Confocal microscope

Abstract

The present invention relates to a confocal microscope apparatus 100, which comprises a structured light source S, a disk group 200 comprising at least a first rotating disk 5 and a second rotating disk 10 configured to receive a beam of structured light and to transmit the resulting excitation beam to the optical system of a microscope M focused on the plane of a sample C, and an acquisition sensor A configured to detect a fluorescence beam emitted by the plane of the sample C, wherein the disk group is optically interposed between the source S and the plane of the sample C. The first rotating disk 5 and the second rotating disk 10 each comprise a disk-shaped substrate 115 made of an optically transparent material and a mask 116 made of an extremely black material that is opaque to light and has at least one sector provided with one or more holes P1, P2. The apparatus comprises moving means 1, 19, 20; 1, 33 configured to move the first rotating disk 5 and the second rotating disk 10 such that a resulting excitation beam is implemented by the passage of a structured light beam through at least one opening L resulting from the overlap of holes, each opening L resulting from the overlap of a first hole P1 in the first rotating disk 5 and a second hole P2 in the second rotating disk 10, and further comprising the moving means 1, 19, 20; 1, 33. The area of ​​the opening L is variable by adjusting the relative positions of the first rotating disk 5 and the second rotating disk 10.

Description

Technical Field

[0001] The present invention relates to fluorescence structured light microscopy techniques, specifically confocal microscopy, and to a confocal microscope apparatus for acquiring and processing images. Such a microscope apparatus implements a multi-point scanner system having a pinhole with a rhomboid shape together with an adaptively variable aperture that is compatible with different acquisition systems. The proposed apparatus makes it possible to significantly increase the spatial resolution of a sample by removing halos caused by scattered light from out-of-focus planes of the sample itself.

Background Art

[0002] As is known, structured light microscopy techniques are based on illuminating a sample to cause fluorescence excitation according to a configuration or pattern that is not uniform but rather clearly defined so as to obtain additional spatial information by the interaction between an illumination pattern and the sample under inspection. After acquiring one single interaction image (i.e., a sub-frame), that image is stored and the illumination pattern is moved to its next position. Then, multiple subsequent sub-frames are continuously acquired until the sample is fully covered, and then, by using a de-convolution algorithm that specifically depends on the type of pattern used, it moves on to the synthesis of the sub-frames in the final image of the plane under inspection.

[0003] More specifically, known types of confocal techniques are divided into two major categories: a point scanner characterized by a single pinhole of variable size, and a spinning disk characterized by the presence of a plurality of pinholes of fixed size.

[0004] In the latter section, a rotating disk confocal microscope is inserted, consisting of a disk with several pinholes (literally "pinholes" in Italian). The disk rotates at an extremely high speed (approximately 5,000 ÷ 10,000 revolutions per minute), and the illumination that needs to reach the sample passes through these pinholes.

[0005] More generally, in the field of optics, the term pinhole refers to an extremely small, precise hole used as a spatial filtering element. Specifically, a pinhole can generate an optical spatial structure with a shape similar to that of the pinhole itself. In terms of applications in microscopy, the presence of one or more pinholes allows for the removal of out-of-focus contributions, enabling the acquisition of more detailed images of the sample.

[0006] The illumination is parallelized before reaching the rotating disk. The objective lens focuses multiple scanning points onto the sample in a specific area by implementing parallel illumination. Each sample portion is illuminated multiple times, typically by providing a frame rate equal to 1,000 ÷ 3,000 fps in a single full rotation of the disk. The frame rate is the frequency of frames, a measure of how quickly a series of frames appear in one second, and is also abbreviated as frames per second (fps). Even if it is easy to reach the standard video speed (approximately 30 fps), it is limited by the signal-to-noise ratio (spatial filtering of out-of-focus contributions), as with other scanning techniques.

[0007] Even if a high frame rate is achieved by parallelizing the lighting (multiple spot concept), the rotating disk has one or two fixed-size pinholes, and these pinholes can interfere with the optical path by reducing the field of view (FOV), or the visual window, or in other words, the area that is captured by the camera and actually displayed.

[0008] The ability of a device implementing multi-point scanner technology to spatially filter out-of-focus contributions depends on the shape and size of the pinhole, and the size of the pinhole is • Wavelengths used, • The optical system of a device linked to an objective lens, especially for confocal microscopes. • Optical systems of one or more devices connected to a device to which a multi-point scanner system is attached. It is strictly functional in that respect.

[0009] The factors described above, combined with the single-point resolving capability of the system (confocal device and connected devices) for acquiring the overall image, mean that such capability cannot be precisely controlled or precisely predicted, as it requires final adjustment of the chain or a portion of the chain.

[0010] An alternative configuration is represented by a single scanning point solution, i.e., a laser scanning device using a single pinhole that can be easily modified.

[0011] Variable pinholes allow for optimal resolution for each objective lens used, and therefore such solutions are valued despite their slower speed compared to spinning disks. On the other hand, multiple pinholes in a spinning disk allow for extremely high acquisition speeds and a wide field of view. These are essential features for microscopy as they enable the acquisition of important data more effectively.

[0012] However, the limitations of this technique are linked to the fixed size of the pinhole, which does not always guarantee optimal resolution. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] European Patent No. EP3362836B1 [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] Therefore, the technical problem addressed and solved by the present invention is to provide a solution for reducing the time required to acquire interaction images of samples while maintaining high resolution, thereby avoiding the drawbacks described with respect to known techniques. [Means for solving the problem]

[0015] Such problems are solved by the microscope apparatus described in claim 1.

[0016] Preferred features of the present invention are described in the dependent claims.

[0017] The present invention offers several important advantages. The proposed microscope system combines two known confocal solutions, such as laser scanning and spinning disks, by combining the advantages obtainable with variable aperture pinholes with the multiple pinhole technology of spinning disks, thereby ensuring that high-importance image data can be acquired rapidly.

[0018] According to a first advantageous embodiment of the microscope apparatus of the present invention, the present invention relates to and provides the use of a plurality of overlapping pinholes for accommodating light transmitted by an illumination beam having a variable or better adjustable spread. The transmitted light has a variable size depending on the relative arrangement of the overlapping pinholes on each disk.

[0019] Within the present invention, transmitted light refers to the minimum surface area for the passage of illumination that needs to reach the sample.

[0020] The proposed device is particularly versatile because it has the ability to adapt to the noise compensation requirements at any given time, covering a wide range of specifications to enable its integration into different systems.

