0054] The wavefront of the wave generated by Eq. 3 has a conical shape since in the Fourier space all values disappear for k values that do not lie on the circle with ^^ଶ^ െ ^^௫ᇲଶ ൌ ^^௬ᇲଶ^ ^^௭ᇲଶin the coordinate system where x’ is the coordinate axis of the beam waist. If now thewave in the center of the field ^^^^௫൫^^ᇱ ൌ 0,^^௬ᇱ,^^௭ᇱ൯ is propagated by adding a propagationoperator in the direction of the beam waist the following equationEq. 7 ^^^ ൫^^ᇱ^௫ ^,^^௬ᇱ, ^^௭ᇱ൯ ൌ With the restriction of the Eq. 6Eq. 8 ^^^ ൫^^ᇱ, ^ ᇱ ^^^ᇲ௫ᇱ^௫ ^^௬ᇱ,^^௭ᇱ൯ ൌ ^^^௫൫^^ ൌ 0, ^^௬ᇱ, ^^௭ᇱ൯^^ .

[0055] This holds also for the non-Fourier-transformed version of the equation. Eq. 8 shows that this generic setup of the excitation beam with an arbitrary wave ^^^^^⃗^^always generates a non-diffracting beam. This means that the intensity is constant in the direction of the propagation axis ^̂^. In case that ^^^^^⃗^^is chosen to generate a waist, the intensity distribution of this waist is constant along the propagation axis. The parameter to be chosen is the angle of the wave front conus

[0056] The other parameter to be chosen is the intensity distribution of ^^^^^⃗^^. The beam waist direction would be typically within the imaging plane and a symmetry of the intensity withrespect to the object plane ^^^ᇱ ൌ ^^ ൌ 0^would be advantageous. It is advantageous to definethe generating beam in polar coordinates ^^^^^⃗^^ ൌ ^^^^^^, ^^, ^^ᇱ^ with ^^ ൌ 0 and ^^ ൌ ^^ relatesto the the object plane. Than a symmetric beam would be generated by ^^^^^^, ^^, ^^ᇱ^ ൌ^^^^െ^^, ^^, ^^ᇱ^.

[0057] Bessel beams and sectioned Bessel as described in [COMMUNICATIONS PHYSICS | https: / / doi.org / 10.1038 / s42005-020-00458-3 (Luise Köbele , Alexander Rohrbach) Fig.1b)] are examples for such beams where the propagation axis of the beam waist is colinear with the optical axis of the excitation objective and the wave has rotational / mirror symmetry with respect to the Propagation-detection-plane (PD-plane (3), see Figure 1). This means that in the state of the art beams with symmetry with respect to the Propagation-detection-plane (PD-plane (3), see Figure 1) and with respect to the object plane obey ^^^^^^, ^^, ^^ᇱ^ ൌ ^^^^െ^^, ^^, ^^ᇱ^and ^^^^గ ଶെ ^ ^^, ^^, ^^ᇱ^. One aspect of the invention reduces TWI beam tovalues of |^^| ^ గଶ or even to lower values:Eq. 10 |^^| ^ ^^ ^గ ^^௫ଶ.

[0058] This has the effect, that the illumination of the waist is only from one side and thus very limited or zero interference occurs within the waist in ^^′ and also ^^ direction.

[0059] If ^̂^ is the unit vector along the waist in the coordinate system depicted in Figure 1, the conical phase in the pupil could be written as In a plane perpendicular to the axis of the scanocular ^⃗^^ ൌ ^^^^, ^^, ^^^ with ^̂^ ൌ ^cos ^^ , sin ^^ , 0^Eq. 13 ^^^^⃗^^^ ൌ ^^^^ cos ^^ ^ ^^ sin ^^^^^^ cos^^ ^^^^^^ sin ^^ ^ ^^ cos ^^^ଶ ^ ^^^ sin ^^^ଶ ^ ^^^ cos ^^^ଶ ^^^ sin^^ൌ ^^^^ cos ^^ ^ ^^ sin ^^^^^^ cos^^ ^^^^^^ sin ^^ ^ ^^ cos ^^^ଶ ^ ^^ଶ ^^^ sin^^Eq. 14 tan^^ ൌ ௭ᇲ௭௬ᇲൌ ି௫ೞ ^୧୬ ఊା௬ ୡ୭^ ఊ

