Rescanning optical system and method

Abstract

The present disclosure relates to a rescan optical system for scanning a sample light spot over an imaging plane of an imaging system in order to form an image of a sample. The system comprises an illumination optical system for directing and optionally focusing illumination light at the sample, thereby providing an illumination light spot at the sample. The illumination light spot causes sample light. The system further comprises a detection optical system for focusing at least a portion of the sample light onto the imaging plane of the imaging system, thereby creating a sample light spot on the imaging plane. The system further comprises a light directing element for scanning the illumination light spot over and / or through the sample and for descanning the sample light from the sample and for scanning the sample light spot over the imaging plane of the imaging system. The detection optical system is configured to direct the descanned sample light back to the light directing element along an optical path extending from the light directing element, such that the light directing element is able to perform the scanning of the sample light spot over the imaging plane. In addition, - the optical path is provided with a prism configured to invert and / or reverse the sample light, and / or - the rescan optical system comprises one or more optical elements that cause or are configured to cause at least two foci of the sample light in the optical path, and / or - the optical path is provided with a sample light deflection prism configured to deflect the sample light without inverting the sample light and / or configured to deflect the sample light without reversing the sample light.

Description

Technical Field

[0001] This disclosure relates to rescanning optical systems and methods, and particularly to such systems and methods, wherein a so-called static sample optical path of the rescanning system is provided with a prism and / or wherein the optical system includes one or more optical elements for generating at least two focal points in the static sample optical path. Background Technology

[0002] A re-scan confocal microscope is known from the paper "Re-scan confocal microscopy: scanning twice for better resolution" by De Luca GM, Breedijk RM, Brandt RA, et al., published on October 25, 2013. Biomed OptExpress. 2013; 4(11):2644–2656. doi:10.1364 / BOE.4.002644 (hereinafter referred to as "De Luca"). This microscope has two units: 1) a standard confocal microscope with a set of scanning mirrors that have dual functions: scanning excitation light and de-scanning sample light, and 2) a re-scanning unit that "writes" light through a pinhole onto the camera. The characteristics of the microscope can be controlled by controlling the angular amplitude ratio of the corresponding scanning mirror and the re-scanning mirror.

[0003] In such a rescanning system, it is crucial that the scanning mirror and the rescanning mirror move synchronously. Preferably, each scan of the scanning mirror begins exactly simultaneously with the corresponding scan of the rescanning mirror and ends separately. If the scanning and rescanning mirrors are not synchronized, the resulting image quality will be low. As will be understood, at higher scanning speeds, the mirrors move faster and the acceptable margin of the absolute error in synchronization becomes smaller. Because the degree of synchronization of the mirrors in De Luca's system is limited, the scanning speed is also limited.

[0004] To overcome such synchronization problems, a single scanning mirror can be used to scan both the excitation light, more generally called the illumination light, and the fluorescence light, more generally called the sample light. For example, Roth et al., Optical photonreassignment microscopy (OPRA). Optical Nanoscopy 20132:5 (hereinafter referred to as "Roth"), disclose an optical photon reassignment microscope. In this microscope, a laser emits 488 nm illumination light, which is directed to a dichroic beamsplitter that reflects the illumination light onto a scanning unit. The scanning unit scans the illumination light onto an objective lens, which focuses the illumination light onto the sample. The returning fluorescence light is then directed back to the scanning unit and de-scanned. Subsequently, the fluorescence light is separated from the illumination light using a dichroic beamsplitter. After this separation, the fluorescence beam passes through an adjustable detection pinhole, which can be used to achieve confocal sectioning. The pinhole is positioned between two lenses that amplify the fluorescence beam. After expansion, the fluorescence beam is rescanned using the same scanning system and projected onto the camera via a lens.

[0005] The disadvantage of the latter microscope setup is that the optical setup is quite large because the scanning and rescanning use the same scanning system. That is, the fluorescence beam descanning is constantly guided around the scanning system and then directed to the scanning system, which rescans the fluorescence beam onto the camera. Therefore, there is a need in the art for a more compact optical rescanning system. Summary of the Invention

[0006] Therefore, one aspect of this disclosure relates to a rescanning optical system for scanning a sample spot on an imaging plane of an imaging system to form an image of the sample. The system includes an illumination optics system for guiding and optionally focusing illumination light onto the sample, thereby providing an illumination spot at the sample. The illumination spot generates sample light. The system also includes a detection optics system for focusing at least a portion of the sample light onto the imaging plane of the imaging system, thereby generating a sample spot on the imaging plane. The system further includes a light guiding element for scanning the illumination spot over and / or through the sample and de-scanning the sample light from the sample and scanning the sample spot over the imaging plane of the imaging system. The detection optics system is configured to guide the de-scanned sample light back to the light guiding element along an optical path extending from the light guiding element, such that the light guiding element can perform the scanning of the sample spot over the imaging plane. Additionally,

[0007] - The optical path is equipped with prisms configured to invert and / or reverse the sample light, and / or

[0008] - The rescanning optical system includes one or more optical elements that will be configured to cause at least two focal points of sample light in the optical path, and / or

[0009] - The optical path is equipped with a sample light deflecting prism configured to deflect the sample light without inverting it and / or to deflect the sample light without reversing it.

[0010] If the rescanning optical system includes one or more optical elements that will be configured to cause at least two focal points of sample light in the optical path, then preferably, the optical path is provided with these one or more optical elements, for example, in the sense that these one or more optical elements are positioned in the optical path.

[0011] The optical path can be understood as being configured to reverse and / or invert the sample light an appropriate number of times, so that the sample spot is scanned in the correct orientation above the imaging plane of the imaging system. What constitutes the correct orientation will be explained in more detail below.

[0012] Moving the illumination spot over the sample can also be called scanning. Moving the sample spot over the imaging plane of the imaging system can also be called rescanning. In a rescanning optical system, the same mirror is used for both scanning and rescanning, which prevents synchronization problems. If two separate mirrors are used for scanning and rescanning respectively, these two mirrors may become out of sync for some reason, as explained in the background section, which will degrade the obtained image.

[0013] The inventors have realized that it is impossible to implement an arbitrary number of mirrors in an optical rescanning system in which the scanning mirror and the rescanning mirror cannot move independently of each other. This is the case, for example, when the scanning mirror and the rescanning mirror are the same mirror and / or when there is some kind of mechanical connection between the scanning mirror and the rescanning mirror. The scanning mirror and the rescanning mirror can be mechanically connected in the sense that the scanning mirror is formed by one reflective surface of a multifaceted (e.g., double-faced) reflector and the rescanning mirror is formed by another reflective surface of a multifaceted (e.g., double-faced) reflector.

[0014] As will be explained below, in a rescanning system, it is important that the sample spot is scanned in the correct orientation above the imaging plane. Now refer to... Figure 3 To explain this principle, the left-hand side of this figure schematically illustrates what happens at sample 22 when illumination spot 24 is scanned over or through it. Illumination spot 24 is not infinitely small, but has a spatial intensity distribution due, among other things, to the finite numerical aperture of the illumination system. An example of the light distribution of the illumination spot is high intensity at its center and low intensity further away from the center. As an example, graph 52 schematically shows the intensity I along line 54.