[0021] The apparatus uses at least two high-speed rotating disks on which multiple (e.g., photolithographic) apertures or pinholes are obtained and arranged according to a predetermined pattern. The pattern can follow an Archimedes spiral path, and the pinholes can be configured as a continuous spiral slit. The geometric shape, configuration, and additional technical features of the pinholes and each rotating disk, as well as the structure and operating mode of the entire microscope apparatus, are in accordance with those described in European Patent No. EP3362836B1, the entire patent of which is incorporated into this patent application. However, the proposed apparatus differs from the technical solution of the patent described immediately above. This is because the proposed apparatus comprises two or more rotating disks, each with at least two pinholes, preferably having a polygonal geometric shape, placed on each rotating disk, which partially overlap, resulting in a variable shape of the transmitted light through which the illumination that needs to reach the sample passes. Each pinhole pattern is obtained on different disks, a total of at least two disks positioned along the optical trajectory followed by light from the illumination source to the sample to be displayed.

[0022] According to the first preferred embodiment, the pinholes present in each rotating disk have a polygonal, particularly a quadrilateral, preferably a rhomboid geometric shape (Figs. 1 and 2), although higher-order polygonal geometric shapes can advantageously be used. Here, a polygon means a broken line formed by joining n non-adjacent consecutive line segments that sequentially connect n + 1 points, where no two consecutive points coincide. Alternatively, pinholes can be used that have at least partially curved perimeters, such as having a circular, elliptical, or oval shape, or perimeters with alternating straight and curved portions.

[0023] When two rhomboids are slid relative to each other, considering that the two rhomboids have overlapping surfaces that are rhombuses even if they have different dimensions from the first rhomboid while maintaining symmetry along two directions in the X - Y plane after aperture adjustment (Fig. 3), it is preferred that at least two overlapping pinholes have a rhomboid shape.

[0024] As described, at least two rotating disks with pinhole patterns are arranged facing each other, that is, at least partially overlapping along the trajectory through which illumination is transmitted from the light source to the sample. According to an additional preferred embodiment, the disks are arranged in sequence along such a trajectory with a very short mutual distance. The mutual distance is about 0.10÷0.50 mm defined to compensate for mechanical uncertainties during operation, such as the form of implementation and the influence of, for example, flatness defects and / or related vibrations, as well as adjustment of the device after assembly and use.

[0025] According to an additional advantageous aspect, in order to further strengthen the device in terms of optical performance and mechanical stability, the presence of immersion oil (specific to the field of optics and microscopy) is provided in the space between at least two disks. The oil is transparent to light and is suitable for compensating the refractive index differences of materials that the light beam traverses along its optical path to the sample, such as the glass of the first disk, the layer of the oil itself, and the glass of the second disk (Figs. 4a and 4b).

[0026] According to a further preferred aspect, in order to ensure the accurate maintenance of the shape resulting from the overlap of the pinholes, the two disks are moved by a single motor fixed (keyed) to the first of those disks, preferably a brushless motor. The motor is configured to synchronously pull the second disk through various mechanical connections.

[0027] Depending on the relative movement desired between the disks and the various mechanical connections used, several different configurations of the device have been developed.

[0028] The first configuration of the present invention enables the relative movement of the disks according to a translational movement. Such a translation can specifically be - implemented as a linear displacement of the axis of rotation of one disk relative to the axis of rotation of at least another disk, so that the two disks do not consequently become coaxial, and the kinematic continuity between the two disks can be implemented by a magnetic coupling, an elastic coupling, or an Oldham coupling, respectively, or - implemented as a linear displacement (the configuration shown in Figs. 60a and 60b where the internal white arrows represent the translation) for sliding one disk relative to the other while maintaining the coaxiality between the two disks by integrally pulling the second disk relative to the first disk. Thus, the axis of rotation is indicated by the internal cross, and such a movement can be implemented by a prism guide and an axial cone pusher.

[0029] Alternatively, a configuration is proposed, as shown in Figures 59a and 59b, in which relative angular displacement (represented by the white arrows inside) between coaxial disks is implemented along the circumference by, for example, an interposed conical pusher.

[0030] For each configuration, the relative displacement between the two disks implements a gradual increase or decrease in the opening resulting from the overlap of the pinholes, and in a preferred deformation, for example, the overlap can be adjusted between approximately 5 and 65 μm.

[0031] Preferably, a rotational speed of 10,000 RPM is obtained for each disk, which enables the device to reconstruct the entire image of the camera's FOV at a speed faster than the acquisition speed while maintaining instantaneous cancellation of out-of-focus contributions during rotation.

[0032] Other advantages, features, and modes of use of the present invention will consequently become apparent from the following detailed description of some embodiments, which are shown as examples rather than for the purpose of limitation.

[0033] Refer to the diagram in the enclosed drawing. [Brief explanation of the drawing]