[0060] The aperture of the scanocular limits the z values which can be utilized. This depends on the numerical aperture ^^^^^௫of the scanocular and its focal length ^^ௌை. In a paraxialapproximation ^^^^௫ ൌ ^^^^^௫ ^^ௌை , with ^^^ ൌ െ^^ ^^ௌை , where ^^ is the refraction index of themedium. The maximum overall numerical aperture can be utilized if the wavefront in the apex of the pupil, i.e. at the pointEq. 15 ^⃗^^^^௫ ൌ ^െ^^ ^^ௌை, 0,^^^^^௫ ^^ௌை^ points towards the center of the imaging field. This is the case if the partial derivative డ డ௬^^^^⃗^^ ൌ 0 at this point. With the introduction of and the partial derivative of Eq.13 the following formula for ^^ெ^௫can be derived which maximizes the use of the pupil:Eq. 17 sin ^^ெ^௫ ൌ ^^sinβ^ଶ െ ^tan^^^ଶ^cos^^^ଶ

[0061] Note that useful optimized values of ^^ெ^௫can only derived from Eq.17 if^^ ^ ^^. The area of the pupil contributing to the waist is limited to the region of interest in theimage plane has an elliptical shapes in the pupil plane (13).

[0062] It is also part of the invention that a lower portion of the provided numerical aperture isused ^^^^^^^^^ ^ ^^^^^௫^. In this case ^^ could still be calculated using Eq. 17 with a modifiedvalue of ^^ according Eq.16 inserting ^^^^^^^^. In this cases it is also versatile to use lowervalues of ^^ which also include ^^ ൌ 0.

[0063] In the case where ^^ ^ ^^ and an optimized ^^ெ^௫ can be calculated according to Eq. 17,Eq.13, one can calculate the maximum value inserting ^⃗^^^^௫from Eq.15 in Eq.14 leading to As sectioning of the resulting TWI beam is mainly dependent on ^^^^^௫one can reduce ^^ெ^௫by increasing β (see examples in Figure 7 and Figure 9). Values of β are not linked to the ^^^^^௫. Increasing β also increases ^^ெ^௫. This will be the angle between the TWI beam and the scan direction. High values up to 60° or higher might be beneficial cases where the object area has high aspect rations.

[0064] One aspect of the invention is that the symmetry with respect to the PD-plane ^^^ ൌ 0^ isbroken. This is always the case if ^^ ് 0 as e.g. outlined in Eq. 17. But this is also the case if^^ ൌ 0 and if the intensity is asymmetric to the PD-plane as outlined in Eq. 10. One mainaspect is illustrated in Figure 4 (c) along with Bessel (a) and sectioned Bessel beams (b), where the wave distribution in the Fourier-plane (13) is shown together with the intensity distribution in the center of the image in a plane parallel to the excitation pupil plane (E-plane (2), see Figure 1). It is obvious that the choice of the intensity distribution in the pupil on the elliptical illumination line and the local angular distribution affects the length of the waist along x’, sectioning capability and intensities of potential side lobes of the central intensity maximum. An intensity modulation within the image plane can be suppressed and the excitation scheme is robust against a jitter of the scanner with respect to the rolling shutter motion. There is no principal limitation of the length of the waist. Limitations relating to Rayleigh length are not present. The full potential of the scanocular (5) in the direction perpendicular to the object plane (1) to provide sectioning can be exploited if Eq.17 is considered.

[0065] Figure 6 shows calculated properties of the TWI beam. Here an excitation wavelength of488 nm, water as an immersion medium (n = 1.33), ^^^^^௫ ൌ 0.5, ^^ ൌ 33.83° and ^^ெ^௫ ൌ26.31° according to Eq. 17 is used. According to Eq. 18 ^^ெ^௫ ൌ 39.2°, (a) shows theintensity distribution of the TWI beam in the y-z plane with the central beam profiles in ^^ and ^^ direction. (b) shows the intensity profile in ^^ direction as an integral over the rangeെ1µ^^ ^ ^^ ^ 1µ^^ which is imaged to the active area of the rolling shutter detector. A Gaussprofile fitted to the profile shows a FWHM of 430 nm in z-direction. As an example a given detection magnification of 20x and a detector pixel size of 4 µm would render the number of active lines of the camera to ଶ^ ଶµ^ ൌ 10. (c) shows the intensity in the Fourier-space,comparable to Figure 4 (c). If as shown in Figure 6 (d) the intensity is smoothed by a truncated gaussian intensity distribution with respect to the azimuthal angle ^^ side-lobs could be suppressed without a big loss of sectioning. All other parameters are identical to those used for the calculation represented by Figure 6 (a-c).