[0015] Figure 3The diagram illustrates line by line how the illumination spot 24 is scanned from left to right over particles 56 and 58. Each line shows the position of the illumination spot relative to particles 56 and 58 at specific times t1, t2, t3, t4, etc. In this text, t1 precedes t2, t2 precedes t3, t3 precedes t4, and so on. First, particle 56 is illuminated, then particle 58 is illuminated. These particles are excited after being illuminated and subsequently emit sample light 28. In this embodiment, particles 56 and 58 are fluorescent particles and sample light 28 is fluorescent light.

[0016] As described, the illumination spot 24 has a spatial light intensity distribution, which in this example has a higher intensity at its center and a lower intensity at its edges. Therefore, as this illumination spot 24 moves over the fluorescent particle, the particle will first be illuminated at a lower intensity, then at a higher intensity, and then again at a lower intensity.

[0017] The right-hand side of the figure schematically illustrates three different examples A, B, and C of what might happen at each moment on the imaging plane 44 of the imaging system 46. Each row shows the sample light 28 present on the imaging plane for a specific moment. In principle, the lower the intensity of the illumination of a particle, the lower the intensity of the sample light generated from that particle. This is in Figure 3 This is reflected in the figure because the resulting sample light 28a(t1) has a lower intensity than the sample light 28b(t3). The lighter hue indicates lower intensity in the figure.

[0018] Figure 3 An image 60 of the illumination spot 24 on the imaging plane is also shown. This image may also be referred to as the illumination spot image. The illumination spot image 60 is positioned on the imaging plane 44, where the reflection of the illumination spot from the sample would be positioned if the illumination light were not blocked by a filter that only allows the sample light to pass through. Figure 1 and 2 In this context, such filters are implemented as dichroic mirrors 12. The “image of the illumination spot” should not be interpreted as the illumination light actually incident on the imaging plane 44 of the imaging system 46. The center 62 of the illumination spot image 60 indicates the center of the image and can therefore be understood as the “image” of the high-intensity central region in the illumination spot 24.

[0019] Additionally, center 62 is also the center of what may be referred to herein as sample spot 63. For clarity, sample spot 63 is indicated only in truncated form in column B. The sample spot at a given time can be understood as covering an area on the imaging plane, where if the entire illuminated portion of the sample will emit sample light at that given time, then that area on the imaging plane will receive the sample light. The sample spot is not infinitely small and also exhibits a spatial light intensity distribution. Furthermore, it should be understood that this light intensity distribution changes as the illumination spot 24 moves over and / or through the sample. For illustration, if the illumination spot 24 does not illuminate any photoexcited particles, then the intensity of the sample spot is zero everywhere in its spatial distribution. This situation occurs, for example, at time t1, when the illumination spot 24 has not yet reached particle 56. On the other hand, if the illumination spot 24 illuminates the particles in the sample with its maximum intensity, i.e., if the high-intensity portion (e.g., the center) of the illumination spot 24 covers the particles, then the sample spot has a relatively high intensity. This occurs, for example, at time t3 when the center of illumination spot 24 illuminates particle 56 and at time t6 when the center of illumination spot 24 illuminates particle 58. Additionally, at time t2, illumination spot 24 weakly illuminates particle 56, which produces a so-called off-axis sample light 28 in the imaging plane. At t2, sample spot 63 exhibits an asymmetric light intensity distribution in the sense that sample light 28d is not concentric with sample spot 63. In a rescanning optical system, the choice of the orientation of the image 60 of the illumination spot and therefore the direction in which the sample spot moves above the imaging plane should consider the following: the direction of movement of the illumination spot above and / or through the sample and the orientation of the image of illumination spot 60 at imaging plane 44 (this can also be referred to as the orientation of the sample spot).

[0020] The sample spot 63 (and thus the illumination spot image 60) is scanned above the imaging plane, from left to right in example A and from right to left in examples B and C. Arrow 61 serves as a guide for the eye to show that the sample spot moves at a constant speed above the imaging plane.

[0021] At any given time, if illumination spot 24 is approaching (or moving away from) any particle illuminated at that given time, which results in sample light 28 at a specific location in the imaging plane at that given time, then the image 60 of the illumination spot (and sample spot 63) should also be approaching (or moving away from) that specific location. In this document, any particle illuminated at a given time is typically illuminated by a low-intensity edge of the illumination light intensity distribution.

[0022] Therefore, it can be understood that at any given time, the direction of movement of the illumination spot relative to the illuminated particles that cause the sample light at a specific location in the imaging plane at that given time should therefore be substantially the same as the direction of movement of the image 60 of the illumination spot (and sample spot 63) relative to said specific location in the imaging plane.

[0023] If the center of illumination spot 24 is close to or far from a particle, then illumination spot 24 can be understood as being close to or far from a particle. Similarly, if the centers 62 of illumination spot image 60 and sample spot 63 are close to or far from their positions in the imaging plane, then illumination spot image 60 (and sample spot 63) can be understood as being close to or far from that position. One element being close to or far from another element can be understood as any relative movement from one element to another, decreasing or increasing the distance between them. Therefore, "close to a particle" should not be interpreted as limited to "moving directly toward the particle." The illumination spot passing by a particle at a certain distance, meaning the center of the illumination spot does not cover that particle, can also be described as the illumination light approaching that particle.

[0024] In Examples A (from left to right) and C (from right to left), the direction of movement of sample spot 63 is correct, while in Example B (from right to left), it is incorrect. This can be determined based on the principles above. In Example A, at t2, illumination spot 24 illuminates particle 56. Therefore, at imaging plane 44, sample light 28a appears at a position on the imaging plane that is to the right of the center 62 (of illumination spot image 60 and sample spot 63) at t2. At t2, illumination spot 24 is approaching particle 56. Therefore, the correct direction of movement of illumination spot image 60 and sample spot 63 in the imaging plane is towards the position where sample light 28a appears, i.e., to the right.

[0025] For each example, the total spatial intensity distribution 64 in the imaging plane 44 is shown. The aggregated spatial intensity distribution 64A for A shows that the image has not suffered significant distortion. However, distinguishing the two particles 56 and 58 from the result 64A can be problematic because there appears to be only a single high-intensity region at the center of the aggregated light distribution 64A, rather than two separate high-intensity regions. Example A corresponds to the case described in De Luca where the optical system exhibits a scan factor M = 1, as in De Luca's... Figure 1 As shown in B / 1C.

[0026] The difference between Example B and Example A is that the illumination spot image 60 is moved in opposite directions (from right to left), while the orientation of the illumination spot image 60 (and therefore the orientation of the sample spot 63) is the same for both Examples A and B. Therefore, the aggregated light intensity distribution 64B is highly distorted. Of course, this effect is worse when many particles are illuminated simultaneously.

[0027] Example C differs from Example A in that the sample spot 60 moves in opposite directions (from right to left) and the orientation of the sample spot 63 is different. In Example C, the orientation of the sample spot is reversed (or inverted, depending on how the horizontal plane is defined) relative to the sample spot in Example A. This positions the sample light 28c to the left of the center 62 of the sample spot, whereas for A, the sample light 28a is positioned to the right of the center 62 of the sample spot. Example C produces the same image quality as Example A. However, the focused light distribution 64C is reversed / inverted relative to the focused light distribution 64A. Example C also corresponds to the case described in D Luca where the system exhibits a sweep factor M = 1.

