Microscope for transmitted light contrasting

Abstract

To provide a microscope for imaging a sample by a transmitted light contrasting method.SOLUTION: A microscope 100 includes: an objective lens holder or changer 114 configured to place an objective lens 106 or objective lenses of multiple objective lenses on an optical axis 120 of the microscope; a lens system 102 for forming an intermediate image 104 of an exit pupil 108 of any one of the objective lenses placed on the optical axis, where the intermediate image is formed at a conjugated plane 124 conjugate to the exit pupil; and a control device 118 configured for automatically positioning a modulation element 110 on the optical axis located at a position of the conjugated plane or related to the conjugated plane.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates substantially to a microscope for imaging a sample by at least transmitted light contrast. [Background technology]

[0002] In microscopy, transmitted light contrast can be used to convert the phase information of an object into intensity modulation, which can then be detected by a detector. This allows for the observation of thin, unstained specimens, particularly in biological imaging in cell biology. This is usually achieved using a modulation element, such as a phase ring (ring diaphragm) or modulator. Such modulators can typically relate to amplitude modulation, i.e., the attenuation of light passing through the microscope objective lens. These modulators must be positioned in a plane conjugate to the exit pupil of the microscope objective lens to ensure homogeneous contrast and translationally invariant imaging across the field of view. [Overview of the project] [Means for solving the problem]

[0003] In view of the above-mentioned shortcomings and problems, an improved method for providing a modulator to a microscope is needed. According to embodiments of the present invention, a microscope having the features of claim 1 is proposed. Advantageous further developments form the subject of the dependent claims and subsequent descriptions.

[0004] Embodiments of the present invention generally relate to a microscope configured to image a sample by at least transmitted light contrast. The microscope comprises an objective lens changer configured to position one of several objective lenses on the optical axis of the microscope. Note that the microscope may also comprise only a single objective lens. In such a case, an objective lens changer would not be necessary, and a simple objective lens holder would suffice. The microscope further comprises a lens system for forming real intermediate images formed on each conjugate plane conjugate to the exit pupil of any one of the objective lenses (or, optionally, a single objective lens) positioned on the optical axis. The microscope also typically comprises an illumination unit that generates an illumination beam path for illuminating the sample to be imaged, with each objective lens positioned on the optical axis generating an imaging beam path for imaging the sample. Preferably, the microscope further comprises a digital image detection module (detector) for detecting an image of the sample to be imaged. Such a digital image detection module may be a camera or comprise a camera (or be included in a camera).

[0005] Microscopes can be either inverted or upright. For example, in an inverted microscope, the object being examined (i.e., the sample) is illuminated from above using transmitted light, and the objective lens is mounted below the microscope stage. In the case of an inverted reflected light microscope, both illumination and observation through the objective lens are performed from below. Such incident light microscopes play a major role, for example, in mineralogy and metallurgy, while inverted transmitted light microscopes are often used to examine or manipulate biological samples. Inverted microscopes allow for good access to the sample being examined because the imaging optics are typically located below the microscope stage, i.e., within the microscope stand.

[0006] In particular, biological samples and thin samples appear almost transparent when viewed by conventional microscopic methods. Such samples typically only have varying optical thicknesses, while the light amplitude is either not attenuated or uniformly attenuated throughout the sample. The optical path difference present when light passes through such a sample (phase object) can be made visible to the human eye by various contrast methods. Of the known contrast methods, such as phase contrast, Hoffmann modulation contrast, relief contrast, Varell contrast, or interference contrast, only phase contrast and modulation contrast, which are typical representatives of contrast methods, will be briefly described below.

[0007] In the case of phase contrast, the upstream annular diaphragm in the illumination beam path is imaged at infinity by a condenser. The illumination beam that passes through the ring diaphragm and penetrates the sample without diffraction ("zero-order diffraction configuration") strikes a phase ring at the back focal plane of the objective lens (coinciding with the exit pupil for telecentric objective lenses commonly used in microscopes). That is, the illumination beam strikes an annular layer, for example, arranged by deposition. A phase difference of λ / 4 is achieved by comparing it with the light rays penetrating the phase plate adjacent to this layer. This enables the following functions: The diffracted light has a phase shift of 180° (λ / 2) relative to the zero order in the case of an amplitude object, whereas in the case of a phase object it is only 90° (λ / 4). Even with this additionally introduced 90° shift of the phase ring, the full 180° shift, i.e., the same phase relationship as the amplitude object, is produced. By additionally weakening the amplitude in the phase ring, the intensity of the zero order is adjusted to the diffraction order. In the intermediate image plane of a microscope, images comparable to amplitude images now arise from interference of diffraction orders.