[0066] The relationship between ^^, ^^ெ^௫, ^^ெ^௫for the conditions chosen for the calculations shown in Figure 6 is plotted in Figure 7. All pairs of ^^ெ^௫, ^^ெ^௫generate an excitation ellipse segment in the Fourier-plane i.e. on the scanner which make use of the full vertical dimension of the aperture. The ellipse segment connects the upper and lower apex of the pupil. The sectioning of the generated light sheets do not strongly depend on the choice of ^^. On the other hand the useful width of the rolling shutter has to be adapted towards larger values if ^^ is enlarged. The TWI beam leaves notch on top and below the beam waist free of excitationlight. The full angle of the notches is 180° െ 2^^ெ^௫. The cone of the detection light which iscollected by the detection objective or objectives should be smaller than the angle of the notches. Figure 7 shows that it is possible to choose a configuration where is reasonable small without compromising the sectioning.

[0067] A version for macro imaging in clearing solution is shown in Figure 8: The parametersare 488nm, n=1.55), ^^^^^௫ ൌ 0.13, ^^ ൌ 12.9° and ^^ ൌ 12.0° with a readout width ofെ5µ^^ ^ ^^ ^ 5µ^^, and According to Eq. 18 ^^ெ^௫ ൌ 22.0°. Figure 8 (d) is the variant withgaussian illumination leading to a side lobe reduced light TWI light sheet. Figure 9 shows relationship between ^^, ^^ெ^௫, ^^ெ^௫for the conditions chosen for the calculations shown in Figure 8.

[0068] Figure 10 shows the robustness of the TWI beam in connection with the rolling-shutter readout. (a) shows the variation of the sectioning profile with a relative shift of the stripe which is integrated by the active area of the rolling-shutter detector. The sectioning is reduced but as shown in (b) the intensity is stable with the relative shift. This would not be the case with Bessel or sectioned Bessel beams and makes the TWI approach very robust.

[0069] Figure 13 (a) and (b) shows a simple but functional version of the tilted waist beam (TWI) in a quasi-continuous form. The incoming collimated beam is hits an axicon (14) at an off axis position (22). An intermediate beam waist (17) is formed with a length which depends on the width of the incoming beam. At this position two sectional apertures (24) can be inserted to confine the intermediate waist. The central beam propagates at an intermediate cone half angle ^^ᇱ(19) with respect to the axis of the axicon. The following lens (15) is placed at a potentially tilted angle ^^′ (18). This angle might have values between 0 and ^^ᇱ. The distance of the lens to the center of the intermediate beam waist (20) is in the current example the focal length of the lens. The following propagation distance (21) leads to a segment of a circular or elliptic focal line on the scanner (6) and telecentric illumination, if the scanner is placed in the focal plane of the lens (15). The beam is than reflected towards the scanocular (5) and generates the image (TWI) of the intermediate beam waist within the object area (1). The tilt angle of the TWI beam with respect to the optical axis of the scanocular scales with ^^′ (18) and the magnification introduced by the lenses (15) and (5). The beam is always asymmetric with respect to the PD-Plane independently of the choice of ^^′. The term Tilted Waist Illumination (TWI) refers to this asymmetry which elucidates the fact that the waist is illuminated predominantly from one side within the object plane (1).

[0070] In all embodiments of the invention, the occurrence of an axicon can be replaced by conical or spheric reflecting surfaces and also toroidal, elliptical and parabolic reflecting surfaces and combination thereof. An aspheric component of one or more of such surfaces might be used to further modify the distribution of light along the TWI axis. This might be utilized to generate a more homogenous (flat top) illumination along the TWI axis leading to an overall homogenous sensitivity over the field of view.

[0071] One realization of a TWI beam setup is shown Figure 14 where a microscope objective (26) is used to generate the TWI beam. Here the position where the scanner (6) is located is imaged into the back focal plane of the excitation objective (26) by means of intermediate imaging optics (5,17,5). The placement of the pivot point in the important to align the TWI beam with the lines of the detector. The limitation of the TWI beams can also be achieved by apertures (24), as shown in Figure 12 (a), near the intermediate image plane (17).

[0072] There are other variants not shown here. One can use two scanners with parallel axis realizing a beam geometry where a pivot point can be addressed which lies at a distance to the surface of the scanners. With this method the use of intermediate imaging optics could be omitted.