[0034] [Figure 1] This is a partial front view of the mask of a rotating disk in a preferred embodiment of the apparatus according to the present invention. [Figure 2] This figure shows an example of pinhole overlap according to the present invention. [Figure 2a] This is a partial cross-sectional view of a preferred embodiment of the rotating disk according to the present invention. [Figure 3] This figure shows an example of a pinhole pattern according to the present invention. [Figure 4a] This figure shows the approximate transition of the structured optical refractive index in the apparatus according to the present invention. [Figure 4b] This figure shows the approximate transition of the structured optical refractive index in the apparatus according to the present invention. [Figure 5a] This figure shows an example of pinhole overlap according to the present invention. [Figure 5b] This figure shows an example of pinhole overlap according to the present invention. [Figure 6a] This figure shows an example of pinhole overlap according to the present invention. [Figure 6b] This figure shows an example of pinhole overlap according to the present invention. [Figure 6c] This figure shows an example of pinhole overlap according to the present invention. [Figure 7] This is a partial cross-sectional view of a preferred embodiment of the apparatus according to the present invention. [Figure 8] This figure shows the change in the average pressure of a rotating fluid. [Figure 9] This is a cross-sectional view of a preferred embodiment of the apparatus according to the present invention. [Figure 10] This is a cross-sectional view of a subset of the first disk in a preferred embodiment of the apparatus according to the present invention. [Figure 11] This is a cross-sectional view of a subset of the second disk in a preferred embodiment of the apparatus according to the present invention. [Figure 12] Figures 10 and 11 show cross-sectional views of partial sets of two subsets. [Figure 13] Figures 10 and 11 show cross-sectional views of the complete set of two subsets. [Figure 14] This is a cross-sectional view of a subset of a second disk in a preferred embodiment of the apparatus according to the present invention, which includes a magnetic coupling. [Figure 14b] This is a cross-sectional view of the apparatus according to the present invention, which includes a magnetic coupling. [Figure 15] This is a cross-sectional view of a subset of the second disk of an apparatus according to the present invention, which includes a magnetic coupling. [Figure 16] This is a cross-sectional view of a subset of the first disk of an apparatus according to the present invention, which includes a magnetic coupling. [Figure 17] This is a cross-sectional view of a subset of the first disk of a preferred embodiment of the apparatus according to the present invention, which includes a magnetic coupling. [Figure 18]This is a cross-sectional view of a subset of the second disk of the apparatus according to the present invention, which is equipped with an elastic joint. [Figure 19] This is a cross-sectional view of a subset of the first disk of the apparatus according to the present invention, which includes an elastic joint. [Figure 20] This is a cross-sectional view of a subset of the second disk of an apparatus according to the present invention, which is equipped with an Oldham joint. [Figure 21] This is a cross-sectional view of a subset of the first disk of an apparatus according to the present invention, which includes an Oldham joint. [Figure 22a] This diagram schematically illustrates the relative translation and rotation between disks that can be implemented using Oldham joints. [Figure 22b] This diagram schematically illustrates the relative translation and rotation between disks that can be implemented using Oldham joints. [Figure 22c] This diagram schematically illustrates the relative translation and rotation between disks that can be implemented using Oldham joints. [Figure 23a] This diagram schematically illustrates the relative translation and rotation between disks that can be implemented using Oldham joints. [Figure 23b] This diagram schematically illustrates the relative translation and rotation between disks that can be implemented using Oldham joints. [Figure 23c] This diagram schematically illustrates the relative translation and rotation between disks that can be implemented using Oldham joints. [Figure 24a] This diagram schematically shows the overall sliding motion of the device. [Figure 24b] This diagram schematically shows the overall sliding motion of the device. [Figure 25] This is a cross-sectional view of a preferred embodiment of the apparatus according to the present invention. [Figure 26] This figure shows the details of the apparatus in Figure 25. [Figure 27] This figure shows the details of the apparatus in Figure 25. [Figure 28] This is a cross-sectional view of a preferred embodiment of the apparatus according to the present invention. [Figure 29] This figure shows the details of the device in the assembly configuration shown in Figure 25. [Figure 30] This figure shows the details of the device in the assembly configuration shown in Figure 25. [Figure 31] This figure shows the details of the device in the assembly configuration shown in Figure 25. [Figure 32] This figure shows the details of the device in the assembly configuration shown in Figure 25. [Figure 33] This figure shows the details of the device in the assembly configuration shown in Figure 25. [Figure 34] This figure shows the details of the device in the assembly configuration shown in Figure 25. [Figure 35] This figure shows the details of the device in the assembly configuration shown in Figure 25. [Figure 36] This figure shows the details of the device in the assembly configuration shown in Figure 25. [Figure 37] This diagram schematically shows the opening that results from a configuration involving relative linear displacement of the disk's rotation axis. [Figure 38] This diagram schematically shows the configuration of the relative linear displacement of the disk while maintaining coaxiality. [Figure 39] This figure shows the details of the sliding mechanism related to the motion of the equilibrium mass. [Figure 40] This figure shows the details of the sliding mechanism related to the motion of the equilibrium mass. [Figure 41] This figure shows the details of the sliding mechanism related to the motion of the equilibrium mass. [Figure 42] This figure shows the details of the sliding mechanism related to the movement of the flange supporting the second disc. [Figure 43] This figure shows the details of the sliding mechanism related to the movement of the flange supporting the second disc. [Figure 44] This figure shows the details of the sliding mechanism related to the movement of the flange supporting the second disc. [Figure 45] This is a left side view of a preferred embodiment of the apparatus according to the present invention. [Figure 46] This is a 60° cross-sectional view of a preferred embodiment of the apparatus according to the present invention. [Figure 47]This is a 90° cross-sectional view of a preferred embodiment of the apparatus according to the present invention. [Figure 48] This is a top cross-sectional view of a preferred embodiment of a device in which protection is not required. [Figure 49] This is a side cross-sectional view of a preferred embodiment of a device for which protection is not required. [Figure 50] This figure shows the detailed construction of the configurations shown in Figures 48 and 49. [Figure 51] This figure shows the detailed construction of the configurations shown in Figures 48 and 49. [Figure 52] This figure shows the relationship between the rotation of the first disk (lower disk) and the rotation of the second disk (upper disk) in the embodiments shown in Figures 48 and 49. [Figure 53] This figure shows the relationship between the rotation of the first disk (lower disk) and the rotation of the second disk (upper disk) in the embodiments shown in Figures 48 and 49. [Figure 54] This figure schematically shows the comparison sequence between the rotation of the first disk and the rotation of the second disk in the embodiments of Figures 48 and 49. [Figure 55] This figure shows a detailed top view of an additional preferred embodiment of a device for which protection is not required. [Figure 56] This figure shows a detailed top view of an additional preferred embodiment of a device for which protection is not required. [Figure 57] This figure shows an example of how to determine the size of a pinhole. [Figure 58] This figure shows a representative configuration of a preferred embodiment of the microscope apparatus according to the present invention. [Figure 59a] This diagram schematically shows a configuration in which the first disk and the second disk are coaxial and have angular displacement. [Figure 59b] This diagram schematically shows a configuration in which the first disk and the second disk are coaxial and have angular displacement. [Figure 60a]This diagram schematically shows a configuration in which the first and second disks are coaxial and have sliding linear displacement, and the second disk is pulled integrally with respect to the first disk. [Figure 60b] This diagram schematically shows a configuration in which the first and second disks are coaxial and have sliding linear displacement, and the second disk is pulled integrally with respect to the first disk. [Modes for carrying out the invention]

[0035] The thickness and curvature shown in the above diagram should be considered purely illustrative, and are generally exaggerated and not necessarily proportional.

[0036] Various embodiments and variations of the present invention will be described below with reference to the figures above.

[0037] Similar components are indicated by the same symbol reference in different diagrams.

[0038] In the detailed description below, the various embodiments and variations described are likely to be used in interchangeable combinations.

[0039] For technical information outside the focus of this patent application and not described below, references are made to European Patent No. EP3362836B1, the entirety of which is incorporated into this patent application.

[0040] Referring to the configuration in Figure 58, the confocal microscope apparatus having several rotating disks according to the present invention, shown by reference nominal reference 1000, first includes a structured light source S configured to generate a beam of structured light that is illuminated by a fundamental light beam and is focused on a first plane.

[0041] The apparatus comprises a group of disks, including at least a first rotating disk 5 and a second rotating disk 10, configured to receive a beam of structured light and to transmit the resulting excitation beam to the optical system of a microscope M, which is focused on the plane of a sample C from which an image needs to be acquired. The group of disks is optically interposed between the light source S and the plane of the sample C. According to already known techniques, multiple lenses L, mirrors Sp, and dichroic mirrors Sd can also be present to appropriately guide and process the light emission and can be arranged along the optical path that extends from the light source S to the sample C and then toward the image acquisition means, i.e., the optical sensor 117.

[0042] As shown in Figure 2A, each disk between the first rotating disk and the second rotating disk is made of an optically transparent material and comprises a disk-shaped substrate 115 having a first plane 115a and a second plane 115b on opposite sides, and a mask 116 disposed on one surface between the first surface and the second surface and having at least one sector with one or more holes PH, i.e., pinholes.