[0008] Details with a higher refractive index than the surrounding area appear darker in this image. Naturally, the phase ring can be cut into a ring aperture so that the ring aperture is mapped onto the phase ring. The phase ring is usually located inside the objective lens pupil, within the objective lens itself. Therefore, special objective lenses are often used for phase contrast, in which case the phase ring is integrated (e.g., by vapor deposition on the lens).

[0009] The combination of a dielectric layer and a metal layer is typically used to construct a phase contrast layer. The dielectric layer (e.g., silicon oxide) can be used to adjust the phase shift, while the metal layer (e.g., chromium) can be used to set the desired transmittance.

[0010] The selection of an appropriate phase ring (and therefore, the associated ring aperture) depends not only on the objective lens but also on the sample being inspected, characterized by its respective transmittance and phase shift. Furthermore, the size of the phase ring should be dimensionally determined according to the desired resolution or contrast. Special lenses with integrated phase rings cannot flexibly accommodate such different requirements. This would require the supply of a large number of special lenses, which would require considerable effort and high cost. Therefore, in practice, general-purpose special lenses are not universally applicable standard solutions and often fail to produce the desired results in special cases.

[0011] For so-called modulation contrast, a plate is placed on the pupil plane of the observation beam path, with plate strip-like regions of different transparency (typically 0%, 20%, and 100%) provided on it. Such a plate is typically called a modulator or modulation element. In this case, since the change in diffraction pattern does not occur symmetrically with respect to the optical axis of the objective lens, phase objects made visible with such a microscope exhibit a relief effect similar to that that occurs when an object or sample is illuminated obliquely from one side. On the illumination side, there is provided at least one slit-shaped aperture conjugate to the modulator in the imaging beam path. This is typically imaged onto the modulator strip on the imaging side at average transmittance. These slit-shaped apertures are usually located inside the focusing disk, and a unique illumination slit is provided for each magnification.

[0012] Such modulators (or modulation elements) must be positioned in a plane conjugate to the exit pupil of the microscope objective lens to ensure uniform contrast and translationally consistent imaging across the field of view. Typically, such modulation elements are positioned within the parallel region of the field imaging beam (ortho imaging beam) but (simultaneously) within the focal point of the pupil imaging beam (conoscopy imaging beam). Therefore, for example, moving the objective lens changes the axial position of the pupil image within the region of the modulation element.

[0013] A means to achieve this, according to embodiments of the present invention, is to provide a microscope having a control device. The control device is configured to automatically position the modulation elements (modulators) on the optical axis, either at the position of each plane or at a position related to each conjugate plane, i.e., within a specific range around the conjugate plane along the optical axis. Thus, manual positioning is not required. Rather, the correct placement of the modulators is provided to the microscope user without any specific interaction. A modulation element positioning device (e.g., a holder with a slider) can be provided to position and move / position the modulation elements. The control device is then connected to the modulation element positioning device for automatic control.

[0014] Preferably, the control device is configured to automatically move the modulation element in an axial direction parallel to the optical axis, i.e., along the optical axis. This allows for proper positioning of the modulator along the optical axis according to specific requirements, and allows for the use of a single modulation element for different objective lenses or different classes of objective lenses having different exit pupil positions along the optical axis. There is no need to provide a slider or the like with several modulation elements positioned at different axial positions along the optical axis.

[0015] Advantageously, the control unit is connected to an objective lens holder or changer to receive information about each objective lens positioned on the optical axis. This makes it possible to determine the correct required (axial) position of the modulation element based on the currently used objective lens having a specific exit pupil position.

[0016] Further advantages are obtained if the control device is connected to the microscope's focus drive to receive information about the axial (or axis) position of each objective lens. The focus drive is configured to move each objective lens, positioned on the optical axis, in an axial direction parallel to the optical axis. In this way, any axial movement of the currently used objective lens can be instantaneously used to determine the required axial position of the modulation element. It should be noted that such a focus drive can be integrated with the objective lens holder or changer, i.e., a common device can be provided that changes focus and provides axial movement for changing objective lenses.

