Multi-immersion microscope objectives with minimal refractive surface

Abstract

The present invention relates to an immersion microscope objective (10) for examining a sample (S) in an immersion medium (M), comprising: at least one concave mirror (3), at least one optical element (1) including an aspherical surface (2) facing the at least one concave mirror (3), and an internal space (4) disposed between the at least one concave mirror (3) and the aspherical surface (2), the internal space (4) being configured to be filled with the immersion medium (M) such that the immersion medium (M) contacts the at least one concave mirror (3) and the aspherical surface (2). According to the invention, the aspherical interface (2) is shaped such that when the refractive index n of the immersion medium (M) increases or decreases by at least 0.025, the working distance (7) of the immersion microscope objective (10) changes by less than 1%.

Description

Technical Field

[0001] The present invention relates to an immersion microscope objective for examining samples in an immersion medium, and a microscope including such an objective. Background Technology

[0002] In microscopes such as laser scanning microscopes, a beam of light is used to examine a sample, which is continuously guided to different parts of the sample while optical signals from the sample response are acquired by a photodetector and a data acquisition system [1]. The signals generated by the sample can be due to reflection, scattering, polarization, fluorescence excited by single or multiphoton light, and other physical processes such as harmonic generation and Raman scattering. To produce an image of the entire sample, the beam or sample is scanned across the entire region of interest by translating the sample (stage scanning) or by translating the beam by an optical scanning system. Such scanning systems can utilize galvanometer scanning mirrors, acousto-optic deflectors, polygon scanners, and other optical elements. The beam deflection introduced by the scanning system is manipulated by a control system linked to the data acquisition system. Typically, the scanning beam is delivered to the microscope objective by an intermediate optical system (e.g., scanning lens and tube lens), which focuses the beam onto the sample (see Figure 1 ).

[0003] In addition to raster scanning, other scanning modes can be used to examine samples (e.g., line scanning, spiral scanning, and random scanning), which allows for faster acquisition of biological, chemical, or physical events within the sample with a reduced number of target points during the scanning cycle, which in turn allows for higher repetition rates of the scanning mode. Many laser scanning microscopes also offer the possibility of using a beam of light to optically stimulate or modify a sample or a subregion thereof, for example, through laser microsurgery, light trapping, optogenetics, demasking, photobleaching, or fluorescence bleach recovery (FRAP) [1]. Many laser scanning microscopes utilize techniques to generate optical section images, i.e., to produce datasets that appear equivalent to mechanically sectioned samples imaged using the same modality. Optical sectioning is achieved by reducing the background signal from portions of the sample that are far from the focal zone of the microscope objective. This reduction can be achieved using optical devices, such as by using a confocal pinhole in a confocal microscope [1] to exclude light from areas outside the focal zone, or by irradiating the sample so that little or no signal is generated outside the focal zone of the microscope objective, for example, by using side illumination in a light sheet microscope. Optical sectioning can also be achieved by computationally processing the acquired data using methods such as deconvolution. Furthermore, optical slicing images can be generated using nonlinear light-matter interactions, such as multiphoton microscopy methods, including two-photon, three-photon, and four-photon microscopy, second and third harmonic generation, coherent anti-Stokes Raman scattering (CARS), and stimulated Raman scattering (SRS). In these methods, multiple photons need to interact with molecules within the sample, thus the resulting signal is nonlinearly dependent on the illumination intensity and has a power higher than unit power. For example, the two-photon signal depends on the square of the illumination intensity. When the illumination intensity drops sharply outside the focal region of the illumination beam, the effective excitation volume in the multiphoton process is spatially confined to a three-dimensional space surrounding this focal region, producing the optical slicing effect. Nonlinear excitation processes can be used to generate fluorescence using pulsed picosecond or femtosecond lasers operating in the near-infrared spectral region (700–2500 nm), which can be detected by photodetectors (e.g., cameras, photomultiplier tubes, or photodiodes) operating in the visible region. In many multiphoton microscopy methods (such as two-photon and three-photon microscopy), it is sufficient to detect light generated inside the sample using a nonlinear process by a photodetector that collects as much light as possible from the sample (non-scan detection).

[0004] A key challenge in the design of modern laser scanning microscopes lies in the presence of various immersion media to which the microscope optics require calibration. This is particularly important when imaging samples treated with tissue ablation techniques [2], which chemically make biological tissues transparent and thus permeable to visible light. By removing tissue components such as lipids that act as scatterers, ablation techniques reduce scattering and homogenize the refractive index of the entire sample. Currently, there are several such ablation techniques, such as Scale [3], 3DISCO [4], iDISCO [5], vDISCO [6], uDISCO [6], CLARITY [7], [8] and CUBIC [9] (see also...) Figure 2 As the final step in each cleaning process, the sample is placed in a liquid medium whose refractive index is balanced by diffusion.