[0043] The apparatus includes a moving mechanism, which is configured to move the first and second rotating disks so that the resulting excitation beam is implemented by the passage of a structured light beam through at least one aperture resulting from the overlap of pinholes, as shown in detail 1005 of Figure 58. Each aperture results from the overlap of each first hole in the first rotating disk and each second hole in the second rotating disk. The area of ​​the apertures is variable by adjusting the relative positions of the first and second rotating disks according to modes which will be described in more detail below.

[0044] The moving means substantially comprises a motor preferably configured to be integrally coupled to a first rotating disk, and a motion transmission means preferably configured to kinematically couple the motor to a second rotating disk.

[0045] A preferred configuration of this device includes a moving means configured to move a first rotating disk and a second rotating disk in accordance with their relative translational motion. Specifically, the device includes a motion transmission means configured to translate the second disk relative to the first disk.

[0046] For example, the motion transmission means is configured to perform linear displacement of the rotation axis of the second disk relative to the rotation axis of the first disk, or to continue to perform sliding linear displacement of the second disk relative to the first disk while the rotation axes of each disk remain aligned.

[0047] Alternatively, a modified form of the apparatus is described in which the motion transmission means is configured to perform angular displacement of the second disk relative to the first disk while the rotational axes of each disk remain aligned, but this is not included within the scope of protection.

[0048] As described, each disk has a substrate, which is also disk-shaped, made at least partially, preferably entirely, from an optically transparent material such as glass. Each substrate has a first plane and a second plane opposite it, and is separate from a mask which is integrally coated or positioned between the two planes. The mask also has a disk-shaped form. The mask, substrate, and disk all have the same overall dimensions, in other words, the main surface of each disk may have the same extent as the surface of the respective substrate and the surface of the respective mask. At least two of the disks may have the same overall dimensions of their main surfaces, and their main surfaces correspond to the dimensions of their respective circular surfaces.

[0049] Each mask has at least a sector having a plurality of through-holes or openings (hereinafter more simply pinholes) arranged according to a predetermined pattern. Such holes are optionally implemented by photolithography. Each patterned mask or the outer surface of the mask is made of or entirely coated with a material that is highly opaque to light, preferably having a light absorption rate of more than 80%, such as an extremely black material that is opaque to light.

[0050] The pinholes are arranged on each disk according to a predetermined path, such as an Archimedes spiral path (Figures 1 and 58; examples of spiral paths are highlighted with dashed lines and indicated by the letter SA). The disks are arranged such that each mask at least partially overlaps along the optical trajectory traced by structured light directed toward the sample by the light source (Figure 2). The geometric configuration and pattern of the pinholes on each disk are such that the shape of the transmitted light passing through, resulting from the overlap of the pinhole openings along the optical trajectory traced by the structured light, is variable in accordance with the relative motion of the disks themselves. Thus, the overlapping area between pinholes is actually variable over time or better adjustable depending on the specific application.

[0051] The aperture area or passage lumen for illumination is determined by at least partial overlap of at least two pinholes placed on different disks. In either case, the overlap of pinholes on all disks arranged along the optical trajectory followed by the sample by illumination always determines the conditions required for light to pass through the disks and finally reach the sample.

[0052] The pinholes preferably have a polygonal, particularly quadrilateral, geometric shape, but higher-order polygonal shapes can be advantageously used. In a polygon, a polyline is defined as a polyline formed by the joining of n non-adjacent consecutive line segments that sequentially connect n+1 points, where no two consecutive points coincide. According to a preferred configuration, the pinholes may have different dimensions depending on whether the pinhole is contained in a first disk or a second disk, and / or depending on their distance from the rotation axis of each disk.

[0053] Given that, for two rhombic pinholes sliding or rotating relative to each other (from an initial configuration in which such pinholes are perfectly superimposed), the rhombic shape is preferred, considering that the surface of the opening resulting from the overlap of those rhombic pinholes will be a regular rhombus, even if it has different dimensions from the initial rhombus. Furthermore, such a resulting rhombic opening preserves symmetry along two directions of the plane XY, on which the disks are positioned for any adjustment of the opening, i.e., any relative displacement of the pinholes, and any relative displacement of at least two disks.

[0054] As described, at least two rotating disks are arranged facing each other, i.e., at least partially overlapping along the optical trajectory of structured illumination from the light source to the sample, and preferably parallel to each other. Preferably, the disks are arranged sequentially along such a trajectory at an extremely short distance between them, for example, between about 0.00 and 0.50 mm. The disk group is optionally configured to provide a transmission oil-tight housing interposed between the first rotating disk and the second rotating disk, and more simply, the aforementioned gap between the disks is completely filled with immersion oil.

[0055] Furthermore, the apparatus comprises an acquisition means, preferably an acquisition sensor, configured to detect a fluorescence beam emitted by the plane of the sample, and a set of lenses configured to optically conjugate the plane under consideration (of the first plane, the plane of the disk, and the plane of the sample).

[0056] Furthermore, the apparatus includes optical means configured to transmit a structured light beam from a light source to the plane of a sample, and to transmit the fluorescence beam emitted by the plane of the sample to an acquisition means. A moving means may be provided, configured to move the structured light source so as to move the structured light beam in the first plane and the rotating disk in each of the second planes.

[0057] The apparatus also includes a central processing unit configured to receive multiple partial acquisitions of the sample plane from an acquisition means and to process such partial acquisitions into a final image of the sample plane by a structured light microscope deconvolution algorithm. The central processing unit may include one or more graphics processors, i.e., GPUs.

[0058] Furthermore, the image acquisition and processing methods in structured optical confocal microscopy that can be implemented by the apparatus described herein can be summarized as follows: - The rotating disks are intersected by a structured light beam, thereby obtaining a resulting excitation light beam, which allows a variable-sized light beam corresponding to the motion of the pinholes on each disk to pass through the aperture. - The resulting excitation beam is focused onto the sample plane, - The steps of acquiring multiple partial acquisitions of the sample plane and constructing such multiple partial acquisitions in an image of the sample plane by processing them with a structured light microscopy deconvolution algorithm, To provide.

[0059] Preferred embodiments of the present invention are described below, and two rotating disks exist, merely as examples and not for the purpose of limitation.

[0060] Referring to the sequence of Figures 6a to 6c, the pinholes preferably have a rhombic geometric shape, where D1 represents the rhombic diagonal aligned along axis Y in a two-dimensional plane reference system XY on which each disk is located, and D2 represents the rhombic diagonal aligned perpendicular to D1, i.e., along axis X. By considering two pinholes P1 and P2 of different rotating disks having the same rhombic geometric configuration and the same size and orientation in the plane XY (where diagonal D1 is parallel to axis Y), it can be noted that by moving the disks as described above, the overlapping area of ​​the pinholes is varied, and the illuminated transmitted light L also has a variable spread. For example, starting with a configuration in which two pinholes completely overlap, where the transmitted light is a rhombus with a size equal to the size of the pinhole itself, to accommodate different acquisition needs, the transmitted light remains rhombic but its size is reduced to preferably within the range of 5 μm ÷ 65 μm by a displacement Δ along axis X. The motion of the disk can be implemented both continuously and in separate steps, preferably with an increasing amplitude of variation in the diagonal D2 of the transmitted light equal to 5 μm. In other words, the relative displacement Δ of the disk along axis X is sequentially equal to 5 μm each time.