[0028] In summary, it is clear that the orientation of the illumination spot image 60 at the imaging plane 44 plays a crucial role. After all, the illumination spot image in the imaging plane 44 can be reversed and / or inverted relative to the sample light generated from the sample. The orientation of the sample spot 63 affects the position of the sample light 28 relative to the sample spot 63, particularly relative to the center 62 of the sample spot 63. The sample light 28 can, for example, be positioned to the right or left of the center 62, depending on the number of reversals. A reversal, also known as an x-flip, can be understood as a 180-degree rotation about a vertical line. For example, the sample light can be positioned below or above the center of the sample spot, depending on the number of reversals. An inversion, also known as a y-flip, can be understood as a 180-degree rotation about a horizontal line. As explained above, given a specific direction of movement of the illumination spot, the position of the sample light relative to the illumination spot image determines which direction the illumination spot image should move.

[0029] When a rescanning optical system includes separately controllable scanning and rescanning mirrors, such as the system shown in De Luca, then proper scanning and rescanning orientations can be easily achieved. If the resulting image is highly distorted, it is likely due to incorrect movement of the illumination spot image. In this case, simply reversing the movement of one of the mirrors will achieve correct, high-quality imaging.

[0030] Unfortunately, this correction is impossible when the scanning mirror and rescanning mirror cannot be controlled independently, for example, because the system includes a mirror that functions as both a scanning mirror and a rescanning mirror. If individual control is not possible, then a specific movement of the illumination spot on and / or through the sample corresponds one-to-one with the movement of the illumination spot image on the imaging plane of the imaging system.

[0031] A typical rescanning system has two dynamic paths and two static paths. The first dynamic path can be understood as the optical path from the scanning mirror to the sample and back to the scanning mirror. The second dynamic path can be understood as the optical path from the rescanning mirror to the imaging plane. The first static optical path can be referred to as the static illumination path and can be understood as the optical path followed by the illumination light from the source to the scanning mirror. The second static optical path can be referred to as the static sample optical path and can be understood as the optical path followed by the sample light from the scanning mirror back to the rescanning mirror. Therefore, when the same mirror is used for both scanning and rescanning, the static path is the path followed by the sample light from the (re)scanning mirror back to the (re)scanning mirror. Whenever "optical path" is mentioned in this disclosure, it refers to the static sample optical path unless otherwise stated or unless the context clearly indicates otherwise.

[0032] If a rescanning system, which includes a single mirror as both a scanning and rescanning mirror, is found to be moving the sample spot in the wrong direction, adding a mirror to the system's dynamic path will not solve the problem. After all, adding a mirror to the dynamic path would not only cause the mirror to further alter the orientation of the sample spot, but would also reverse the (re)scanning direction. Clearly, an incorrect pair (i) of scanning direction and (ii) sample spot orientation cannot be corrected by reversing the scanning direction and orientation of the sample spot.

[0033] Based on the above considerations regarding the orientation and scanning direction of the sample light, redesigning Roth's optical setup to reduce its size is not straightforward. For example, it's not possible to simply redesign the static path and remove the mirrors. As explained above, this would produce a distorted image because it changes the orientation of the illumination spot image 60 in the imaging plane without changing its direction of movement within the plane. Therefore, the direction of movement of the illumination spot image 60 would be incorrect.

[0034] The inventors have realized that a more compact optical setup is possible by implementing prisms and / or one or more optical elements for forming at least two focal points in a static sample optical path and / or sample light deflecting prisms configured to deflect the sample light without inverting and / or reversing it. That is, these measures avoid the need for a third mirror in the static sample optical path and achieve a compact optical setup. It should be recognized that, theoretically, mirrors can be placed very close together to make the optical system more compact. However, in practice, this is very cumbersome and technically challenging. In practice, if many mirrors are positioned very close to each other, the support structure of one mirror often blocks the beam reflected from another mirror. Moreover, the more mirrors present in the static path, the more difficult it is to properly align them. To illustrate, if the sample light in the static sample optical path is reflected from a first mirror, then from a second mirror, and then from a third mirror to a rescanning mirror, then the correct orientation of the third mirror depends on both the orientation of the first mirror and the orientation of the second mirror. If a prism is implemented instead of a third mirror, for example, after a second mirror or a pair of lenses, then only the first and second mirrors need to be properly aligned, and the prism or lens pair can be simply placed in the optical path between the second mirror and the rescanning mirror. Preferably, such a prism or lens pair does not change the direction of the sample light in the sense that the light incident on the prism / lens pair has the same propagation direction when it hits the prism / lens pair as it leaves the prism / lens pair. Therefore, implementing a prism and / or optical elements (such as lenses for creating at least two focal points) makes it possible to reduce the size of the rescanning optical system while simplifying the optical setup.

[0035] Preferably, the sample light is reversed an even number of times, such as zero, two, four, or six times, rather than an odd number of times, such as not three times, throughout its journey along the static sample light path, if such reversal causes the orientation of the sample spot to flip around a line perpendicular to the scanning direction of the sample spot, then the line lies in the imaging plane of the imaging system.

[0036] Preferably, the sample light is inverted an even number of times, such as zero, two, four, or six times, rather than an odd number of times, such as not three times, throughout its journey along the static sample light path, if such inversion causes the orientation of the sample spot to flip around a line perpendicular to the scanning direction of the sample spot, then the line lies in the imaging plane of the imaging system.

[0037] To illustrate, if the sample spot is scanned in only one direction, for example, if the illumination spot is linear and covers the entire sample under study, and if reversing (or inverting) the sample light in the static sample light path causes the orientation of the sample spot to flip around a line perpendicular to the scanning direction, which lies in the imaging plane of the imaging system, and if inverting (or reversing) the sample light in the static sample light path causes the orientation of the sample spot to flip around a second line parallel to the scanning direction, which also lies in the imaging plane, then the number of times the sample light in the static sample light path is reversed (or inverted) should be even, for example, zero or two times, and the number of times the sample light in the static sample light path is inverted (or reversed) can be any number of times.

[0038] As another example, if the sample spot is scanned in two directions, x and y, for example, if the illumination spot is circular and scanned over the sample under study in both x and y directions, and if the reversal (or inversion) of the sample light in the static sample light path causes the orientation of the sample spot to flip about a line perpendicular to the x direction, which lies in the imaging plane of the imaging system, and if the inversion (or reversal) of the sample light in the static sample light path causes the orientation of the sample spot to flip about a second line perpendicular to the y direction, which also lies in the imaging plane, then the number of reversals of the sample light in the static sample light path should be even, for example, zero or two, and the number of inversions in the static sample light path should also be even.