[0017] To achieve the required position of the modulation element, the control device preferably includes data representing the axial position of the focus drive, a defined mechanical reference plane, for example, the position of the exit pupil of the objective lens (currently positioned on the optical axis) relative to the mounting thread, and additional calibration data. As a result, when the focus drive moves the objective lens along the optical axis or when the objective lens is changed, the physical position of the image of the exit pupil changes, and therefore the plane conjugate to the exit pupil also changes. The control device recalculates the physical position of the image of the exit pupil and then uses the calibration data to reposition the modulation element axially within the plane conjugate to the exit pupil.

[0018] To avoid overhead due to mechanical wear and synchronization, a criterion for repositioning the modulation element can be implemented. That is, the control device according to an embodiment of the present invention is configured to move the modulation element axially only when the displacement of the axial position of each conjugate surface (for example, in the case of a change in the axial position of the conjugate plate due to a change in the objective lens for focal change) exceeds a predetermined threshold. For example, in a microscope with a reference focal length of 200 mm and a field of view (diagonal) of 25 mm, the numerical aperture for pupil imaging is 0.0625. Therefore, the depth of focus of the pupil image is 140 μm (for a typical wavelength of 546 nm). Therefore, mechanical movement is only required when the cumulative correction exceeds such a threshold of 140 μm in this example. In typical high-magnification imaging, this is typically only when searching for a specimen or changing the objective lens.

[0019] Preferably, the control device is configured to automatically move the modulation element in a lateral direction with respect to the optical axis. Further, the control device is preferably also configured to determine the lateral displacement of the modulation element according to quantitative image quality analysis. The quantitative image quality analysis particularly includes at least one of the quantitative reproducibility of the image impression (e.g., contrast impression, i.e., the exact amount of intensity modulation due to various phase changes, which is a transfer function in a mathematical sense. The transfer function converts the phase and amplitude characteristics of an object into image information) and the quantitative reproducibility of the contrast transfer function. That is, the lateral positioning of the modulation element can also be used in conjunction with calibration data to enable the quantitative reproducibility of the image impression and the contrast transfer function for quantitative analysis. This applies not only to the same microscope but also to different microscopes of the same type. The lateral positioning of the modulation element may be left to the user due to subjective variations in the image impression or may be used at a predetermined calibrated position.

[0020] The contrast transfer function converts the amplitude and phase changes through or within the sample into intensity contrast in the image. This intensity contrast, on the one hand, ensures the comparability of images of different samples, and on the other hand, is quantitatively reconstructed in order to use the same contrast transfer function in the quantitative reconstruction of the phase. The latter has also been studied, for example, in Michael Chen, Lei Tian, and Laura Waller, 3D differential phase contrast microscopy, Biomed. Opt. Express 7, 3940 - 3950 (2016). These transfer functions are, for example, referred to as H in Equation (11) in this paper. For example, by appropriate reformulation of Equation (11), it is possible to determine the real and imaginary parts of the refractive index (phase change and absorption) of the sample from the transfer function and the images from transmitted bright-field microscopy and modulation contrast microscopy or images from different implementations of modulation contrast microscopy.

[0021] According to a preferred embodiment, the control device is configured to replace the first modulation element with another second modulation element by lateral movement of the first modulation element out of the optical axis and lateral movement of the second modulation element onto the optical axis. Thereby, for example, the use of phase and modulation contrast elements or different modulation elements of the same type becomes possible. Depending on the situation, this may require changing or adapting the illumination of the microscope so that the microscope is adapted to the second (exchanged) modulation element. For example, the illumination should be adapted such that an appropriate aperture is used for each modulation element. Depending on the modulator (or modulation element), the width or length of the slit aperture must match the modulator. The combination of the objective lens and the condenser determines the magnification of the aperture diaphragm plane at the surface where the modulator is arranged. If the magnification of the objective lens is different and the image of the aperture diaphragm remains the same size, the size of the aperture will be changed. Then, when another (different, e.g., rotated) modulator is used, the aperture is adjusted accordingly (e.g., rotated) so that it is adapted to the new modulator.

[0022] Preferably, the modulation element comprises a transparent section arranged such that when a predetermined lateral displacement of the modulation element deviates from its position on the optical axis, the imaging beam path passes through the transparent section of the modulation element. This means no modulation. In particular, the control unit is configured to move the modulation element laterally by a predetermined lateral displacement to deactivate the modulation element. The predetermined displacement is typically determined by the diameter of the pupil. Thereby, for example, a change in the imaging mode from a contrast method to fluorescence imaging where no modulation is required becomes possible. The user does not need to manually remove such a modulation element. By a specific selection of the predetermined lateral displacement, it is possible to minimize the required movement range and optimize the switching speed. In particular, the use of the transparent portion enables a short movement range.