[0061] Specifically, to implement such relative displacements in the range of 5 μm, it is preferable to use a micro-positioning piezoelectric actuator having the following desirable characteristics. - Strokes that include a range of approximately 100-200 μm - Closed-loop, single-axis stroke control type - Scanning step frequency of 40-50 μm - 100nm repeatability - 100nm resolution - A weight of 100 ÷ 200 g moved for a maximum displacement equal to 50 mm. - Power supply / interface, analog voltage only, 0-10V, OEM

[0062] In particular, in the pass-through area related to a 25 mm FOV, the optimal shape of the pinholes that better ensures the repeated overlapping behavior across the entire helical pattern is, as shown in Figure 5a, a quadrilateral shape, preferably a square shape, with diagonals parallel to the axes X and Y.

[0063] As shown in the sequence of Figures 6a to 6c, two pinholes P1 and P2, which are quadrilateral polygons, are arranged to reduce the delta of approximation to a circle, which represents the ideal shape of light passage related to the point scanner system. Even when they move relative to each other (Figure 5b), they ensure that the final shape of the transmitted light determined by the overlap of these pinholes is maintained, as they are also quadrilateral polygons that are symmetric with respect to the axes X and Y of the incident plane and preferably have a tolerance of ±1 μm.

[0064] In a preferred modification, the disk has two sets of mappings, each consisting of two helices and five helices, with each helix having a pitch equal to 250 μm and a distance equal to 250 μm between one pinhole and the next in a particular helix. The nominal FOV is preferably equal to 25 mm. It is preferable to generate a pinhole mapping with a starting diameter equal to 34.5 mm by employing one of the spinning disks described in European Patent No. EP3362836B1 as the base structure, and taking into account the uncertainty delta for the maximum diameter of the possible coupling flange of 33.6 mm, and preferably, as a precaution, by using a brushless motor with a diameter that falls between 28 mm and 30 mm, for example. In this case, the outer diameter of the final FOV is consequently equal to 84.5 mm. Finally, the constructed outer diameter of the disk can be fixed in the range that falls between 88.5 and 92.5 mm and can be optionally extended up to 100 mm.

[0065] As stated, compensation for refractive index variations between the components of a rotating kinetic chain is performed by immersion oil interposed between the rotating disks. The immersion oil is interposed between the two disks for optical and mechanical reasons. The optical reason is to harmonize the refractive index variations encountered when the beam intersects the confocal module, specifically the air-glass-air-glass-air module. The mechanical reasons are to improve the mutual sliding between the two disks, to compensate for possible planar defects and manufacturing tolerances, and to limit variations in the thickness of the gap between the two disks during its own assembly and operation; these mechanical reasons are better understood as adjustment.

[0066] Referring to Figure 7, which partially details one embodiment of a device 100 having two rotating disks, an example of a preferred embodiment of a sealing lock system for rotating disks suitable for preventing oil from leaking out of the air space between the two disks is shown, comprising a lower disk 5 (i.e., the first disk), an outer sealing bottom 6 flange, an outer sealing O-ring 7, an outer sealing upper flange 8, an outer retaining clip 9 (6 × 60°), an upper disk 10 (i.e., the second disk), and an inner sealing O-ring 14.

[0067] This configuration ensures that the thickness of the oil in question is kept uniform and dynamically limited by avoiding leakage and spillage. Therefore, - Positioning of the inner gasket 14 - Positioning of outer gasket 7 - Sealing and bonding of flange 6 to lower disc 5 - Sealing and bonding of flange 8 to upper disc 10 - Sealing and bonding of the support flange 13 to the upper disc 10 - External throttling of the disc by elastic clips 9 (there may be 4 or 8 clips) to counteract the centrifugal force of the oil while the disc is rotating. A sealing circuit was obtained by this method.

[0068] The oil thickness is approximately 0.30 ± 0.20 mm, and the law

number

[0069] Here, the integration extremes are the inner and outer radii of the oil housing itself, supplied per unit weight under standard conditions. At a disk speed of 10,000 RPM, P = 0.9 MPa = 9 kg / cm². 2 = A force of 450g is obtained per centimeter of straight line sealing at the edge.

[0070] The components that implement the variable aperture device are rotated synchronously at a rotational speed of 10,000 RPM or more in a steady state. The preferred external dimensions of the proposed device are listed below, with height × width × depth = 160 × 110 × 41 mm, i.e., - Height: 80 ÷ 250 mm - Width: 60 ÷ 170 mm - Depth: 20 ÷ 70 mm That is the case.

[0071] Once a speed level is established for a point scanner system where the disk rotation speed is 10,000 RPM or higher in a steady state, a brushless motor is preferred because it can easily drive the moving torque that can overcome both friction between components and the polar inertia of the components, and lastly, it can ensure a reduced size in terms of diameter and length along the axis of rotation. The solution for the motor is as follows: - Type: DC brushless motor - Electrical specifications: 24V, 30W - Maximum continuous torque: 37.3 mNm - Steady-state rotation: 15000 RPM

[0072] The following describes embodiments illustrating preferred configurations of the present invention that implement different modes of mutual displacement between rotating disks.

[0073] Relative linear displacement of the disk's rotation axis The overall configuration of this device provides linear displacement of the rotation axis of one disk relative to another disk, with the continuity of motion guaranteed by a magnetic coupling, elastic coupling, or Oldham coupling.

[0074] An Oldham coupling is a mechanical coupling useful for transmitting motion between two parallel shafts. In each time range, the angle of rotation of the crankshaft is exactly equal to the angle of rotation of the driven shaft, and therefore the speed ratio between the two shafts is constant and equal to 1. The coupling mainly consists of a bearing element having a straight axis and two grooves (griffs) perpendicular to each other to form a cross. A shoe integral with one of the shafts engages with each groove. By rotating the crankshaft with the shoe at the end, the bearing cross is moved, thereby moving the second shaft. The two shafts terminate with two discs on which two diameter grooves are mounted. An intermediate bearing body also has a disc shape and is interposed between the two discs. As mentioned above, the intermediate bearing body has two orthogonal prism projections that engage with the two grooves at each terminal.

[0075] The convenience of using this joint depends on the extreme proximity of the two shafts between which power is transmitted. Larger distances result in significant work losses due to friction and extremely low yields, but it becomes convenient when the two shafts start in a perfectly coaxial position and one of the two shafts is subjected to displacement during operation.

[0076] This structure is common to all three configurations, and in terms of operating modes, the two disks are considered to be completely independent in their motion and locking points.

[0077] Referring to Figure 9, a first embodiment of the present invention is shown, in which the continuity of motion is guaranteed by a magnetic coupling, and the linear displacement of the rotation axis of one disk relative to the other disks is observed.