[0039] Reversal (or inversion) in a static sample light path is achieved, for example, by reflection of the sample light from a mirror. A point focal point in a static sample light path causes both reversal and inversion of the sample light. A line focal point in a static sample light path causes either reversal or inversion of the sample light, depending on the orientation of the line focal point and assuming that the line focal point is not oriented such that it flips the orientation of the sample light spot around a line at a non-90-degree, non-zero-degree angle with the scanning direction. Such a line focal point can be achieved, for example, by providing a static path with two cylindrical lenses. Preferably, at least two focal points caused by one or more optical elements cause at least two reversals or at least two inversions. For example, if there are two and only two focal points caused by optical elements, then one focal point can be a line focal point and the other can be a point focal point. These two focal points can also be, for example, two line focal points, both causing reversal of the sample light. These two focal points can also be, for example, two line focal points, both causing inversion of the sample light. The two focal points can also be two point focal points.

[0040] The light guiding element can be a movable reflector configured to reflect illumination light and sample light, such as a movable mirror. The reflector can be movable in the sense that it can rotate about a rotation axis. Additionally or alternatively, the light guiding element can be an acousto-optic modulator (AOM) or an electro-optic modulator (EOM). Such deflectors can be used as reflectors with a controllable reflection direction.

[0041] The light guiding element can be understood as being configured to scan the illumination spot over and / or through the sample and simultaneously de-scan the sample light from the sample and simultaneously scan the sample spot over the imaging plane of the imaging system. Note that scanning the sample spot over the imaging plane can be referred to as rescanning. Of course, as used herein, since the speed of light is not infinite and, for example, since photoexcited molecules in the sample remain excited for a period of time before they decay and emit photons, if two events, such as scanning the illumination spot and rescanning (generated by the scanning illumination spot) the sample spot, are said to occur simultaneously, then in reality, one event may occur shortly after the other. This is the case, for example, with scanning and rescanning, because the sample light must travel from the sample to the rescanning mirror via a static path.

[0042] It should be recognized that at least one or more (e.g., all) of the two focal points can be line focal points. Additionally or alternatively, at least one or more (e.g., all) of the two focal points can be point focal points. A point focal point can be understood as both reversing and inverting the sample light, while a line focal point only reverses or only inverts the sample light, depending on the orientation of the line focal point.

[0043] Preferably, the prism configured to invert and / or reverse the sample light is a prism configured to provide inline inverted or reversed sample light. This can be understood as the propagation direction of the sample light hitting the prism being the same as the propagation direction of the sample light leaving the prism and / or the propagation direction of the sample light leaving the prism not being laterally displaced relative to the propagation direction of the sample light hitting the prism. "Laterally" can be understood as a direction perpendicular to the propagation direction of the light.

[0044] Preferably, one or more optical elements also provide inline inversion and / or reversal. Additionally, preferably, one or more optical elements are transmission optical elements in the sense that they transmit at least the wavelength of interest (i.e., the wavelength of the sample light).

[0045] Any optical element described herein, such as a sample light deflecting prism configured to deflect sample light, can be understood as altering the propagation direction of the sample light. This can be understood as the sample light having a first propagation direction just before hitting the optical element, and a second propagation direction different from the first propagation direction as it leaves the optical element.

[0046] Optionally, the rescanning system includes a light source, such as the light source described herein. Optionally, the rescanning optical system includes a microscope system, such as the microscope system described herein. Optionally, the rescanning optical system includes an imaging system, such as the imaging system described herein.

[0047] In this embodiment, the optical path is provided with

[0048] - Two, and only two, lenses, and

[0049] - Two, and only two, mirrors for reflecting the sample light, and

[0050] - The prism is configured to invert and / or reverse the sample light.

[0051] This embodiment advantageously ensures that the sample spot is scanned in the correct direction.

[0052] In this embodiment, the prism configured to invert and / or reverse the sample light is a Dove prism. This is advantageous because it can be easily aligned and the Dove prism does not change the direction of the light path.

[0053] In this embodiment, the prism configured to invert and / or reverse the sample light is a Pechan prism.

[0054] The prism configured to invert and / or reverse the sample light can also be a Schmidt-Pechan prism, an Abbe-Koenig prism, a Porro-Abbe prism, or a double Porro prism.

[0055] In one embodiment, the optical path includes a sample light deflecting prism configured to deflect the sample light without reversing it and / or to deflect the sample light without inverting it. An advantage of this embodiment is that it allows for a reduction in the number of optical elements in a static optical path. For illustration, instead of mirrors and (in-line) prisms configured to invert and / or reverse the sample light, a single sample light deflecting prism can be used to deflect the sample light without reversing or inverting it. This allows for even more compact optical setups. Such a sample light deflecting prism can be, for example, a Bauernfeind prism or a pentaprism. It should be appreciated that the optical path can include multiple sample light deflecting prisms configured to deflect the sample light without reversing it and / or to deflect the sample light without inverting it.

[0056] In this embodiment, one or more optical elements include at least three lenses, preferably at least four lenses. Using three lenses, when properly positioned and with an appropriate focal length, two sample light focal points can be easily created.

[0057] In this embodiment, the optical path is provided with

[0058] - Four, and only four, lenses, and

[0059] - Two, and only two, mirrors that reflect the sample light.

[0060] For four lenses, designing a setup that produces two focal points may be simpler, as this allows for two simple telescopes in a static path, each formed by two lenses. One of these telescopes can be used to focus the sample light onto a pinhole or optical slit. In this embodiment, preferably, the optical path does not include a prism configured to invert and / or reverse the sample light. Advantageously, four lenses provide flexibility regarding how to achieve the desired optical magnification for the rescanning optics system.

[0061] In one embodiment, the light guiding element is configured to scan a sample spot above the imaging plane in a first scanning direction. In this embodiment, the reversal or inversion of the sample light in the optical path can be understood as causing the orientation of the sample spot in the imaging plane to be flipped about a line perpendicular to the first scanning direction and located in the imaging plane of the imaging system. In this embodiment, the optical path is configured to cause an even number of reversals or inversions of the sample light in the optical path.

[0062] In this embodiment, the light guiding element can also be configured to scan the sample spot in a second scanning direction (e.g., perpendicular to the first scanning direction) above the imaging plane, different from the first scanning direction. In this embodiment, the inversion or reversal of the sample light in the optical path can be understood as causing the orientation of the sample spot in the imaging plane to flip around a line perpendicular to the second scanning direction and located within the imaging plane of the imaging system. In this embodiment, the optical path is configured to cause an even number of reversals or inversions of the sample light in the optical path.

[0063] It should be recognized that optical elements cause reversal and / or inversion of the sample light in the static optical path, and that an appropriate number (i.e., an even number) of reversals can be achieved by properly combining these optical elements in the static optical path (and an appropriate number of inversions are also required if the sample light spot is scanned in two dimensions). For illustration, a pair of lenses typically causes one reversal and one inversion of the sample light (therefore, a single lens can be understood as causing 0.5 reversals and 0.5 inversions of the sample light), a pair of spherical mirrors causes three reversals (therefore, a single spherical mirror can be understood as causing 1.5 reversals), a reflecting mirror causes one reversal, a pentaprism causes no reversal or inversion but deflects the sample light, a prism used as a reflecting mirror causes one reversal of the sample light, a Porro prism causes no reversal or inversion of the sample light, and a pair of cylindrical mirrors can be understood as causing three reversals of the sample light (therefore, a cylinder can be understood as causing 1.5 reversals). Additionally, the Dove prism causes one reversal (or inversion, depending on its orientation) of the sample light, the Pechan prism causes one reversal and one inversion, the Abbe-Koenig prism causes one reversal and one inversion, the Porro-Abbe prism causes one reversal and one inversion, the double Porro prism causes one reversal and one inversion, and a cylindrical lens can be understood as causing 0.5 reversals (or 0.5 inversions, depending on its orientation). As described, such building blocks can be appropriately combined in a static optical path to achieve an even number of reversals, and, if necessary, an even number of inversions. This will refer to... Figure 1-2F To explain in more detail.