[0073] A person skilled in the art can adapt the scanning scheme in various variants: Scanning could also be realized by a relative movement of the excitation lens (26 or 05) including parts of the excitation optics with respect to the object and of the detection optics. In certain cases the image of the TWI beam waist would move relative to the detector. De-scanning mechanisms and or Time-delayed-integration cameras could be also used.

[0074] Further variants of the invention includes variations of the conus angle ^^^^^ᇱ^ along the waist and means to dynamically change those variation. This variable conus angle has to be inserted in

[0075] Eq.11. One solution depicted in Figure 15 is to leave the telecentric condition in the object space of the scanocular (5) or excitation objective (26). This allows the beam waist to be concentrated or expanded in the direction of ^^ᇱto adapt to the size of the sample or subarea to be examined. It would be also advantageous if a magnification changer or changing of detection objectives are implemented. The conus angle ^^^^^ᇱ^is related to the useful width of the active detection region (number of active detector lines in the rolling shutter mode).

[0076] It is also part of the invention to change the characteristic of the TWI beam dynamically especially to change ^^^^^௫towards smaller values ^^^^௨^^. Figure 16 shows a possibility to move the point where the excitation beam hits the axicon radially by a synchronous move of the axicon (14) and a mirror (39) parallel to the axis of the axicon. The parallel movement maintains the position of the intermediate TWI beam (17) but reduces the intermediate cone half angle ^^ᇱ. This can be utilized to increase the width of the TWI beam. The width of the rolling shutter can then also be increased. The dependency of a useful width of the rolling shutter to ^^ᇱis approximately quadratic.

[0077] The wave according to the invention can be generated by means of various means. Among those are Spatial-light-modulators (SLM) either in one or in double path as well as diffractive optical elements (DOE). Classic optical elements to be used are also axicons. Details are explained within the drawing descriptions.

[0078] In another embodiment of the invention more than one excitation laser source is used. The shaping of the wavefront could be done by using separated diffractive optical elements of different areas on the same diffractive optical element. It is advantageous that the single sided elliptical shape of the wavefront is relatively small. This makes it possible to utilize reasonably small diffractive optical elements for shaping several laser sources. The combination of the light sources could be done by dielectric beam combiners, prisms of just geometrically is the angle ^^ is chosen individually with identical values to ^^ for each wavelength. Than the elliptical shaped beams hit the scanner plane at different positions but identical positions of the TWI beam would be obtained. The different wavelength could also hit the scanner at different angles resulting in different but parallel positions of the TWI beams for each wavelength. It is advantageous to utilize more than one camera and split the light onto the different cameras by means of dielectric beam splitters. Then simultaneous multi-color fluorescence imaging could be performed by synchronizing the different cameras to the different TWI beams of the different excitation lasers. The fact that the tilt angle of the TWI beam could be small allows for an almost complete suppression of cross talk between the channels.

[0079] In another embodiment shown in Figure 17 of the invention, two light sheets are generated within the same sample and also more than one objective (7) used to image on more than one camera. The imaging would be typically from opposite sides. The two beam waists (8) of the excitation sheets can be arranged to be parallel or collinear also in the case where the two axis of the illumination objectives (5) are not colinear.

[0080] In this embodiment, at least two excitation laser light beams (4) have the same wavelength are used to incoherently illuminate the excitation beam waists (TWI) from opposite directions. This can be achieved by splitting one excitation laser light beam (4).

[0081] The tilt ^^ of the TWI beam with respect to the illumination axis would be typically half of the angle between the axis of the two illumination objectives (5). The advantage is that a small imaging chamber (29) which is inserted between the two detection objectives could be moved parallel to the TWI beam and the lines of the two detectors and multiple detection areas (10) could be addressed. The preparation of the wavefront and intensity distribution of the incoming beams (4) is not shown in the figure. The setup might be equipped with two scanners (6). The scans of the two TWI beams can be performed simultaneous or subsequently. Alternatively it is possible but not shown in the figure to use only one scanner and split the light out of the scanner in two portions. This splitting could be performed either by a beam splitter or by splitting the a symmetric wave into two portions resulting in a splitwhere according to Eq. 10 |^^| ^ ^^^^௫ ^గ ଶ contributes to one of the TWI beams and|^^ െ ^^| ^ ^^గ ^^௫ ^ଶ contributes to the second TWI beam. The same could be also done by first splitting the beam and then scan the two beams by two individual scanners. The basic setup without the use of TWI beams is known from the system provides by VIVENTIS (Lausanne).