[0023] In a more preferred embodiment, the modulation element is embedded in a planar parallel plate having an anti-reflective coating. That is, the substrate (of the modulation element) itself can be a planar parallel plate having an anti-reflective coating. This reduces reflections that could potentially interfere with light in imaging.

[0024] Another or additional means to reduce such reflections or their effects is when the modulation element (plate) is tilted with respect to the optical axis, particularly when the exit pupil image is in a parallel section of the beam path (though this is not necessarily required). Additionally or alternatively, the control device may be configured to tilt the modulation element, for example, by a specific arrangement of the modulation element holder. This allows for tilting only when necessary. Tilting the modulation element with respect to the optical axis means, in particular, that the modulation element is tilted by a value of 1° to 5° from a plane perpendicular to the optical axis, for example.

[0025] Preferably, the illumination unit includes a focusing lens aperture changer for generating each collimated illumination beam path for each aperture, and a control device is connected to the focusing lens aperture changer to illuminate the sample with a predetermined spatial illumination spectrum. This makes it possible to illuminate the specimen with a predetermined spatial illumination spectrum required for the contrast method. The focusing lens aperture changer includes, in particular, a focusing lens and focusing aperture diaphragm changer (i.e., a changer for changing the focusing lens and / or aperture diaphragm). This means that the condenser may have a fixed lens, while the aperture diaphragm may be changed (e.g., by changing the aperture diameter or other dimensions) to provide respective illumination (see also the further note above regarding illumination adaptation). However, generally, the focusing lens may also be adapted or changed. More sophisticated methods or means of aperture control, such as a DMD (Digital Micromirror Device) system, an LED (Light Emitting Diode) array, etc., can also be implemented.

[0026] In a more preferred embodiment, the control device is connected to the digital image detection module to detect intensity images for display, documentation, and / or quantitative reconstruction of sample phase modulation and / or sample amplitude modulation. See the above interpretation, particularly with respect to the reconstruction of phase modulation and / or amplitude modulation. Furthermore, the control device is preferably configured to determine at least one of the phase transfer function and amplitude transfer function of the microscope's optical system for reconstructing a quantitative phase image of the sample. When used in a calibrated position and with known spatial illumination distribution and design data of the optical system, the control device can calculate the imaging characteristics of the optical system, for example, in the form of phase transfer function and amplitude transfer function. If different imaging characteristics are realized, for example, two contrast methods with different settings of modulation contrast and brightfield or modulation contrast, the system can use the diversity of imaging characteristics and each transfer function to reconstruct a quantitative phase image of the sample.

[0027] Such reconstructions can be based on a forward imaging model that uses linear phase and amplitude transfer functions acting on the complex (phase and amplitude) refractive index distribution of the object (sample), yielding a number of intensity images for at least two different imaging configurations. These at least two relations can be coupled into a system of linear equations that can be solved for the real and imaginary parts of the refractive index distribution (where the number of pixels is a multiple of two unknown variables and at least two known variables). If noise is present and so-called Tikhonov-type regularization is used, this can be achieved by a suitable adaptation of the Wiener-filter reconstruction approach described in equation (11) of Michael Chen, Lei Tian, ​​and Laura Waller, 3D differential phase contrast microscopy, Biomed. Opt. Express 7, 3940-3950 (2016). Of course, more sophisticated reconstruction methods can be envisioned, as already suggested in the cited literature.

[0028] In summary, embodiments of the present invention make it possible to optimize the usefulness of the contrast method, particularly in automated multimodal microscope systems. This is achieved, in particular, by automated modulated contrast microscopes or their control devices (or methods), which automatically position the modulator in a plane conjugate to the exit pupil of the objective lens, and also take into account the mechanical movement of the objective lens by a focusing nosepiece, as is commonly used in inverted microscopes, and automatically remove the modulator for the beam path for brightfield or fluorescence imaging, enabling quantitative positioning of the modulator, and thus enabling quantitative prediction of image formation within the theory of partial interference imaging.