[0064] In this embodiment, the illumination spot is a linear illumination spot. This embodiment enables rapid scanning of samples.

[0065] In one embodiment, the system includes a beam splitter configured to separate illumination light from sample light. The beam splitter is configured, for example, to reflect illumination light toward the sample and allow sample light to pass through, or to reflect sample light and allow illumination light to pass through. Preferably, a filter is positioned in the optical path.

[0066] In an embodiment, the beam splitter is a dichroic filter, such as a dichroic mirror, used to separate the sample light from the illumination light.

[0067] In this embodiment, the optical path includes a pinhole and / or an optical slit. This embodiment improves the system's optical slicing capability. The pinhole and / or optical slit are preferably configured to block sample light from areas of the sample that are not in the focal point of the scanning lens. Preferably, the optical path includes optical elements configured to focus sample light at the pinhole or optical slit.

[0068] In an embodiment, the imaging system is configured to integrate sample light incident on the imaging plane over time. The imaging system may be configured to integrate sample light incident on the imaging plane during a single scan of the sample. Then, preferably, the imaging system is configured to integrate the incident sample light over a time period equal to the duration of a single scan. The imaging system may be configured to integrate sample light incident on the imaging plane during multiple (e.g., repeated) scans of the sample. Then, preferably, the imaging system is configured to integrate the incident sample light over a time period longer than the duration of a single scan (e.g., equal to five scan cycles). The imaging system may be configured to integrate sample light incident on the imaging plane during a portion of a scan of the sample. Then, preferably, the imaging system is configured to integrate the incident sample light over a time period shorter than the duration of a single scan. As used herein, a single scan of the sample can be understood as an illumination spot illuminating each location of the region of interest on or within the sample once, while, for example, two scans of the sample can be understood as illumination spots illuminating each location of the region of interest twice, and so on.

[0069] In an embodiment, the rescanning optics system is configured to move an illumination spot over and / or through the sample at a first velocity and to move a sample spot over the imaging plane at a second velocity, such that the second velocity is different from, preferably higher than, and more preferably approximately twice the baseline velocity. Hereinafter, the baseline velocity is defined as the first velocity multiplied by the optical magnification of the rescanning optics system. This embodiment allows for increased resolution of the acquired image, as explained in De Luca.

[0070] In one embodiment, the system includes an objective lens configured to collect sample light from the sample and focus the sample light onto the main imaging plane of the scanning electron microscope system. In this embodiment, the detection optics system can be configured to image an image from the main imaging plane onto the imaging plane of the imaging system, preferably having an optical magnification of approximately 0.5, which would mean that the sample light spot is on the main image plane (see [link to relevant documentation]). Figure 1 and 2 The image in the main image plane 16) is twice the size of the image of the sample spot at the imaging plane 44 of the imaging system. This embodiment allows for a sweep factor of M=2 while scanning the illumination light above the main imaging plane and the sample light above the imaging plane 44 at equal speeds.

[0071] One aspect of this disclosure relates to a method for scanning a sample spot over an imaging plane of an imaging system to form a sample image using a rescanning optical system as described herein. The method includes positioning a light guiding element over and / or through the sample to scan the illumination spot, descanning the sample light from the sample, and scanning the sample spot over the imaging plane of the imaging system. Additionally or alternatively, the method includes guiding the sample light through an optical path.

[0072] Unless otherwise expressly stated, elements and aspects discussed with respect to a particular embodiment or with respect to a particular embodiment may be suitably combined with elements and aspects of other embodiments. Embodiments of the invention will be further described with reference to the accompanying drawings, which will schematically illustrate embodiments according to the invention. It will be understood that the invention is not limited in any way to these specific embodiments. Attached Figure Description

[0073] Various aspects of the invention will be explained in more detail with reference to the exemplary embodiments shown in the accompanying drawings, wherein:

[0074] Figure 1 A rescanning optical system including a prism is shown according to an embodiment;

[0075] Figure 2 A rescanning optical system comprising one or more elements is shown, which generate at least two focal points in the static sample optical path;

[0076] Figure 2A-2C The illustration shows a further rescanning optical system according to a corresponding further embodiment, wherein the optical path is provided with a prism configured to invert and / or reverse the sample light;

[0077] Figure 2D-2F The illustration shows a further rescanning optical system according to a corresponding further embodiment, wherein the optical path is provided with a prism configured to deflect the sample light without reversing the sample light and / or configured to deflect the sample light without inverting the sample light.

[0078] Figure 3 The illustration shows the scanning of the illumination spot and the rescanning of the sample spot according to an embodiment;

[0079] Figure 4 The illustration shows the scanning of an illumination spot with a high sweep factor and the rescanning of a sample spot according to an embodiment.

[0080] Figure 5 The illustration shows the scanning of a line-shaped illumination spot and the rescanning of a sample spot according to an embodiment. Detailed Implementation

[0081] In the figures, identical reference numerals indicate identical or similar elements.

[0082] Figure 1 A rescanning optical system 2 for forming an image of sample 22, such as a rescanning microscope system 2, is illustrated according to an embodiment. This optical system 2 includes a light source 4 configured to generate illumination light 6. However, it should be understood that the rescanning optical system itself does not include a light source. The rescanning optical system includes, for example, components for receiving the illumination light, such as an input connection for an optical fiber to provide the illumination light. The light source 4 may be a laser, such as a solid-state laser or a diode laser. The light source 4 may include a collimator (not shown) configured to generate a parallel beam with a relatively large diameter, wherein the cross-section of the beam preferably has a uniform intensity distribution. The collimator may include one or more negative lenses and one or more positive lenses to generate an extended collimated beam. Such a collimator in… Figure 6A The image is shown (see reference numeral 70). In this example, the diameter of the beam generated by the light source 4 is 10 mm. The sample light 6 is typically the light that excites photons in the sample 22, and therefore can also be referred to as the excitation light. However, the sample light can also be generated by other effects, such as reflection, Raman effect, Billouin radiation, etc. The illumination light 6 is reflected from the reflector 8 and strikes the cylindrical lens 10, which focuses the illumination light onto the light guiding element 14, thereby creating a horizontal line at the reflector 14. In this document, horizontal can be understood as parallel to the indicated xz plane. The optical path distance between the cylindrical lens 10 and the aperture of the light source 4 is preferably as close as possible to the focal length of the cylindrical lens 10.