[0082] In another embodiment of the invention shown in Figure 18, the use of a scanocular is omitted. Here the beam generated by an axicon (14) or other means to provide the required wavefront and intensity distribution is scanned by means of a two reflections on the same scanner. Here a variant is shown where the beam hits the scanner (8) from opposite sites. The intermediate reflections on the mirrors (30) and the second reflection on the scanner leads to an compensation of the scan angle. Thus a pure parallel movement of the TWI beam is achieved. The setup is thus very simple and does not suffer from distortions of a scanocular. High TWI beam numerical apertures are achievable for a large field of view. This setup might be ideal for macro-light sheet imaging. Alternatively but not shown in the Figure the scanner motor might be equipped with two mirrors mountet opposite to each other such that the beam is reflected first by one of the mirrors and then by the other one. Also here the scanner angle is compensated and a parrallel movement is achieved.

[0083] The invention can also be applied if the angle between the excitation and detection objective differs from 90°. Here often a compensation of the distortion due to a tilted image plane has to be utilized. This can cause keystone distortion of the image. The problem here is, that the lines simultaneously illuminated by the TWI beam on the sensor might rotate in the image space while scanning. One embodiment of the invention also deals with this restriction. The TWI could be adapted to a keystone distorted image on the detector to be able to synchronize with the rolling shutter by leaving the telecentric condition in the image space of the scanocular (see Figure 19).

[0084] The invention also includes the use of light sheet microscopy with generate the light sheet and perform imaging through the same objective (Dunsby, C. Optically sectioned imaging by oblique plane microscopy. Opt. Express 16, 20306 (2008), see Figure 20).

[0085] In another embodiment of the invention STED is utilized: The Excitation-TWI beam at wavelength 1 is used to excite the sample whereas a second modified STED-TWI beam at a wavelength 2 is used to deplete the fluorescence by deexcitation of the excited molecules. Themodification of the second beam is a ^^ step in the phase at ^^ ൌ 0 leading to a destructiveinterference in the plane of the TWI beam of the wavelength 1. The realization is similar to that done by Luise Köbele , Alexander Rohrbach (citation see above). The difference is that with the present invention using a Excitation-TWI beam and STED-TWI beam no lateral structure is introduced. This makes the setup robust and it is easier to utilize saturation of the STED-TWI beam to limit the emission of fluorescence to a thin layer within the TWI beam. The cone angles ^^ and azimuthal intensity distributions could be selected differently for Excitation-TWI and STED-TWI beam. This could be used to optimize the impact of the STED-TWI beam. One could for example use a Gaussian envelope for the Excitation-TWI beam to generate a Gaussian focus which is than squeezed down by the two symmetric maxima of the STED-TWI beam resulting in a side lobe free high resolution sectioning. Figure 11 shows a STED-TWI beam cross-section (a) and ^^-profile taking the rolling shutterreadout into account (b). The parameters for the calculation are ^^ௌ்ா^ ൌ 560^^^^, ^^ ൌ 1.33,^^^^^௫ ൌ 0.5, ^^ ൌ 33.83° and ^^ ൌ 26.3° , ^^^^௫ ൌ 42,46°, a Gaussian envelope wasadditionally also applied. (c) shows an Excitation-TWI beam with the identical parameters but^^ௌ்ா^ ൌ 491^^^^ together with the STED-TWI beam (b) and an exemplary depleted TWIprofile. There is no principal limitation in the potential length of the TWI beam. Adding a truncated Gaussian envelope in ^^ – direction especially to the STED-TWI beams helps to generate a robust deexcitation since the unwanted minima between the sidelobes are reduced. In another embodiment of the invention could be used to generate a layer of predominantly dual color activated photo-initiators as used in the XOLO printing system. The scanned tilted light sheet could then be synchronized with a perpendicular illumination with a structured activation light e.g. with the use of a DLP-Projector or light structured by a fast scanning acousto-optical deflector. The cross-section of the TWI activation beam and the projected polymerization beam would then be identical over the full length of the TWI beam. The intensity of the TWI beam could be adapted along the TWI beam without infringing the volume which is closer to the source and is thus less absorbed. A completely homogenized printing is than possible. TWI light sheet activation and imaging could be combined to guarantee a long homogenic activation and a high resolution quality control of the printed volume. DESCRIPTION OF THE DRAWINGS / OF EMBODIMENTS

[0086] Figure 1 describes the general geometrical layout of the optical system with the Image plane XY (1) referred to as I-Plane, Excitation pupil plane YZ (2) referred as E-plane, Propagation – detection plane (XZ) (3) referred as PD-plane, Excitation laser beam (4), Scanocular (SO) (5), Scanner (6), Detection objective (DO) (7), which is used to describe the state of the art and invention.