[0005] The refractive index can vary significantly depending on the medium used. For example, the 3DISCO, vDISCO, and uDISCO schemes use BABB media (a 50 / 50 vol / % mixture of benzyl alcohol and benzyl benzoate), which results in a final refractive index n. d =1.559

[10] , while iDISCO uses refractive index n d =1.562

[10] dibenzyl ether. Here, n d The refractive index is measured at 587,562 nm along the d-line of sodium. On the other hand, CLARITY uses immersion fluids with lower refractive indices, such as refractive index-matching solutions with a refractive index of 1.45[8],

[11] . Additionally, there is another type of tissue processing technique called extended microscopy

[12] , which uses tissue expansion as a chemical amplification process and utilizes water as the final immersion medium. Water (n d =1.333) is also commonly used as an embedding agent for in vivo microscopy of living cells and whole organisms such as developing embryos. For living samples, other immersion media such as silicone oil and gradient density media such as 2,2'-thiodiethanol have been proposed

[13] . 2,2'-thiodiethanol as a 60% stock solution has a refractive index of 1.429 and is commercially available under the trade name OptiPrep. Depending on the mixing ratio with water, the refractive index can be adjusted to n d =1.333 to n d =RI 1.429, adjusted accordingly. Due to the refractive index n d and dispersion (by Abbe number V) dThe significant differences in quantitation make the design of microscope objectives compatible with all these media highly challenging. Furthermore, high-resolution depth imaging in living or clean samples requires high numerical apertures (NA) and long working distances to allow sufficient mechanical distance between optics (e.g., the front lens of a refractive objective) and the sample itself. These two requirements further complicate the optical design of microscope objectives used in this application.

[0006] For example, a series of refractive microscope objectives are disclosed in US 9,195,040, which are designed to operate in conjunction with an immersion medium having a numerical aperture of NA 0.9-0.95 and a working distance of up to 8 mm and a variable refractive index in at least a portion of the range of 1.33-1.52.

[0007] In addition, US 9,477,073 discloses an immersion microscope objective with a working distance of up to 10 mm and an NA of 1.0, and a variable refractive index ranging from 1.4 to 1.52.

[0008] In addition, US 10,330,908 discloses a series of microscope objectives with an NA of 0.6 and a working distance of 20 mm in a medium with a variable refractive index of n = 1.45 to 1.51.

[0009] However, all of these designs require a large number of lens groups to adequately correct aberrations and tight tolerances during manufacturing and assembly, making such objectives very expensive.

[0010] One approach to improving immersion microscope objectives is to use different design forms, such as purely reflective designs or reflective-refracting objectives that use both refractive and reflective elements. This approach has a long optical tradition: the idea that chromatic aberration caused by material dispersion could not be corrected by selecting the glass available at the time led Newton to design reflecting telescopes because they were not subject to chromatic aberration. He also suggested the use of reflecting mirrors for microscopes

[14] . Today, purely reflective microscope objectives are common in NIR spectroscopy and UV semiconductor examination microscopes, however, they are rarely used in biological microscopes. Summary of the Invention

[0011] Based on the above, the problem to be solved by the present invention is to provide a cost-effective microscope objective that is compatible with a wide range of refractive indices and dispersive properties of liquid immersion media.

[0012] This problem is addressed using an immersion microscope objective lens for examining samples in an immersion medium, which includes:

[0013] -At least one concave mirror,

[0014] - At least one optical element, comprising an aspherical surface facing the at least one concave mirror.

[0015] - An internal space disposed between the at least one concave mirror and the aspherical surface, the internal space being configured to be filled with an immersion medium such that the immersion medium contacts the at least one concave mirror and the aspherical surface.

[0016] According to the invention, the non-planar aspherical surface is shaped such that when the second immersion medium resides in the internal space instead of the first immersion medium being arranged in the internal space, the working distance of the immersion microscope objective changes by less than 1%, and the second immersion medium comprises a refractive index n that increases or decreases by at least 0.025 relative to the refractive index of the first immersion medium.

[0017] In other words, according to the present invention, the non-planar aspherical interface is shaped such that when the refractive index n of the immersion medium increases or decreases by at least 0.025 (i.e., n ± 0.025), the working distance of the immersion microscope objective changes by less than 1%. Specifically, the refractive index can be changed by exchanging the immersion medium in the internal space with another immersion medium or by changing the immersion medium present in the internal space (e.g., by changing its composition and / or by changing the temperature of the immersion medium).

[0018] According to another alternative aspect of the invention, the aspherical interface is shaped such that the immersion microscope objective maintains diffraction-limited image quality, wherein when the refractive index n of the immersion medium at the wavelength increases or decreases by at least 0.025, the Strehl ratio is greater than 0.8 for light passing through at least one wavelength of the aspherical surface.

[0019] According to a preferred embodiment of the invention, the refractive index n of the first and / or second immersion medium is in the range of 1.0 to 1.6, particularly in the range of 1.3 to 1.6.

[0020] Furthermore, according to embodiments of immersion microscope objectives, the numerical aperture (NA) of the immersion microscope objective is in the range of 0.3 to 1.0 in air.

[0021] According to another embodiment of an immersion microscope objective, the internal space of the immersion microscope objective is configured to be filled with an immersion medium, which is one of the following: a fluid, a gas, a liquid, a gel (i.e., a non-fluid colloidal network or polymer network that expands throughout its volume by a fluid), or a hydrogel (i.e., a gel in which the swelling agent is water). Furthermore, according to embodiments, the immersion medium included by the immersion microscope objective is one of the following: a fluid, a gas, a liquid, a gel, or a hydrogel.

[0022] Furthermore, according to an embodiment, at least one reflector and at least one optical element are rotationally symmetrical about the optical axis. In particular, the vertex of the reflector corresponds to the intersection of the reflecting surface of at least one reflector and the optical axis.