[0078] The assembly of such a preferred variant is carried out as follows. Referring to Figure 10, which shows the assembly of the support components related to the lower disc (i.e., the first disc), the motor 1 is provided with an interface flange 3, and the flange 2 is then fixed to the axis of the motor 1 that generates traction by interference, while the lower disc 5 is placed on the flange 2 to avoid direct metal-to-glass contact by elements 11 and 12. Previously, the oil storage outer flange 6 was bonded to the lower disc 5, while the flange 16 is positioned above the disc by interposing element 15 and then locking the whole with appropriate screws, for example, four M1.6 screws.

[0079] This device is balanced by the correct centering of the disk itself to ensure consistent behavior under all dynamic conditions, and is hardened by the addition of epoxy resin. At the end of assembly, it is fixed to the frame 4. The O-ring 14 for the oil circuit is positioned outward and is the first interface element between the lower and upper systems, and finally, altitude measurements of the disk 5, useful for subsequent assembly, are recorded.

[0080] Separately, the upper disc 10 (i.e., the second disc) is coupled to the support flange 13 (preferably by bonding it to one of the two discs to reduce the gap between the two discs) and to the outer edge of the disc itself, to which the oil storage flange 8 is coupled (bonded), and to which the O-ring 7 is fixed in place to implement the oil outer seal. Finally, the bearing 18 and the associated spacers 21 are inserted to complete the first pre-assembled unit.

[0081] Next, separately, as shown in Figure 11, the assembly of the support components related to the upper disc—the arm 30, the upper bearing 22, and the outer spacer 23—continues by aligning the slit / cut in the spacer 23 with the hole in the outer cylindrical edge of the arm 30 and fixing the desired mechanical coupling to the axis of the system part related to the upper disc. Then, the whole is tightened with the ferrule 26, the piezoelectric actuator 29 is attached to the arm 30, and finally the support body 31 is attached to the piezoelectric actuator 29 to complete the second pre-assembly unit (Figure 12).

[0082] The subsequent steps shown in Figure 13 are for completing the assembly of the upper part of the device by inserting the two aforementioned pre-assembly units, positioning the inner spacer 25 with the same alignment as 23, and tightening the whole assembly with the ferrule 27, as follows: Tightening the ferrule 27 in the configuration with a magnetic coupling is preceded by inserting the magnet 20, the second magnetic disk flange 24, and the grain for securing the magnet 28, starting from the top (Figures 14a-14b).

[0083] As shown in Figures 15 and 16, which illustrate the two aligned upper and lower subassemblies, the upper system can rotate along its own axis (only when pulled, as it is not coupled to its own motor) and can also translate laterally along the axis of the piezoelectric actuator 29.

[0084] Finally, by keeping the fixed route accessible, the connecting elements are positioned within the base system, and each of these elements consists of the following:

[0085] - For the magnetic coupling (Figure 17), its elements consist of pre-assembly elements 19 and 20 fixed by interference with the motor axis, respecting the defined angular orientation. The magnets, as in the upper sub-assembly, are assembled in even numbers with alternating polarity to improve the auto-centering capability by utilizing the attractive force between the upper and lower magnet supports and the repulsion between adjacent magnets on the same support flange.

[0086] - For the elastic joint (Figures 18 and 19), the configuration is the same as that already described for the magnetic joint, and by utilizing the flexibility of aluminum in combination with an accordion-shaped geometric form that increases adaptability to different positions of the axis, the continuity of motion is ensured by a single unit of the elastic joint 33 by tightening the grains related to the lower axis. This is a simple solution, but it has a margin for vibration.

[0087] - For Oldham joints (which are better described below and shown in Figures 20 and 21), the elements consist of a series of pre-assembled components shown in 34, 35, 36, 37, 38, 39, and 40, fixed by interference with the shaft. Disc springs are preferred because they allow for a higher preload on the overall dimensions. The addition of spheres makes it possible to reduce friction during the sliding phase between the components of the joint and between the discs themselves, and the springs allow for a reduction of the existing backlash to extremely low values. The sequence of Figures 22a–22c and 23a–23c schematically illustrates the relative translation and rotation that can be implemented by such joints.

[0088] Furthermore, with respect to the configuration already illustrated, and referring again to Figure 9 as an example, after the assembly of each joint is complete, the oil is deposited according to a rate per unit weight until it reaches an estimated amount having a thickness of 0.20-0.30 mm on the upper surface of the disc 5, while taking care to position the disc on a plane so that the oil spreads first by gravity and then by a spatula to evenly distribute the oil.

[0089] In this way, the device is fully assembled by utilizing appropriate seats provided on the joints and axial interface elements of the two sub-assembly units, paying attention to the precise positioning of the inner seal 14 and outer seal 7 for the oil. Further miniaturization of the configuration is possible by centrifugal expansion of the upper outer flange 8 and lower outer flange 6, and radial insertion of elastic clips in the peripheral area of ​​the disc suitable for accommodating the centrifugal pressure of the rotating oil.

[0090] The choice to develop several connection types stems from the need to encompass different product ranges in terms of overall cost, overall dimensions, and performance. Thus, each joint covers different specifications in terms of speed, friction and resistance torque, level of manufacturing complexity, potential for custom design, dynamic vibration, and dynamic shear. The sequence in Figures 24a and 24b schematically illustrates the overall sliding of the apparatus for a maximum stroke preferably equal to 150 μm.

[0091] Table 1 is reported below, summarizing the performance of each joint configuration with respect to the linear displacement between disks (V is the lowest rating, and VVV is the highest rating). [Table 1]

[0092] In conclusion, as is clear from Table 1, each of the three joint configurations is reliable in the ultimately determined form. The simplicity of being able to switch from one configuration to another during the assembly of the device made it possible to develop a common production line for the most intricate components, and to reflect the different characteristics of the joint selected for the final configuration in dedicated lines.

[0093] Maintaining the relative linear displacement and coaxiality of the disks. Such a configuration of the device provides linear displacement of the rotating disks relative to each other, while maintaining the same single axis of rotation for both disks, ensuring continuity of motion between the two disks. As shown in Figure 25 and more clearly in Figure 28, the sliding linear displacement of the upper and lower disks can be implemented by prism guides, whose motion is actuated by vertical conical pushers 66.

[0094] The prism guides are preferably located on elements 53 and 60, allowing the second disk to be pulled integrally with the first disk while maintaining a single axis of rotation.

[0095] Within the present invention, a prism guide means a pair of geometric contours suitable for creating constraints (called prism pairs), which allow translation of the body according to the assigned direction by preventing rotation and displacement of the body in a direction perpendicular to the direction detected by the prism guide itself.

[0096] This solution allows for a favorable improvement in the specifications for maintaining the variable shape of the pinhole compared to the previously described configuration, by reducing the relative motion parameters while introducing a kinematically more complex system with an instability of only 1 degree.