[0087] Figure 2 describes the geometry of the elongated excitation beam waist (8) which might have a tilt angle ^^ (9) against the excitation direction X . The image area (10) is aligned with the waist direction (8). The area (11) corresponding to the rolling shutter of the camera propagates corresponding to the rows of the sensor in a direction Y’ (12) which is perpendicular to the waist direction (8) in the telecentric case. The excitation direction might be tilted with respect to the optical axis of the excitation objective.

[0088] Figure 3 shows the illumination plane with a tilted series of illumination beamlets (25) according to the invention. The waists of the beamlets are arranged along a line. The Figure also depicts the area corresponding to the rolling shutter of the camera (11).

[0089] Figure 4 (a) shows a Bessel beam with the rotational symmetric intensity distribution and a strong central maximum but relatively strong secondary maxima (lower left sketch). Upper sketches shows a schematic sketch of the Fourier transform of the pupil function. The illumination in the pupil is concentrated on a circle. The area suitable for rolling shutter selection is depicted by the arrow. It has to be confined with the central maximum. Figure 4 (b) shows a sectioned Bessel beam where in segments of the illumination circle are blanked out. This results in less prominent secondary maxima, enlarges the suitable are for rolling shutter selection but also shows less defined sectioning since the height of the pupil is not fully used. Figure 4 (c) one embodiment of the beam according to the invention where a tilted axis of the waist of the beam is chosen in combination with a conus angle ^^ relating to an illumination in the pupil as a section of an ellipse which reaches between the upper and lower apex of the pupil.

[0090] Figure 5 (a) shows the geometry of a continuous TWI beam in the object area (10) in a schematic sketch. At the endpoints (28) of the TWI area the light propagates from a segment of a circle with the segment angle ^^ through the beam waist (8) to the other side of the waist and forms at the end of the object area again a mirrored segment with identical dimensions. In the center of the object area (27) the profile is symmetric with the incoming light at one side and the outgoing light at the other side. This longitudinal and transversal asymmetry is the main aspect of the invention.

[0091] Figure 6 to Figure 9 show simulated data for the y-z cross section of the TWI beam together with vertical and horizontal central profiles (a), sectioning profiles taking a rolling shutter readout into account without including the effect of the detection point spread function.

[0092] Figure 10 shows the robustness of the TWI beam in connection with the rolling-shutter readout. (a) shows the variation of the sectioning profile with a relative shift of the stripe which is integrated by the active area of the rolling-shutter detector. The sectioning is reduced but as shown in (b) the intensity is stable with the relative shift.

[0093] Figure 11 shows an example where the invention is used to produce a robust STED deexcitation of out of plane excitation of a TWI excitation beam.

[0094] Figure 12 shows a discontinuous variant of a TWI beam where a series of 11 beams here with a flat top illumination of the pupil are superimposed according to Eq. 1 with a tilt angle^^ ൌ 0.2 ^^^^^^, ^^^^^௫ ൌ 0.13, ^^ ൌ 1.55, ^^ா௫ ൌ 488 ^^^^ and a axial distance of the foci of43µm. The width of the readout area was set to 18µm. (a) shows the y-z cross section and a schematic representation of the Fourier-space (left) which shows that the discrete TWI beam also renders a discrete set of elliptical shaped areas (an extended radial structure) in the Fourier-space. This version generates softer shadows since a larger portion of the pupil is utilized. (b) shows a top view on the central plane intensity of the TWI beam. (c) shows the homogeneity of the illumination taking the scanning and rolling shutter readout into account. (d) shows the sectioning achieved under conditions. The different profiles represent different positions between the center focus and half way to the next focus showing the overall stability of the TWI excitation also in the discrete case. There is no principle limitation to the number of applied foci and sectioning is not compromised if the TWI beam is extended. If the multiplexing of the beam is generated by means of a diffractive optical element or SLM one can correct for individual aberrations which could occur especially due to the fact that the individual beams propagate different distances within the medium. Other aberrations such as field curvature, tilt and all effects that are linked to the field coordinates could be corrected. It would be also possible to dynamically adjust the length of the TWI beam by means of an SLM but also by means of edge apertures in an intermediate image space (see (24) as an example in Figure 12). This dynamic adaptation might help to reduce the total dose of light to the sample and apply light only where it is needed. This adaptation might be linked to individual planes which are scanned or also to the scanner movement itself.