[0023] According to a preferred embodiment of an immersion microscope objective, at least one reflecting mirror comprises a spherical shape.

[0024] Furthermore, in one embodiment, the at least one reflector is one of a plurality of reflectors included in an immersion microscope objective, wherein each of the plurality of reflectors is configured to contact the immersion medium when the immersion medium resides in the internal space.

[0025] According to yet another embodiment, the immersion microscope objective includes another aspherical surface shaped to compensate for spherical aberration caused by at least one mirror.

[0026] In this regard, in one embodiment, the other aspherical surface is formed by the at least one optical element and faces away from the aspherical surface facing the at least one mirror.

[0027] The optical system underlying this embodiment is called a Schmidt telescope or Schmidt objective.

[15] In this case, the other aspherical surface is typically described by a polynomial of order 10 or higher:

[0028] z(y) = a1y 2 +a2y 4 +a3y 6 +a4y 8 +a5y 10

[0029] According to reference

[16] , for the correction of spherical aberration up to the third order, the other aspherical surface can be defined as:

[0030]

[0031] Here, y0 is the maximum radius of the correction surface, n is the refractive index of the correction element, and R is the radius of at least one mirror. If higher orders of spherical aberration correction are required (e.g., 5th, 7th, and 9th orders), the above polynomial needs to be extended with higher terms, the coefficients of which can be found numerically using optical design software. In this optimization process, off-axis image quality is usually balanced with on-axis performance; consequently, the coefficient a of the aspherical surface... i They often deviate from the stated regulations.

[0032] When a standard Schmidt objective is in operation in air, the at least one mirror and the at least one optical element are separated by an air gap. In a solid Schmidt objective

[16] , this gap is filled with a solid medium with a refractive index of n. The surface diagram of the other aspherical surface used to correct spherical aberrations up to the third order can be described as

[16] :

[0033]

[0034] If at least one reflector is a sphere of radius R, then the surface shape will distort the incident parallel wavefront into a shape that cancels out spherical aberration. In this case, according to an embodiment of the invention, the aforementioned aspherical surface facing at least one concave mirror is preferably shaped as follows:

[0035]

[0036] To satisfy the aforementioned condition, namely that the non-planar aspherical surface is shaped such that the working distance of the immersion microscope objective changes by less than 1% when the second immersion medium resides in the internal space instead of the first immersion medium being arranged in the internal space, the second immersion medium comprises a refractive index that increases or decreases by at least 0.025 relative to the first immersion medium. In the paraxial approximation, if a polynomial form z(y) for another aspherical surface has been found, the shape w(y) of the aspherical surface facing at least one concave mirror is preferably given by the following formula:

[0037]

[0038] According to an embodiment of the invention, n is the refractive index of the correction element. As a result, the shape of the aspherical surface facing at least one concave mirror is a scaled version of the shape of the other aspherical surface. In itself, the aspherical surface facing at least one concave mirror will be insufficient to correct the spherical aberration of at least one concave mirror and therefore cannot represent the shape of the conventional Schmidt corrector according to

[15] and

[16] . However, it does represent the precise shape of the wavefront inside the optical element facing at least one mirror. If the optical surface forming the interface between the two media is shaped to resemble the incident wavefront, any light passing through such an interface will not experience angular deviation due to refraction when it passes through perpendicularly. Since no refraction occurs at this interface, it does not contribute any optical power to the objective lens and does not produce additional aberrations (including defocus). As a result, the non-planar aspherical surface is shaped such that when the second immersion medium resides in the internal space instead of the first immersion medium being arranged in the internal space, the working distance of the immersion microscope objective changes by less than 1%, and the second immersion medium includes a refractive index that increases or decreases by at least 0.025 relative to the refractive index of the first immersion medium.

[0039] According to an alternative embodiment, the other aspherical surface is formed by another optical element of the immersion microscope objective.

[0040] In particular, in one embodiment, the immersion microscope objective includes a lens group comprising a plurality of lenses, wherein a first optical element forms a lens of the lens group, and / or wherein another optical element forms a lens of the lens group.

[0041] According to another preferred embodiment, the immersion microscope objective includes a sample holder configured to hold a sample such that the sample is located in the internal space between at least one mirror and the aspherical surface facing at least one mirror.

[0042] Another aspect of the invention relates to a microscope comprising an immersion microscope objective according to the invention.

[0043] According to a preferred embodiment of the microscope, the microscope is one of the following:

[0044] - Wide field-of-view microscopes, especially those including single-element or multi-element tube lenses.

[0045] - Light sheet microscopes, particularly those comprising single-element or multi-element tube lenses,

[0046] - Two-photon fluorescence microscopy

[0047] - Three-photon fluorescence microscopy

[0048] - Four-photon fluorescence microscopy

[0049] - Second harmonic generation microscope

[0050] - Third harmonic generation microscope

[0051] - Fluorescence confocal microscope

[0052] -Reflection confocal microscope

[0053] - Polarizing microscope

[0054] - Coherent anti-Stokes Raman scattering (CARS) microscopy,

[0055] - Stimulated Raman scattering (SRS) microscopy. Attached Figure Description

[0056] The embodiments of the invention, as well as further features and advantages, are described below with reference to the accompanying drawings, wherein...