[0083] Before the illumination light 6 is incident on the light guiding element 14, it is reflected by the dichroic mirror 12. Preferably, the dichroic mirror 12 is positioned at a 45-degree angle relative to the propagation direction of the incoming illumination light 6, such that the propagation direction of the illumination light 6 changes by 90 degrees upon reflection.

[0084] The light guiding element 14 reflects the illumination light 6 toward the scanning lens 15, which is configured to focus the sample light onto the main image plane 16 (also referred to as the interface plane 16) of the microscope 17, which includes the tube lens 18 and the objective lens 20. The rescanning optics system itself does not include the microscope. It can, for example, be configured to establish an optical connection with the microscope. In the depicted embodiment, the scanning lens 15 creates a vertical line at the main image plane 16. Herein, vertical can be understood as parallel to the indicated y-axis. The distance between the scanning lens 15 and the light guiding element 14 is approximately equal to the focal length of the scanning lens 15. In the example, the effective focal length of the scanning lens 15 is approximately 75 mm. The scanning lens 15 has, for example, a diameter of 2 inches.

[0085] Each microscope can have a different focal length for the tube lens 18. The distance between the main image plane 16 and the tube lens is preferably equal to the focal length of the tube lens.

[0086] In the rescanning microscope system described herein, and similarly for any microscope system, objective 20 is preferably the NA-limiting component of the entire optical system. NA stands for numerical aperture.

[0087] Objective lens 20 is configured to focus illumination light onto sample 22, thereby generating an illumination spot 24 on or within sample 22. Illumination spot 24 generates sample light 28 from sample 22. This sample light 28 can be illumination light reflected from the sample. However, typically, illumination light 28 induces photoluminescence in the sample, such as fluorescence and / or phosphorescence, and generally, sample light 28 is photoluminescent, such as fluorescence and / or phosphorescence.

[0088] Objective lens 20 is configured to capture sample light 28 from the sample. On its return journey, from objective lens 20 to dichroic filter 12, sample light 28 travels along the same path as illumination light 6 on its path to the sample. In other words, from objective lens 20 to dichroic filter 12, the sample light path is substantially the same as the illumination light path.

[0089] In this embodiment, the sample light 28 passes through the filter 12. Simultaneously, the filter reflects any illumination light already reflected from the sample 22. Therefore, the filter 12 can be understood as separating the illumination light 6 from the sample light 28.

[0090] After passing through filter 12, the sample light is incident on lens 30. Preferably, the distance between lens 30 and light guiding element 14 is approximately equal to the focal length of lens 30. In this example, the focal length of lens 30 is 80 mm.

[0091] Lens 30 focuses sample light 28 onto optical slit 34 via mirror 32. In this example, the optical slit has a width of approximately 50 micrometers and a length of 2-3 cm. After the sample light 28 passes through optical slit 34, it is reflected by mirror 36 and meets lens 38. The optical distance between optical slit 34 and lens 38 is preferably equal to the focal length of lens 38. Lens 38 can be identical to lens 30.

[0092] After lens 38, the sample light is incident on prism 40, which inverts and / or reverses the sample light. This prism can be as follows: Figure 1The Dove prism shown is an example. Alternatively, it can be any other type of prism, such as a Pechan prism, a Schmidt-Pechan prism, an Abbe-Koenig prism, a Porro-Abbe prism, or a double Porro prism. These other types of prisms can be positioned between lens 38 and rescanning mirror 14, just like the Dove prism 40. In order to move the sample spot in the correct direction, prism 40 is configured to reverse and / or invert the sample light, and therefore also reverse and / or invert the sample spot 63 in imaging plane 44.

[0093] After prism 40, the sample light is again incident on the light guiding element 14. The optical distance between lens 38 and light guiding element 14 is preferably close to (e.g., equal to) the focal length of lens 38. In this embodiment, the sample light 28 hits the light guiding element 14 at an angle different from the angle at which the illumination light 6 hits the light guiding element. When the sample light 28 hits the light guiding element 14, the light guiding element 14 acts as a rescanner to scan the sample light 28 on the imaging plane 44 of the imaging system 46.

[0094] therefore, Figure 1 The static sample optical path in the embodiment is provided with

[0095] - Two, and only two, lenses, and

[0096] - Two, and only two, mirrors for reflecting the sample light, and

[0097] -The prism.

[0098] Imaging systems include, for example, cameras, such as CCD cameras. The imaging plane preferably comprises a plurality of pixels arranged in a predetermined manner (e.g., in a 2D grid).

[0099] Before the sample light 28 reaches the imaging plane 44, the sample light 28 is incident on the rescanning lens 42, which is configured to focus the sample light onto the imaging plane 44. Preferably, the distance between the light guiding element 14 and the lens 42 is equal to the focal length of the lens 42. In an embodiment, the lens 42 is the same as the lens 15. The lens 42 may have a focal length of 75 mm.

[0100] The optical magnification of a rescanning microscope system can be understood as the optical magnification provided by the combination of objective lens 20 and tube lens 18 (referred to as M in De Luca). micrThe optical magnification M2 of the detection optical system is determined by scanning lens 15, lenses 30 and 38, and rescanning lens 42. In the depicted embodiment, the optical magnification M2 of the detection optical system is given by the following formula: M2 = (f_30 * f_42) / (f_15 * f_38), where f_x represents the focal length of lens x.

[0101] It should be recognized that the velocity v_16 of the sample spot image in the main image plane 16 is given by the product of the first velocity v_1 (i.e., the actual velocity of the illumination spot 24 above and / or through the sample 22) and the optical magnification of the combination of the objective lens 30 and the tube lens 28, that is, v_16 = v_1 x M micr .

[0102] Then, in the depicted embodiment, the baseline velocity v_B is determined by v_B = v_1 x M. micr x M2 is given.

[0103] In one embodiment, the second velocity (i.e., the velocity at which the sample spot moves above the imaging plane 44) is approximately twice the baseline velocity, v_2 ≈ 2 x v_B. Note that the second velocity is equal to the velocity at which the image 60 of the illumination spot in the imaging plane 44 moves above the imaging plane 44. This can be achieved, for example, by configuring the detection optics such that it images the image in the main image plane 16 onto the imaging plane 44 of the imaging system 46 with an optical magnification of approximately 0.5, M2 ≈ 0.5. In this way, if v_16 equals v_2, then the second velocity is twice the baseline velocity.

[0104] As described in De Luca, the so-called sweep factor M is preferably approximately equal to 2. In the depicted embodiment, the corresponding amplitudes of the scanning and rescanning mirrors are the same, of course, because the same mirrors are used for both scanning and rescanning. Therefore, in the depicted embodiment, the sweep factor M is given by M = (f_38) / (f_30).

[0105] exist Figure 1 In the illumination optical system, there are a reflector 8, a lens 10, a dichroic mirror 12, a scanning mirror 14, and a scanning lens 15. Figure 1 The detection optical system includes a scanning lens 15, a scanning mirror 14 (which can also be referred to as a de-scanning mirror 14 in the context of detection), a dichroic mirror 12, a lens 30, a reflecting mirror 32, an optical slit 34, a reflecting mirror 36, a lens 38, a prism 40, a scanning mirror 14 (which can also be referred to as a re-scanning mirror 14 in the context of detection), and a lens 42.