[0095] Figure 13 (a) and (b) shows a simple but functional version of the tilted waist beam (TWI) in a quasi-continuous form. The incoming collimated beam is hits an axicon (14) at an off axis position.

[0096] Figure 14 shows an embodiment of the invention where the excitation light is imaged from the pivot point of the scanner (6) to the back-focal plane of an excitation objective (26).

[0097] Figure 15 shows an embodiment of the invention where the collimation of the excitation light can be adapted at the plane of the scanner introducing a variable propagation distance (21). This allows the TWI-beam waist to be concentrated or expanded in the direction of ^^ᇱto adapt to the size of the sample or subarea to be examined.

[0098] Figure 16 shows a solution to change the intermediate cone half angle ^^ᇱwithout changing the position of the intermeditate TWI beam (17).

[0099] Figure 17 shows an embodiment of the invention where two light sheets are generated within the same sample and also more than one objective (7) is used to image on more than one camera. The two excitation beams propagate in this example from opposite sides but with an angle with respect to each other. The TWI direction of both beams are in line.

[0100] Figure 18 shows a variant that does not use of a scanocular. A parallel movement of the TWI beam is achieved by a double reflection of the incoming beam on the scanner (8). Similar results could also be realized by subsequently using two synchronized scanners with parallel scan axis.

[0101] Figure 15 shows an example where the telecentric condition in the object space of the scanocular (5) has been left by an increased distance of the propagation distance (21) with respect to the telecentric setup shown in Figure 13. The angle ^^ varies along the beam waist of the TWI beam in the sample. Here the light is concentrated along the waist and illuminates a shorter portion in the sample. The opposite could be achieved by elongating the propagation distance (21).

[0102] Figure 19 shows another embodiment of the invention, where 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. Here the tilt angle is according to the state of the art corrected by an external tilted intermediate image plane. This results in a keystone distorted image on the detector. Parallel lines in the object are not imaged to parallel lines on the detector. This can be compensated by moving the pivot point of the scanner along the optical axis of the excitation lens leaving the telecentric condition.

[0103] Figure 20 shows an embodiment of the invention where the light sheet is generated and imaging is performed through the same objective. The potential correction of the keystone distortion is addressed in a similar way explained above.

[0104] Figure 21 shows an embodiment of the invention where an additional tilt angle (39) between the TWI beam and the detection plane is introduced. This can be realized if for example the interval of ^^ values is chosen to be asymmetric with respect to the image plane using. Here the TWI beam is tilted against the image plane which compromises the sectioning if an extended range of lines of the rolling shutter readout is activated. The advantage is that one can illuminate the sample from one hemisphere. This allows for a light sheet readout within a microtiter plate without tilting the detection objective or the image plane with respect to the cover glass of the microtiter plate. The light sheet is generated parallel to the cover glass. The excitation could be through an high NA objective or with additional optics from the side. The only limitation is the angle of total reflection on the cover glass. Low refraction index materials like FEP could replace the cover glass and allows to choses small values for the tilt angle (39). The setup can be regarded as a scanning halo microscope with the difference that the rolling shutter can be utilized to suppress out scattered light. Sectioning of less than 1 µm is feasible. The setup could also be used for lower magnification but larger working distance detection objectives with a separate excitation optics placed at an angle with respect to the image plane. This angle might also be zero with the axis of the excitation objective within the object plane. The lower half of the excitation objective would be used to still illuminate the object plane from below.

[0105] The z’ direction (35) might also be additionally tilted in a similar this embodiment not shown in the figure with respect to the optical axis z of the detection objective such that z’ z. The tilt could be with respect to x’ such that the TWI would be still perpendicular to the z but the scanning translation would be in the x’y’ plane tilted against the axis of the detection objective.