[0057] Figure 1 The general layout of a laser scanning microscope known in the prior art is shown;

[0058] Figure 2A table is shown that includes an overview of common cleaning methods and immersion media;

[0059] Figure 3 A wavefront W of arbitrary shape is shown encountering an interface between two different media, the interface being sufficiently similar to the wavefront itself such that the wavefront W' continues to propagate in the second medium in a non-offset manner, wherein for every position along the wavefront, the normal vector of the interface is perpendicular to the wavefront, thus no refraction occurs, and the surface is minimally refractive (left). Furthermore, Figure 3 The general concept of an immersion microscope objective using a concave mirror according to the present invention is shown (right);

[0060] Figure 4 An embodiment of a multi-immersion microscope objective with minimal refractive surface according to the present invention is shown;

[0061] Figure 5 An embodiment of a multi-immersion microscope objective according to the present invention, combined with a multiphoton microscope, is shown;

[0062] Figure 6 An example of a multi-immersion microscope objective combined with a wide field-of-view microscope is shown;

[0063] Figure 7 An embodiment of a multi-immersion microscope objective combined with a light sheet microscope is shown;

[0064] Figure 8 An example of a multi-immersion microscope objective combined with a confocal microscope is shown;

[0065] Figure 9 A cross-sectional view of an embodiment of a multi-immersion microscope objective is shown;

[0066] Figure 10A-10D It shows Figure 9 RMS wavefront plots of different immersion media and wavelengths in the illustrated embodiments;

[0067] Figure 11 A cross-sectional view of another embodiment of a multi-immersion microscope objective is shown;

[0068] Figure 12A-12D It shows Figure 11 RMS wavefront plots of different immersion media and wavelengths in the illustrated embodiments;

[0069] Figure 13 A cross-sectional view of another embodiment of a multi-immersion microscope objective is shown; and

[0070] Figures 14A-14D It shows Figure 13 RMS wavefront diagrams of different immersion media and wavelengths in the illustrated embodiments. Detailed Implementation

[0071] This invention relates to an immersion microscope objective 10, which can be used with various different immersion media M. In particular, such as... Figure 3 The right side and Figure 4 The diagram illustrates a general embodiment of an objective 10 according to the invention, which includes at least one concave mirror 3 having a working distance 7, the working distance being the distance between the focal point F of the microscope objective 10 and the vertex 3a of the concave mirror 3, at least one optical element 1 including an aspherical surface 2 facing the at least one concave mirror 3, and an internal space 4 disposed between the at least one concave mirror 3 and the aspherical surface 2, the internal space 4 being configured to be filled with an immersion medium M such that the immersion medium M contacts the at least one concave mirror 3 and the aspherical surface 2, wherein the aspherical interface is shaped such that when the refractive index n of the immersion medium increases or decreases by at least 0.025 (e.g., due to the exchange of immersion medium M with another immersion medium M), the working distance of the immersion microscope objective 10 changes by less than 1%.

[0072] The preferred high-order aspherical surface 2 can be formed from the transparent correction plate 1. Furthermore, this correction plate 1 can have another aspherical surface 5, which is adapted to counteract the spherical aberration of at least one reflector 3 to provide excellent image quality.

[0073] As described above, if a polynomial form z5(y) for the other aspherical surface 5 has been found for the paraxial case (e.g., through numerical optimization in an optical design procedure), then the shape z2(y) of the higher-order aspherical surface 2 is preferably selected according to the following formula:

[0074]

[0075] If n is the refractive index of the material of the correction plate 1, then the shape of the higher-order aspherical surface 2 is a scaled-down form of the shape of another aspherical surface 5. In itself, the aspherical surface 2 facing at least one concave mirror will be insufficient to correct the spherical aberration of at least one concave mirror, and therefore cannot represent the shape of the conventional Schmidt corrector according to

[15] and

[16] .

[0076] If the medium M between the correction plate 1 and the reflecting mirror 3 is replaced by a solid with a refractive index of n, the numerical aperture (NA = nsinα) increases by a factor of n, which further improves the microscope's resolving power. Additionally, the image brightness scale is n. 2 If the space between the correction plate 1 and the mirror 3 is not filled with solid material, but with a liquid immersion medium M, the system is used as an immersion objective lens 10.

[0077] Advantageously, in this design, when the immersion medium M (e.g., a fluid) is replaced with an immersion medium having a different refractive index and dispersion, the mirror 3 does not introduce changing aberrations (especially chromatic aberration). This is because the law of reflection at the interface (θ1 = θ2) does not include any dependence on the wavelength-dependent refractive index (λ) of the medium, such as Snell's law of refraction at the interface between two media: n1sinθ1 = n2sinθ2. This design concept can be used to transform any mirror-based telescope or microscope design (e.g., a Schwarz double-mirror objective) into an immersion objective 10. However, since a portion of the excitation optics (if used in conjunction with laser scanning) or the detection path (if used in conjunction with an eyepiece or camera) is typically placed in the air, a window is necessary to separate the immersion portion of the microscope from the rest of the optical path. When light rays originating at or directed to an off-axis position in a rotationally symmetric optical system strike this surface, transverse chromatic aberration occurs, which is the dominant chromatic aberration in such an immersion mirror system.