[0097] The embodiments described above will be explained in more detail below with further specific reference to Figure 25.

[0098] For convenience, the configuration will be explained by highlighting the differences from the previously described configuration. The motor 1 is preferably provided with an interface 3, and a flange 52 is secured to the axis of the motor 1 that generates traction by interference, while the lower disc 5 rests on the flange 52, and the flange 52 prevents direct metal-to-glass contact by elements 11 and 12. Previously, the oil storage outer flange 6 was bonded to the disc 5, but now the flange 56 is positioned above the disc 5 by interposing element 15 and fastening the whole with appropriate screws, for example, four M1.6 screws.

[0099] The device undergoes balancing and bonding only at the end of assembly, after which the disk 5 is centered and flattened. This is followed by the insertion of the mass 54 into the outer prism guide mounted on 56 by positioning the mass 54 as far outward as possible (Figures 29 and 30).

[0100] Separately, the upper disc 10 is bonded to the support flange 53 and the outer edge of the disc itself, then the oil storage flange 8 is bonded, and the O-ring 7 is fixed thereon, thereby implementing the outer seal and completing the upper pre-assembly unit (Figures 31-33). The elements shown in Figure 26 are assembled to the flange 52 and then oriented downward. Subsequently, the two pre-loading springs 67 of the second disc are positioned, and finally, the support flange 60 with the elements of detail B described above pre-assembled is inserted into the second prism guide. The flange 60 thus assembled then allows the pre-assembled subset of the second disc (components shown as 10, 53, 8, and 7) to be fixed in place.

[0101] The rotational and balancing components further include an upper balancing mass 55 having related pins and screws, a preload spring and grain 51 for relieving backlash, a preload spring and grain 50 for radial centering of masses 54 and 55, and preferably a preload spring and grain 68 for a second disk.

[0102] Referring further to Figure 34, the piezoelectric actuator 29, the conical pin 66, the pin support 69, the "L"-shaped arm 70, and the support components for the piezoelectric actuator 31 are assembled separately, and these components form the upper conical push that actually pushes the internal displacement mechanism. The conical pin refers to a tapered cylindrical element whose end is narrowed like a cone.

[0103] Referring to Figures 35a and 35b, the operating principle of the configuration with an external sliding joint is schematically described below. A piezoelectric actuator 29, which is integral to the external frame through components 31 and 70 and moves vertically, pushes a pin 66 integral to the external frame by pushing two subassemblies shown in Figure 31 (the subassembly is integral to the lower disk 5) and Figure 33 (the subassembly is integral to the upper disk 10). As the pin 66 moves downward, the flanges 53 and 60 and the disk 10 integral to those flanges move to the right (the contact point is shown to the right of the pin axis) by maintaining contact with the two bearings shown in Figure 33. The lower disk 5 remains on the original axis without translation, but the system as a whole needs to balance the mass that has moved to the right (with respect to the axis of rotation). This balance is achieved by the transfer of motion from the pin 66 to the masses 54 and 55 on the left side of the axis, the displacement of which is shown by horizontal arrows in Figure 35a.

[0104] At a distance of δ_r_cam=K from the center of rotation of the mechanism in Figure 31, indicated by the cross, pin 66 pushes the rotating cam through point contact as shown in Figure 31. At a distance of δ_r_mass=n×K (n=amplification factor of the displacement arm) from the center of rotation, indicated by the cross, masses 54 and 55 make contact instead. The displacement increases due to the fact that the mass of the upper disk 10 and the mass of the elements associated with the upper disk 10 are greater than the masses 54 and 55. Therefore, masses 54 and 55 are given by the following equation: esyst=0;→[eleft=mleft×δleft]=[eright=mright×δright]mleft mright=δright δleft→4gr20rg=15 / =δright n×δright→n=5 To ensure the equilibrium given by (nominal dynamic eccentricity is zero), a larger amount of movement is required.

[0105] Details of the contact distance between the cams are shown in Figure 36. According to this mechanism, as the upper disk 10 rotates by changing its angular position but not its radial position, the second disk ensures a constant desired opening of the pinhole in all helices by respecting the equilibrium dynamic conditions of the mass (where the mass is moved proportionally, unlike inversely proportional displacement). As shown in the comparison between Figure 37, which shows the opening resulting from a configuration involving relative linear displacement of the disk's rotation axis, and Figure 38, which relates to a configuration involving relative linear displacement of the disk while maintaining coaxiality, such a configuration is consequently more mechanically complex and requires more precise preload adjustment than the preceding configuration, but is more stable in maintaining the stacked shape.

[0106] For example, Figures 39 to 41 show details of the sliding mechanism related to the motion of the equilibrium mass, Figures 42 to 44 show details of the sliding mechanism related to the motion of the flange supporting the second disk, and Figures 45 to 47 show the assembled device as a whole, which are left side view, cross-sectional view at 60°, and cross-sectional view at 90°, respectively.

[0107] Maintaining relative angular displacement and coaxiality of the two disks The proposed alternative configuration of the apparatus is based on the angular displacement of the disks, i.e., the relative displacement along the circumference. According to such a solution, the two disks are assembled coaxially with a backlash of approximately a few micrometers due to micro-mechanical precision machining, and are moved by a conical pusher as in the configuration described above (maintaining the linear displacement of the disks and the same axis of rotation).

[0108] Referring to Figures 48 and 49, the pin 66 is moved by a piezoelectric actuator 29, which is coupled to a fixed reference or frame through components 31 and 70. The pin 66 pushes two spheres 75 that are positioned axially symmetrically and are preloaded by a spring 74 having related grains 17. The flange 72 is integral to the lower disk 5, while the flange 73 is integral to the upper disk 10. The flanges described above are assembled coaxially so that they are rotatable relative to each other on a single axis. However, such motion is constrained by the position of the spheres located in a channel that is half geometrically formed by the lower flange 72 and the other half geometrically formed by the upper flange 73, as shown in Figures 50 and 51.

[0109] The sphere is displaced radially by the descent of the pin 66, reducing the distance between the sliding surfaces shown in Figures 50 and 51, thereby defining controlled relative rotation between the two flanges. Both flanges are pulled asynchronously by a motor 1 fixed to the lower flange 72.

[0110] Figures 52 and 54 show the relationship between the rotation of the first disk (lower disk) and the rotation of the second disk (upper disk), respectively, and Figure 55 shows the relative comparison sequence.

[0111] Additional configurations of the apparatus to implement the relative angular displacement of the two disks and maintain the coaxiality of the two disks by achieving higher speeds and reducing wear between components are shown in Figures 55 and 56. The largest sphere (i.e., the main sphere), which previously made direct contact with the conical pin, is provided with two dome-shaped recesses, each housing two smaller spheres with reduced diameter relative to the main sphere. The last small sphere is positioned between the large sphere and the pin. Thus, by minimizing friction and wear, the large sphere no longer rolls on the surface of the prism guide but is pulled only by the rotation of the entire system. The smaller spheres, made from a suitable wear-resistant material, instead rotate relative to the central pin and crawl on the outer pin by actually displacing the crawling only between the spheres themselves.