[0106] The duration of a single scan can be between 25ms and 10 seconds.

[0107] exist Figure 1 In the embodiment, lenses 30 and 38 cause the sample light to be focused in the static sample optical path, particularly at the optical slit 34. Therefore, the two lenses 30 and 38 cause the sample light to be inverted and reversed (and thus the sample spot to be inverted and reversed at the imaging plane 44). Additionally, mirror 32 causes reversal, mirror 36 causes reversal, and prism 40 causes another reversal of the sample light. Therefore, in the static sample optical path, the sample light is reversed four times and inverted once. In the described embodiment, we can assume that reversal flips the orientation of the sample spot in the imaging plane 44 about the vertical axis, and inversion flips the orientation of the sample spot about the horizontal axis, while the sample spot is scanned only in the horizontal direction. Therefore, the number of reversals of the sample light in the static sample optical path should be even, and the number of inversions can be arbitrary. This is correct since the sample light is reversed four times in the static sample optical path.

[0108] Figure 2 A rescanning optical system according to another embodiment is shown. In this embodiment, the static sample light path includes four and only four lenses and two and only two mirrors that reflect the sample light. In this embodiment, the static sample light path returning from the light guiding element 14 to the light guiding element 14 includes four lenses 30, 38, 48, and 49 and two mirrors 32 and 36. In this particular embodiment, the static sample light path does not include prisms.

[0109] It should be recognized that the two additional lenses 48 and 49 introduce additional inversion and inversion in the static sample optical path, thus affecting the orientation of the sample spot in the imaging plane. Therefore, these lenses ensure that the scanning direction of the sample spot at the imaging plane 44 is correct.

[0110] Specifically, the four lenses 30, 38, 48, and 49 produce two focal points for the sample light in the static sample optical path. Therefore, in the depicted embodiment, the four lenses together cause two reversals and two inversions, and mirror 32 causes a reversal, and mirror 36 causes a reversal. Thus, in this static path, the sample light 28 is reversed four times and inverted twice. In the depicted embodiment, we can assume that the reversals cause the sample light spot to flip around a vertical line, while the sample light spot is scanned horizontally. Therefore, in summary, the number of reversals of the sample light in the static sample optical path should be even, since the number of reversals of the sample light in the static sample optical path is four.

[0111] In this embodiment, the sweep factor M, as defined in De Luca, is given by M = (f_38 * f_49) / (f_30 * f_48). This formula shows the flexibility that the two additional lenses 48 and 49 provide in how to achieve, for example, a desired sweep factor of M = 2. For example, if lens 30 is the same as 38, then a sweep factor of M = 2 can be obtained by choosing a lens 49 with a focal length twice that of lens 48.

[0112] exist Figure 2 In the illumination optical system, there are a reflector 8, a lens 10, a dichroic mirror 12, a scanning mirror 14, and a scanning lens 15. Figure 2 The detection optical system includes a scanning lens 15, a scanning mirror 14 (which may also be referred to as a descanning mirror 14 in the context of detection), a dichroic mirror 12, a lens 30, a reflecting mirror 32, an optical slit 34, a reflecting mirror 36, a lens 38, a lens 48, a lens 49, a scanning mirror 14 (which may also be referred to as a rescanning mirror 14 in the context of detection), and a rescanning lens 42.

[0113] Figure 2A An embodiment in which the optical path includes prisms configured to invert and / or reverse the sample light is illustrated. In this embodiment, a Schmidt-Pechan prism (shown schematically only) is used. As a side note, in this embodiment, two prisms 29a and 29b are used to deflect the sample light. As explained above, the number of reversals in the static optical path in this embodiment should be even. The number of inversions in the static optical path is irrelevant because the sample light is scanned in only one direction, as the sample light spot is linear. Lens pairs 30 and 38 can be understood as causing one reversal (because they create the focal point at slit 34), prism 29a causes one reversal, prism 29b causes one reversal, and the Schmidt-Pechan prism also causes one reversal. Therefore, the total number of reversals of the sample light in the static optical path is four, which is even and therefore correct.

[0114] Figure 2B An embodiment in which the optical path includes a prism configured to invert and / or reverse the sample light is illustrated. In this embodiment, an Abbe-Koenig prism 39b (shown schematically only) is used. In this embodiment, a convex mirror 29c and a regular mirror 36 are used to deflect the sample light. The number of reversals is four: the convex mirror, together with lens 38, causes two reversals, the mirror 36 causes one reversal, and the Abbe-Koenig prism 39b causes one reversal of the sample light. Of course, any other prism that causes one reversal of the sample light can be used in this embodiment.

[0115] Figure 2CAn embodiment in which the optical path includes a prism configured to invert and / or reverse sample light is illustrated. In this embodiment, an Abbe-Koenig prism 39b (shown schematically only) is used. In this embodiment, convex mirrors 29c and 29d are used to deflect the sample light. The number of reversals is four: convex mirrors 29c and 29d cause three reversals of the sample light, and the Abbe-Koenig prism 39b causes one reversal of the sample light. Of course, any other prism causing one reversal of the sample light can be used in this embodiment.

[0116] Figure 2D The illustration shows an embodiment in which a sample light deflecting prism 35 is provided in the static optical path. This sample light deflecting prism 35 is configured to deflect the sample light without reversing it and / or to deflect the sample light without inverting it. In this embodiment, the sample light deflecting prism 35 is a Bauernfeind prism. This prism deflects the sample light without reversing or inverting it. The sample light is reversed a total of twice in the static optical path: once by lenses 30 and 38, once by prism 35, and once by mirror 36.

[0117] Figure 2E The illustration shows an embodiment in which a sample light deflecting prism 37 is provided in the static optical path. This sample light deflecting prism 37 is configured to deflect the sample light without reversing it and / or to deflect the sample light without inverting it. In this embodiment, the sample light deflecting prism 37 is a pentaprism. This prism deflects the sample light without reversing or inverting it. The sample light is reversed a total of twice in the static optical path: once by lenses 30 and 38, once by prism 37, and once by mirror 32.

[0118] Figure 2F Is with Figure 2E The same embodiment, the only difference being that the lens focuses the sample light at the reflector 32 and that the slit exists on the reflector.

[0119] Figure 4 This schematically illustrates what happens if the sweep factor M, mentioned in De Luca, equals 2 (M = 2). For example... Figure 3 As shown in the diagram, the left-hand side illustrates what happens on sample 22. Illumination spot 24 scans over the sample and illuminates the first particle 56, then the particle 58. Additionally, columns D, E, and F illustrate what may occur at imaging plane 44, depending on the direction of movement of the sample spot over imaging plane 44 (and therefore the direction of movement of the illumination spot image 60), and depending on the orientation of the sample spot (and therefore the orientation of the illumination spot image 60).