[0078] Typically, the aspherical correction plate 1 deforms the parallel wavefront outside the objective lens to compensate for the spherical aberration of the primary mirror. As mentioned above, the wavefront reflected from the mirror is independent of the medium into which it propagates. This means that for any possible immersion medium M within the objective lens 10, the single shape of the aspherical correction plate 1 is sufficient to correct the spherical aberration of the mirror 3, provided that no additional refraction (equivalent to additional wavefront distortion) occurs when the wavefront passes through the interface (e.g., the aspherical surface 2) between the correction plate 1 (preferably made of a transparent solid material such as glass or plastic) and the immersion medium M (e.g., the liquid). This can be achieved by shaping the surface 2 separating the correction element 1 and the liquid medium M to resemble the surface through which the wavefront passes. In this case, there is no additional refraction of light (because locally, the wavefront passes through the interface with the surface normal perpendicular to the wavefront) and no additional aberration is produced—the surface is minimally refractive. If the wavefront originates from or points off-axis, there will be a slight deviation from locally normal incidence, and therefore, additional off-axis aberration can occur. However, for sufficiently small angular differences (<11.4° or 0.2 radians), the resulting aberrations are small. Therefore, if the absolute difference between the angle of incidence θ1 and the angle of refraction θ2 at any point where light rays contributing to image formation intersect the surface (and thus obey Snell's law according to n1sinθ1=n2sinθ2) is less than 0.2 radians, then we define the surface as having minimal refraction.

[0079] |θ1-θ2|<0.2 radians

[0080] Therefore, the minimum refractive surface (here, for example, aspherical surface 2) can be used to transmit wavefronts between media (see...). Figure 3The wavefronts W and W' in the spherical surface are used without introducing aberrations, which is a very useful general optical design principle for designing immersion objectives for a wide range of media. This can be seen in the generalization of the well-known aberration-free concentric surface in optical design: if the desired wavefront should be spherical, then the necessary minimum refractive surface is concentric around the focal point—a design method frequently used in high-NA collimators, microscope objectives, and interferometric objectives for optical metrology. Besides the rotationally symmetric aspherical surface used here, one can imagine the usefulness of the minimum refractive free-form surface in off-axis variations of the optical system given here.

[0081] As mentioned above, Figure 4 A basic embodiment / design concept of a multi-immersion microscope objective 10 according to the present invention is shown. In its general form, the disclosed microscope objective 10 comprises a concave mirror 3 in contact with an immersion medium M and a group of one or more optical elements 1, the group having an element whose surface forms an aspherical surface 2 separating the internal space 4 of the immersion medium M / objective 10 from the group. According to one example, the group of optical elements may be formed by a correction plate 1 as previously described or may include such a correction plate (among other optical elements).

[0082] For example, such as Figure 3 and 4 As shown, sample S is immersed in immersion medium M. The front group (e.g., optical elements) 1 reshapes the wavefront entering or leaving objective lens 10, for example, to compensate for aberrations introduced by concave mirror 3. The aspherical surface 2 forming the boundary between the solid medium or optical element in group 1 and the liquid immersion medium M is preferably shaped as a minimal refractive surface (see above). Mirror 3 can be replaced by a combination of mirrors, wherein at least one mirror should be concave, and all mirrors are in contact with the immersion medium M, for example, to fold the beam path for accessibility.

[0083] As an example of this design principle according to the invention, the invention is applied to the design of multiphoton microscope objectives. While in confocal laser scanning fluorescence microscopy, adequate correction of the transverse axis and chromatic aberration within the wavelength bands covering the necessary excitation and emission regions of the spectrum is beneficial for achieving a sufficiently large field of view (FOV), in multiphoton microscopy such as two-photon microscopy, the requirement for color correction is reduced. For example, in a two-photon microscope operating at an 850 nm excitation wavelength and utilizing a 100 fs laser pulse, the full width at half maximum (FWHM) of the excitation spectrum is <10 nm. Since two-photon microscopy can be combined with descattering detection by collecting a large amount of scattered and unscattered emitted light with a photodetector, color correction is not required for the emitted fluorescence. This means that unless multiple excitation wavelengths are used simultaneously, there is no need to correct for transverse chromatic aberration in the visible and near-infrared regions of the spectrum. Furthermore, in extended (or even cleared) samples, the image field is irrelevant because the collected three-dimensional imaging data can be calculated and deformed back into the Cartesian coordinate system.

[0084] Figure 5 An embodiment of a microscope 100 in the form of a multiphoton microscope according to the invention is shown, comprising a multi-immersion microscope objective 10 according to the invention. In this apparatus, light from a light source 101 (e.g., a picosecond or femtosecond laser source operating in the 700-2000 nm wavelength region, or a combination of multiple such sources) is directed to a beam-shaping device 102, which may include functions such as beam spreading, beam intensity control, and beam stabilization, and is then directed to a scanning system 103. The system 103 may include means for splitting the beam into smaller beams and allows for controllable beam control. The scanning beam is then directed to the microscope objective 10, which is filled with the immersion medium M according to the invention. The sample S is immersed in the medium M (in the internal space 4 of the objective 10) via a sample holder 6 and can be translated and rotated using a sample positioning device if desired. The emitted light from the sample S is collected by the microscope objective 10 and separated from the excitation light using an emission dichroic mirror 104. It is then directed to a collecting optics 105 that focuses the emitted light onto a photodetector 106. Sample positioning, beam scanning, and optical detection are preferably electronically controlled.