[0112] For such a configuration, the order of the pinhole shapes must be adapted to the radius in order to compensate for displacement in the circumference. If the rotations involved are equal, this displacement will consequently increase with respect to the distance from the center by covering different arc shapes and different radii. The pinholes of the two disks will consequently have elongated, stretched, and / or scaled shapes, different with respect to their relative distance from the disk centers. The size and ratio of the pinholes may differ between the two disks, for example, directly proportional to the distance from the rotation axis of each disk. The extension of the pinholes is calculated specifically for each of the two disks with the aim of obtaining an overlapping area with a variation of ±10%. An example of size determination in this sense is shown in Figure 57. In this estimation, the construction factor of the lithographic shape is also calculated, i.e., the actual sharp edges are even implemented by a radius chamfer equal to approximately 1 ÷ 2 μm.

[0113] Such a configuration combines the advantages of the proposed kinetic chain's simplicity, which is suitable for implementing variable openings resulting from the overlap of pinholes on different disks, with the technical improvements in micro-precision required to reduce the backlash of coaxial coupling. Finally, while this configuration is consequently very advantageous for maintaining equilibrium under dynamic conditions (balance), it requires a concrete investigation of the pinhole shapes, which are no longer equal at each position but are variable per disk and per radius, compared to previous configurations.

[0114] The present invention has been described herein with reference to preferred embodiments. It should be understood that other embodiments belonging to the same inventive center may exist, as defined by the scope of protection of the claims reported below. [Explanation of Symbols]

[0115] 1 Brushless motor 2. Lower flange of the first disc 3. Motor support plate 4 System Loading Frame 11. Outer O-ring of the first lower disc 12. Inner O-ring of the first lower disc 13 Upper disc mounting second flange 15. First outer upper disc O-ring 16. First upper flange of the disc 18 Lower arm bearing 19 Magnetic disk first flange 20 magnets 21 Intermediate spacer for the bearing of the internal arm 22 Bearing of the upper arm 23 Outer upper spacer 24 Magnetic disk second flange 25 Inner upper spacer 26 Outer upper ferrule 27 Inner upper ferrule 28 Magnet fixed grain 29 Piezoelectric Actuator 30 Piezoelectric Arm 31 Sliding support 34 Lower Oldham flange 35 Torsional preload spring 36 Intermediate Oldham flange 37 Drive ball 38 Disc springs 39 Upper Oldham flange 40 fixed grains 50 Grains of horizontal preload flange 51. Mass of grains under vertical preload 52 Lower flange of the first disc 53 Second upper disc bearing flange 54 Lower balance mass 55 Upper balancing mass 56 First upper flange of the disc 60 Upper disc second intermediate flange 66 Vertical cone pin 70 L-shaped arm 57 Combined Cylinder 58 Cam flange 59 Cam bearing 62-pin contact pins 63 Contact Spacer 64 Contact pin bearing 67 Preload spring 71 Preload grains (2 pieces) 72 Lower flange of the first disc 73 Upper disc bearing second flange 74 Preload springs (2 pieces) 75 Contact balls (2 pieces)

Claims

1. Source of structured light (S), A disk group (200) comprising at least a first rotating disk (5) and a second rotating disk (10), configured to receive the structured light beam and to transmit the resulting excitation beam to the optical system of a microscope (M) focused on the plane of a sample (C), wherein the disk group (200) is optically interposed between the source (S) and the plane of the sample (C). The first rotating disk (5) and the second rotating disk (10) are, A disk-shaped substrate (115) made of an optically transparent material, having a first plane (115a) and a second plane (115b) on opposite sides, A mask (116) having a pattern disposed on one of the first surface (115a) and the second surface (115b), wherein the mask (116) comprises at least one sector having one or more holes (P1, P2), and the outer surface of the patterned mask (116) or the mask (116) is made of an extremely black material that is opaque to light, and A group of disks (200) comprising, An acquisition sensor (117) is configured to detect a fluorescence beam emitted from the plane of the sample (C), Moving means (1, 19, 20; 1, 33) configured to move the first rotating disk (5) and the second rotating disk (10) such that the resulting excitation beam is produced by the passage of the structured light beam through at least one opening (L) resulting from the overlap of the holes (P1, P2), wherein each opening (L) results from the overlap of each first hole (P1) in the first rotating disk (5) and each second hole (P2) in the second rotating disk (10), and the moving means (1, 19, 20; 1, 33) Equipped with, The moving means (1, 19, 20; 1, 33) is configured to move the first rotating disk (5) and the second rotating disk (10) in accordance with their relative translational motion. The area of ​​the opening (L) is variable by adjusting the relative positions of the first rotating disk (5) and the second rotating disk (10). Confocal microscope apparatus (100).

2. The aforementioned means of transport is A motor (1) is configured to be integrally coupled to the first rotating disk (5), Motion transmission means (19, 20; 33) configured to kinematically couple the motor (1) to the second rotating disk (10), Equipped with, The apparatus (100) according to claim 1, wherein the motion transmission means (19, 20; 33) is configured to translate the second rotating disk (10) relative to the first rotating disk (5).

3. The apparatus (100) according to claim 2, wherein the motion transmission means (19, 20; 33) is configured to perform linear displacement of the rotation axis of the second rotating disk (10) with respect to the rotation axis of the first rotating disk (5).

4. The apparatus (100) according to claim 3, wherein the motion transmission means comprises an elastic joint, a magnetic joint, or an Oldham joint.

5. The motion transmission means (66) is configured to perform linear sliding displacement of the second rotating disk (10) relative to the first rotating disk (5), and the use of the prism guide and the axial conical pusher (66) ensures that the rotation axes of the first rotating disk (5) and the second rotating disk (10) remain aligned, according to claim 2 (100).

6. The apparatus (100) according to any one of claims 1 to 5, wherein the holes (P1, P2) have a polygonal, preferably quadrilateral, geometric shape.

7. The apparatus (100) according to any one of claims 1 to 6, wherein the holes (P1, P2) have a rhombic or square geometric shape.

8. The apparatus (100) according to any one of claims 1 to 7, wherein the holes (P1, P2) have different dimensions depending on whether they are located on the first rotating disk (5) or the second rotating disk (10), and / or depending on their distance from the respective rotational axis (R) of the first rotating disk (5) or the second rotating disk (10).

9. The apparatus (100) according to any one of claims 1 to 8, wherein the holes (P1, P2) are arranged in the mask (116) according to an Archimedes spiral pattern.

10. The apparatus (100) according to any one of claims 1 to 9, wherein the group of disks (200) is configured to provide a transmission oil-tight housing interposed between the first rotating disk (5) and the second rotating disk (10).

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