[0002] GLOSSARY 1 Object plane (XY) 2 Excitation pupil plane (YZ) 3 Propagation – detection plane (XZ) (PD-plane) 4 Excitation laser beam 5 Scanocular (SO) 6 Scanner 7 Detection objective 8 X’, Tilted waist direction ^̂^ 9 Tilt angle ^^ 10 Object area imaged 11 Area in the object plane, which is illuminated by the beam waist 12 Y’, perpendicular to X’ and Z = Z’, direction corresponding to the rows of the image sensor. 13 Pupil of the scanocular (5) 14 Axicon 15 Lens 16 Intermediate waist 17 Intermediate waist length ^^′ 18 Intermediate waist to optical axis tilt angle ^^′ 19 Intermediate cone half angle ^^ᇱ20 First propagation distance 21 Second propagation distance 22 Radial beam displacement 23 Detector 24 Sectional aperture 25 Series of individual beams 26 Excitation objective 27 Center of the TWI sheet / object area 28 Begin / end of the TWI sheet / object area 29 Sample chamber 30 Mirror 31 Detection cone 32 Area on the optic contributing to the TWI beam 33 X, axis of the scanning eyepiece 34 Y axis perpendicular to X and Z 35 Z = Z’ axis 36 Tilted waist length 37 Foci of the individual beamlets contributing to the TWI beam 38 Means to form an excitation wavefront and power / amplitude density distribution of the laser beams 39 Out of plane TWI tilt angle 40 Extended radial structure

Claims

CLAIMS 1. Light sheet microscope comprising one or more excitation laser light beams (4); a volume defined by the coordinate system (x’(8), y’(12), z’(35)) containing the object plane (1); a means (38) to form the excitation laser light beams (4) with a wavefront and a 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 means (6) to translate the excitation beam waists in the object plane (1); at least one camera detector having at least one active area; a detection optics (7) to image the emission excited in the object plane (1) including the emission excited by excitation beam waists onto the camera detector and a means to dynamically confine at least one active area on the camera detectors characterized in that the said one or more excitation laser beams (4) are disposed along the x’(8) axis and coherently illuminate the volume from one side with respect to the x’(8),z’(35) plane as one or more tilted excitation beam waists (TWI) and that the emission excited by the one or more excitation beam waists (TWI) along the x’ axis is synchronized with the acquisition by at least one active area of at least one camera detector.

2. Light sheet microscope according to claim 1 characterized in that means (38) generates an excitation beam with a power / amplitude density distribution having the shape of at least one circular or elliptical arc of 10 - 179° in the Fourier transformation of the wavefront and amplitude distribution.

3. 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) forming a distribution of predominantly conical waves interfering to a axially confined and or modulated or discontinuous excitation beam waists (TWI) in the object plane (1).

4. Light sheet microscope according to any of claims 1 to 3 characterized in that at least two excitation laser light beams (4) have the same wavelength are used to generate the excitation beam waists (TWI) from opposite directions.

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

6. Light sheet microscope according to any of claims 1 to 5 characterized in that the means (38) comprises diaphragms, and / or apertures and / or segmented apertures and / or variable density apertures.

7. Light sheet microscope according to any of claims 1 to 6 characterized in that the at least one of the camera detectors is a rolling shutter camera.

8. Light sheet microscope according to any of claims 1 to 7 characterized in that the means to dynamically confine at least one active area on the camera detectors is at least one scanning slit.

9. Light sheet microscope according to any of claims 1 to 7 characterized in that the means to dynamically confine at least one active area on the camera detectors is a scanner in combination with a TDI camera.

10. Light sheet microscope according to any of claims 1 to 9 characterized in that the excitation beams are propagated in the form of a light sheet in the object plane (1) using one or more galvanometric scan mirrors which mirrors the excitation beam one or two times.

11. Light sheet microscope according to any of claims 1 to 10 characterized in that at least two excitation laser light beams (4) having different wavelengths are used to incoherently illuminate the individual excitation beam waists (TWI) in separate portions of the object plane (1).

12. Light sheet microscope according to any of claims 1 to 11 characterized in that at least two excitation laser light beams (4) are used creating at least two images of the emission excited by excitation beam waists (TWI) on at least two active areas of at least one camera detector.

13. Light sheet microscope according to any of claims 1 to 12 characterized in that the means (38) forms a first excitation beam waist (TWI) excite chromophores and with a second excitation beam waist (TWI) having a intensity minimum in the object plane (1) to deexcite the chromophores resulting in a STED light sheet mode.

14. Light sheet microscope according to any of claims 1 to 12 characterized in that the two means (38) form two light sheet in the object plane (1) which are arranged to excite the sample from different directions.

15. Light sheet microscope according to any of claims 1 to 14 characterized in that the generation of the excitation beam waist TWI is generated through the detection objective (7).

Images