[0085] Figure 6Another embodiment of the microscope 100 according to the invention is shown, here in the form of a large field-of-view microscope, comprising a multi-immersion microscope objective 10 with a minimal refractive surface according to the invention. Light from a light source 101, such as a laser, light-emitting diode, or fluorescent lamp, is directed to a beam-shaping illumination optics 107, which can be used for intensity control, beam spreading, beam shaping, or beam scanning, and redirects the excitation light to a beam splitter 108, such as a dichroic mirror. The excitation light is then redirected to the microscope objective 10, which directs the excitation light onto the sample S. The sample S is immersed in the immersion medium M via a sample holder 6 and can be translated and rotated using a sample positioning device if desired. Light emitted from the sample S is collected by the immersion microscope objective 10 and directed to a tube optics module 109, which may contain optical filters to modify the spectrum of the detected light and focus the light onto a camera module 110.

[0086] According to another embodiment of the microscope 100 of the present invention, Figure 7 A light-sheet microscope 100 including an immersion microscope objective 10 according to the invention is depicted. Here, light from a light source 101, such as a laser, is directed to a beam-forming optics 111, which can be used for intensity control, beam spreading, beam shaping, or beam scanning, and redirects the excitation light to an excitation optics 112. After excitation of the excitation optics 112, the beam is shaped into a light-sheet-like structure by timely scanning of the beam or by reshaping the beam profile into a light-sheet. The excitation optics 112 also forms an interface between the immersion medium M and external components and may include means for sealing the immersion medium M relative to the surrounding medium. The sample S is immersed in the medium M in the internal space 4 via a sample holder 6 and can be translated and rotated using a sample positioning device if desired. Light emitted from the sample S is collected by the microscope objective 10 and directed to a barrel optics 109, which may contain optical filters to modify the spectrum of the detected light and focus the light onto a camera module 110. To generate multiple light sheets, the beam-forming optics 111 may include one or more beam-splitting devices that direct the excitation light to several excitation optics modules. This can be used to allow for multi-directional illumination of sample S, for example, to reduce shadow artifacts caused by refraction or absorption of sample features.

[0087] also, Figure 8 An embodiment of a microscope 100 in the form of a confocal microscope according to the present invention is shown, which includes an immersion microscope objective 10 according to the present invention. Specifically, in Figure 8In the illustrated configuration, light from light source 101 (e.g., a continuous-wave laser source operating at wavelengths in the visible region of the spectrum, or a combination of multiple such sources) is directed to a beam-shaping device 111, which may include functions such as beam spreading, beam intensity control, and beam stabilization, and is then directed to a beam-splitting device 113, such as a dichroic beam splitter. The excitation light is then fed into a scanning system 114. This system 114 may include, for example, a device for splitting the beam into smaller beams in a rotating disk confocal microscope, and allows for controlled beam steering. The scanning beam is then directed to the aforementioned microscope objective 10 via an intermediate optical module 115. The intermediate optical module 115 may also be designed to reduce chromatic aberration caused by multi-immersion microscope objectives. Objective 10 is filled with an immersion medium M. The sample is immersed in the medium M via a sample holder 6 and can be translated and rotated using a sample positioning device if necessary. Light emitted from the sample S is collected by the microscope objective 10 and redirected through the optical path. The beam splitter 113 is designed to transmit at least a portion of the light reflected, scattered, or emitted by the sample S, and may utilize a dichroic beam splitter. The collecting optics module 116 then focuses the light onto a size-adjustable confocal pinhole 117. A photodetector 106 then detects the light passing through the pinhole 117. Sample positioning, beam scanning, and light detection are preferably electronically controlled. In variations of this microscope concept, particularly in rotating disk confocal microscopes, the scanning system 114 also includes an array of confocal pinholes.

[0088] Specifically, if the Strelby ratio (the ratio of the peak intensity of the point spread function of the optical system to the maximum intensity using the ideal point spread function) is greater than 0.8, the optical system within the framework of this invention can be considered diffraction-limited. This corresponds to a root mean square (RMS) wavefront error of less than approximately 1 / 14λ = 0.0714λ.

[0089] Furthermore, according to a preferred embodiment of the invention, the immersion medium facing the concave mirror and in contact with the objective lens and / or the aspherical surface defining the objective lens, particularly containing the internal space of the sample, is a rotationally symmetric polynomial aspherical surface, which is described by a polynomial expansion of the radial coordinate y relative to the deviation z of a sphere having radius r:

[0090] z = a1y 2 +a2y 4 +a3y 6 +a4y 8 +a5y 10

[0091] To demonstrate the utility of an optical design with minimal refractive surface area, the following will refer to... Figures 9 to 12DTwo additional embodiments of immersion microscope objectives are described in more detail. These objectives are medium to high NA multi-immersion microscope objectives and are specifically designed to provide diffraction-limited performance in air at FOVs up to 2.8 mm and NAs of 0.5 and 0.85. If these objectives are filled with a liquid immersion medium of refractive index n, the focal position and working distance remain unchanged (because no additional refraction is introduced around the surface of the medium), and the numerical aperture increases by a factor of n. d In liquid media with a refractive index of 1.55, the numerical aperture (NA) reaches 0.8 and 1.33, respectively. With a constant pupil diameter, these designs exhibit constant light-gathering efficiency (or constant Lagrangian invariance) for varying immersion media. This means that the increase in numerical aperture (NA) caused by filling the objective with a medium of refractive index n corresponds to a reduction in the FOV size by a factor of 1 / n. If the medium filling the objective space is non-uniform, the resulting localized wavefront distortion will degrade optical performance.