[0120] Columns D and F illustrate examples where the direction of movement of the illumination spot image is correct. Column E illustrates an example where the direction of movement of the illumination spot image 60 is incorrect. The resulting image 64E in column E is highly distorted. However, the mirror images 64D and 64F each clearly show two high-intensity regions corresponding to particles 56 and 58, respectively. This demonstrates that a sweep factor higher than 1, preferably around 2 (see De Luca), produces a better image in terms of sharpness. Images 64D and 64F obtained with M=2 are superior to images 64A and 64C obtained with M=1. However, in images 64A and 64C, the two separate high-intensity regions corresponding to particles 56 and 58 cannot be distinguished, while in images 64D and 64F, the two high-intensity regions can be distinguished.

[0121] Figure 5 The illustration shows an embodiment where the illumination spot 24 is a linear illumination spot. This illumination spot is, for example, formed by... Figure 1 and 2 The embodiment shown is formed. The advantage of the linear illumination spot is that it enables rapid scanning of the sample.

[0122] This linear illumination spot 24 also has a spatial intensity distribution. Reference numeral 52 indicates the light intensity in the illumination light 24 along line 54.

[0123] and Figure 3 and 4 Similar to China, Figure 5 The right-hand side of the image indicates what might happen at the imaging plane 44 of the imaging system 46 in columns G, H, and I. Specifically, the position of the sample light 28 is shown at different times t1-t7. Image 62 in the imaging plane 44 also indicates the high-intensity region of the linear illumination spot 24. Image 62 indicates the position of the high-intensity region of the illumination light reflected from the sample 22 in the imaging plane 44 if the reflected illumination light were not blocked by a filter (e.g., dichroic mirror 12). Further shown is the (linear) sample spot 63. The dotted line indicates the boundary of the sample spot 63. For clarity, the sample spot 63 is shown only in truncated form in column H. It should be noted that 63 is the image of the illumination spot (…). Figure 5 (not indicated in the text) and the central area of ​​both sample spot 63.

[0124] Figure 6A The diagram shows Figure 1 The optical series of the rescanning optical system. Figure 6A The top section shows the optical series of the illumination light and Figure 6AThe bottom section shows the optical sequence of the sample light. In this paper, 6a and 6b depict the illumination light as observed from two orthogonal directions, respectively. These views differ because the cylindrical lens 10 causes a line focus in 14 instead of a point focus, which ultimately produces a line-shaped illumination spot at the sample.

[0125] In this embodiment, plane 14 (i.e., the light guiding element) is the conjugate plane of the back focal plane 72 of the microscope 17. Additionally, planes 19, 16, and 34 (aperture slits) are the conjugate planes of the focal plane of the microscope 17 (i.e., the plane where the illumination light is focused).

[0126] Figure 6B The diagram illustrates the optical sequence of off-axis sample light from the sample. The optical axis is indicated by 76.

[0127] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the terms “comprising” and / or “including” as used in this specification specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0128] All components or steps plus functional elements in the following claims are intended to include any structure, material, action, and equivalent that, in combination with other claimed elements as particularly claimed, to perform a function. The description of embodiments of the invention is presented for illustrative purposes and is not intended to be exhaustive or limited to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the invention. The embodiments were chosen and described to best explain the principles of the invention and some practical applications, and to enable others skilled in the art to understand the various embodiments of the invention and various modifications suitable for the particular purpose intended.

Claims

1. A rescanning optical system for scanning a sample spot on the imaging plane of an imaging system to form an image of the sample, comprising: An illumination optics system is used to guide and optionally focus illumination light onto a sample, thereby providing an illumination spot at the sample, the illumination spot generating sample light. An optical detection system is used to focus at least a portion of the sample light onto the imaging plane of an imaging system, thereby generating a sample light spot on the imaging plane. The system also includes a light guiding element for scanning the illumination spot over and / or through the sample and descanning the sample light from the sample, and scanning the sample spot over the imaging plane of the imaging system. The detection optical system is configured to guide the sample light to be scanned along the optical path traveling from and back to the light guiding element, such that the light guiding element can perform the scanning of the sample light spot on the imaging plane, wherein... - The optical path includes a prism configured to invert the sample light by flipping its orientation in the imaging plane about a horizontal axis and / or to reverse its orientation in the imaging plane about a vertical axis, and / or wherein... - The rescanning optical system includes one or more optical elements configured to invert the sample light by flipping its orientation in the imaging plane about a horizontal axis and / or to reverse its orientation in the imaging plane about a vertical axis, and also to generate at least two focal points of the sample light in the optical path, and / or wherein... - The optical path includes a sample light deflecting prism configured to deflect the sample light without inverting it and / or to deflect the sample light without reversing it, and the optical path is configured to cause the sample light to reverse or invert an even number of times in the optical path.

2. The rescanning optical system according to claim 1, wherein the optical path is provided with - Two lenses, - Two mirrors for reflecting sample light, and - The prism is configured to invert the sample light by flipping its orientation in the imaging plane about a horizontal axis and / or to reverse its orientation in the imaging plane about a vertical axis.

3. The rescanning optical system of claim 2, wherein the prism configured to invert and / or reverse the sample light in the optical path is a Dove prism, a Pechan prism, a Schmidt-Pechan prism, an Abbe-Koenig prism, a Porro-Abbe prism, or a double Porro prism.

4. The rescanning optical system according to claim 1, wherein the sample light deflection prism is a pentaprism or a Bauernfeind prism.

5. The rescanning optical system according to any one of the preceding claims, wherein The light guiding element is configured to scan the sample light spot above the imaging plane in the scanning direction, wherein The reversal or inversion of the sample light in the optical path causes the orientation of the sample spot in the imaging plane to be flipped around a line perpendicular to the scanning direction and located in the imaging plane of the imaging system.

6. The rescanning optical system according to any one of claims 1-4, wherein the illumination spot is a linear illumination spot.

7. The rescanning optical system according to any one of claims 1-4 further includes a beam splitter configured to separate the illumination light from the sample light.

8. The rescanning optical system according to any one of claims 1-4, wherein the optical path is provided with a pinhole and / or an optical slit.

9. The rescanning optical system according to any one of claims 1-4, wherein the imaging system is configured to integrate the sample light incident on the imaging plane over time.

10. The rescanning optical system according to any one of claims 1-4, wherein the rescanning optical system is configured to move an illumination spot over and / or through a sample at a first speed and to move the sample spot over an imaging plane at a second speed, such that the second speed is higher than the baseline speed, wherein The baseline velocity is defined as the first velocity multiplied by the optical magnification of the rescanning optical system.

11. The rescanning optical system of claim 10, wherein the second speed is twice the baseline speed.

12. The rescanning optical system according to any one of claims 1-4, further comprising: The objective lens is configured to collect sample light from the sample and focus the sample light onto the main imaging plane of the scanning electron microscope system, wherein The detection optical system is configured to image an image in the main imaging plane onto the imaging plane of the imaging system.

13. The rescanning optical system according to claim 12, wherein... The detection optical system is configured to image the image in the main imaging plane onto the imaging plane of the imaging system at an optical magnification of 0.

5.

14. A method for scanning a sample spot on the imaging plane of an imaging system using a rescanning optical system according to any one of the preceding claims to form an image of the sample, the method comprising: The light guiding element is positioned above and / or through the sample to scan the illumination spot, and the sample light from the sample is de-scanned, and the sample spot is scanned above the imaging plane of the imaging system, and / or The guide sample light passes through the optical path.

Images