[0092] In particular, Figure 9 and Figure 10A-10D An embodiment of the multi-immersion microscope objective according to the present invention is shown.

[0093] In n d For materials with a value of 1, such as air, the numerical aperture (NA) in this embodiment is 0.52, and in n... d In materials with a refractive index of 1.333 (such as water), the NA of this design is 0.69. In media with higher refractive indices, such as n... d =1.5579, the system's NA is 0.80.

[0094] It can function as a two-photon microscope objective, with an excitation wavelength range from 800 to 1000 nm, and provides sufficient color correction to operate within a 20 nm wide wavelength band. The immersion medium fills the space between surface 2 and mirror 3. It is compatible with air, fluid, and has variable n... d and V d The position of the image remains stable regardless of the different combinations of immersion media in the solid medium.

[0095] According to Figure 9 Exemplary parameters used in embodiments 10 and 10 are stated in the following two tables:

[0096] Surface number according to Figure 9 surface Radius (mm) Thickness (mm) <![CDATA[n d ]]> <![CDATA[V d ]]> 1 5 468.366 5 1.4584 67.82 2 2 1600.594 35.058 variable variable 3 3 -40 -19.476 reflector reflector image image -19.761

[0097] Surface number according to Figure 9 surface a1 a2 a3 a4 a5 1 5 9.744E-04 -1.124E-05 -1.083E-08 -5.473E-12 -3.876E-14 2 2 3.279E-04 -3.486E-06 -4.951E-09 1.350E-11 -4.465E-14 3 3 image image

[0098] Another embodiment of the immersion microscope objective according to the invention is shown in Figure 11 and 12A -12D.

[0099] In nd In a material such as air where NA = 1, the NA in this embodiment is 0.85, and in n d In materials with a refractive index of 1.333 (such as water), the NA of this design is 1.14. In media with higher refractive indices, such as n... d =1.5579, the system's NA is 1.33.

[0100] It can function as a two-photon microscope objective, with an excitation wavelength range from 780 to 940 nm, and provides sufficient color correction to operate within a 20 nm wide wavelength band. The immersion medium fills the space between surface 2 and mirror 3. It is compatible with air, fluid, and has variable n... d and V d The position of the image remains stable regardless of the different combinations of immersion media in the solid medium.

[0101] According to Figure 11 and 12A Exemplary parameters used in the -12D embodiments are stated in the following two tables:

[0102] Surface number according to Figure 11 surface Radius (mm) Thickness (mm) <![CDATA[n d ]]> <![CDATA[V d ]]> 1 5 85.512 5 1.4584 67.82 2 2 517.546 17.293 variable variable 3 3 -22 -10.366 reflector reflector image image -10.834

[0103]

[0104] Another embodiment of the immersion microscope objective according to the invention is shown in Figure 13 and 14A -14D in.

[0105] In n d In a material such as air where NA = 1, the NA in this example is 0.53, and in n d In materials with a refractive index of 1.333 (such as water), the NA of this design is 0.71. In media with higher refractive indices, such as n... d =1.5579, the system's NA is 0.82.

[0106] It can function as a two-photon microscope objective, with an excitation wavelength range from 780 to 940 nm, and provides sufficient color correction to operate within a 20 nm wide wavelength band. The immersion medium fills the space between surface 2 and mirror 3. It is compatible with air, fluid, and has variable n... d and V d The position of the image remains stable regardless of the different combinations of immersion media in the solid medium.

[0107] Compared to the previous embodiment, this embodiment designates surfaces 5 and 2 as flat (infinite radius). Therefore, the condition that the ratio of the polynomials describing surfaces 5 and 2 should be (n-1) / n can be explicitly tested. Here, n is the refractive index of the correction plate 1 at the center wavelength of the excitation spectrum. In the previous embodiment, this comparison could only be made if the non-zero surface radii were included in the surface polynomials of surfaces 5 and 2.

[0108] According to Figure 13 and 14A Exemplary parameters used in the -14D embodiments are stated in the following two tables:

[0109] Surface number according to Figure 13 surface Radius (mm) Thickness (mm) <![CDATA[n d ]]> <![CDATA[V d ]]> 1 5 unlimited 1 1.4584 67.82 2 2 unlimited 39.068 variable variable 3 3 -40 -19.408 reflector reflector image image -20.667

[0110] Surface number according to Figure 13 surface a1 a2 a3 1 5 2.29856E-03 -1.08328E-05 -1.34800E-08 2 2 7.17691E-04 -3.40284E-06 -4.08693E-09 3 3 image image

[0111] Assuming the refractive index n of the correction plate 1 in this embodiment is... d =1.4525, therefore the ratio of the aspheric coefficients of surfaces 5 and 2 should be (n-1) / n = 0.312 in the paraxial case. The table below shows that this condition is met in this embodiment:

[0112] surface Ideal ratio a1 a2 a3 The ratio of surface 2 to surface 5 0.312 0.312 0.314 0.303

[0113] References

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[0118] [5]N.Renier,Z.Wu,D.J.Simon,J.Yang,P.Ariel,and M.Tessier-Lavigne,“iDISCO:A Simple,Rapid Method to Immunolabel Large Tissue Samples for VolumeImaging,”Cell,vol.159,no.4,pp.896–910,Nov.2014,doi:10.1016 / j.cell.2014.10.010.

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[0122] [9]E.A.Susaki et al.,“Whole-Brain Imaging with Single-Cell ResolutionUsing Chemical Cocktails and Computational Analysis,”Cell,vol.157,no.3,pp.726–739,Apr.2014,doi:10.1016 / j.cell.2014.03.042.

[0123]

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[0124]

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[0125]

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[0126]

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Claims

1. An immersion microscope objective (10) for examining a sample (S) in an immersion medium (M), comprising: - At least one concave mirror (3). - At least one optical element (1) comprising an aspherical surface (2) facing the at least one concave mirror (3). - An internal space (4) is arranged between the at least one concave mirror (3) and the aspherical surface (2), the internal space (4) being configured to be filled with an immersion medium (M) such that the immersion medium (M) contacts the at least one concave mirror (3) and the aspherical surface (2). in The immersion microscope objective (10) includes a working distance (7), which is the distance between the focal point (F) of the immersion microscope objective (10) and the vertex (3a) of the concave mirror (3), wherein the aspherical surface (2) is non-planar and is shaped such that when the second immersion medium (M) resides in the internal space (4) instead of the first immersion medium (M) being arranged in the internal space (4), the working distance (7) of the immersion microscope objective (10) changes by less than 1%, the second immersion medium (M) including a refractive index n that increases or decreases by at least 0.025 relative to the refractive index of the first immersion medium (M).

2. The immersion microscope objective according to claim 1, wherein, The refractive index n of the first immersion medium is in the range of 1.0 to 1.6, and / or the refractive index n of the second immersion medium is in the range of 1.0 to 1.

6.

3. The immersion microscope objective according to claim 1, wherein, When the immersion medium (M) present in the internal space (4) is air, the numerical aperture NA of the immersion microscope objective (10) is in the range of 0.3 to 1.

0.

4. The immersion microscope objective according to any one of the preceding claims, wherein, The immersion medium (M) is one of the following: fluid, gas, liquid, gel.

5. The immersion microscope objective according to any one of claims 1 to 3, wherein, The immersion medium (M) is a hydrogel.

6. The immersion microscope objective according to claim 1, wherein, The at least one concave mirror (3) and the at least one optical element (1) are rotationally symmetrical with respect to the optical axis (A).

7. The immersion microscope objective according to claim 1, wherein the at least one concave mirror (3) comprises a spherical shape.

8. The immersion microscope objective according to claim 1, wherein the at least one concave mirror (3) is one of a plurality of mirrors included in the immersion microscope objective (10), wherein each of the plurality of mirrors is configured to contact the immersion medium (M) when the immersion medium (M) resides in the interior space (4).

9. The immersion microscope objective according to claim 1, wherein, The immersion microscope objective (10) includes another aspherical surface (5) which is shaped to compensate for spherical aberration caused by the at least one concave mirror (3).

10. The immersion microscope objective according to claim 9, wherein, The other aspherical surface (5) is formed by the at least one optical element (1) and faces away from the aspherical surface (2) facing the at least one concave mirror (3).

11. The immersion microscope objective according to claim 9, wherein, The other aspherical surface is formed by another optical element of the immersion microscope objective (10).

12. The immersion microscope objective according to claim 11, wherein, The immersion microscope objective (10) includes a lens group comprising a plurality of lenses, wherein at least one optical element (1) forms a lens of the lens group, and / or wherein another optical element forms a lens of the lens group.

13. The immersion microscope objective according to claim 1, wherein, The immersion microscope objective (10) includes a sample holder (6) configured to hold a sample (S) such that the sample (S) is located in the internal space (4) between the at least one concave mirror (3) and the aspherical surface (2) facing the at least one concave mirror (3).

14. The immersion microscope objective according to claim 9 or 10, wherein, The aspherical surface (2) is defined by the polynomial equation ay=aiyi describing the aspherical surface (2), and the other aspherical surface (5) is defined by the polynomial equation by=aiyi describing the other aspherical surface (5), and the polynomial equations ay and by follow the relation ay=((n-1)n)∙b(y), where n is the refractive index of the optical element (1) at the working wavelength of the immersion microscope objective.

15. A microscope (100) comprising an immersion microscope objective (10) according to any one of the preceding claims.

16. The microscope according to claim 15, wherein, The microscope (100) is one of the following: - Wide field-of-view microscopes, including single-element or multi-element lens tubes. - Light sheet microscopes, including single-element or multi-element lens tubes. - Two-photon fluorescence microscopy, - Three-photon fluorescence microscopy - Four-photon fluorescence microscope - Second harmonic generation microscope - Third harmonic generation microscope - Fluorescence confocal microscopy, - Reflection confocal microscope, - Polarizing microscope, - Coherent anti-Stokes Raman scattering (CARS) microscopy, - Stimulated Raman scattering (SRS) microscopy.

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