[0029] In particular, there is no need to manually insert the modulator into the microscope stand, nor is there any need to use or even provide a manual lever for movement along the optical axis. The user does not need to ensure that such a slider is inserted to position the correct pupil position modulator within the beam path, visual inspection is unnecessary, and manual movement of the modulator along the optical axis to subjectively homogenize the image impression across the field of view is not required. In addition, manual lateral movement of the modulator by the user to generate an image representation to subjective preferences is not required. In addition to avoiding manual user interaction, embodiments of the present invention enable quantitative evaluation of image data and continuous quantitative analysis of contrast data.

[0030] Furthermore, in multimodal microscope systems that include fluorescence imaging, when switching the imaging modality from contrast technology to fluorescence imaging, the user does not need to manually remove a slider. Rather, distortion of the fluorescence image point spread function is automatically prevented, and modulator attenuation is also eliminated. This will result in photon depletion situations, such as a decrease in the number of detected fluorescence photons and a decrease in the signal-to-noise ratio in fluorescence imaging.

[0031] Further advantages and embodiments of the present invention will become apparent from the description and accompanying drawings.

[0032] It should be noted that the features mentioned above and those further described below can be used not only in the combinations shown, but also in further combinations or individually, without departing from the scope of the present invention. [Brief explanation of the drawing]

[0033] [Figure 1] This figure schematically shows a microscope according to a preferred embodiment of the present invention. [Figure 2] This figure schematically shows a modulation element positioning device for a microscope according to a preferred embodiment of the present invention. [Figure 3a] This figure schematically shows different diagrams of the modulation element positioning device shown in Figure 2. [Figure 3b] This figure schematically shows different diagrams of the modulation element positioning device shown in Figure 2. [Figure 4a] This figure schematically shows a modulation element of a microscope according to a different preferred embodiment of the present invention. [Figure 4b] This figure schematically shows a modulation element of a microscope according to a different preferred embodiment of the present invention. [Figure 5a] This figure shows the amplitude and phase transfer functions for modulation contrast microscopy. [Figure 5b] This figure shows the amplitude and phase transfer functions for modulation contrast microscopy. [Figure 6] This figure shows the amplitude transfer function in a bright-field microscope. [Figure 7] This figure shows the steps performed by a microscope control device according to a preferred embodiment of the present invention. [Modes for carrying out the invention]

[0034] Figure 1 shows a microscope 100 according to a preferred embodiment of the present invention in a highly schematic manner. In particular, Figure 1 shows the beam path of the microscope 100 and related (optical) components of particular interest. Note that such a microscope 100 typically also includes a housing, microscope stage, etc., which are not shown for clarity.

[0035] The microscope 100 includes an objective lens 106 positioned on an objective lens changer 114 (also called a nosepiece or turret) via the objective lens changer 114. The objective lens 106 is positioned on the optical axis 120 of the microscope 100 and has an exit pupil 108. Further objective lenses (not shown) may be provided on or attached to the objective lens changer 114, in which case the objective lens changer 114 may be configured to position any one of the multiple objective lenses on the optical axis 120 (indicated by double-headed arrows).

[0036] In addition, a focus drive device 115 is provided, configured to move each objective lens 106, which is positioned on the optical axis 120, in the z-direction, which is parallel to the optical axis 120. The focus drive device 115 can be integrated with the objective lens changer 114.

[0037] Furthermore, the microscope 100 includes a lens system 102, exemplified by two lenses (relay system) for forming a real intermediate image 104 of the exit pupil 108 of the objective lens 106 currently positioned on the optical axis 120 (or any other). The intermediate image 104 is formed on each conjugate plane 124 conjugate to the exit pupil 108.

[0038] Furthermore, the microscope 100 includes an illumination unit 122 that generates an illumination beam path 120.3 for illuminating the sample 128 to be imaged. Each objective lens 106 positioned on the optical axis 120 generates an (ortho) imaging beam path 120.2 for imaging the sample 128. The image of the sample 128 is detected by a digital image detection module (detector) 112, which may be a camera or part of such a camera. Note that an eyepiece may be used (for example, further) as needed.

[0039] The lighting unit 122 includes a fixed focusing lens 117 having an aperture diaphragm. Furthermore, the lighting unit 122 includes a focusing lens aperture changer 126 for generating each collimated illumination beam path for each aperture. Next, the focusing lens aperture changer 126 includes a focusing aperture diaphragm changer 116 for changing the aperture diaphragm (in particular, its dimensions indicated by the double-headed arrow) in addition to the focusing lens 117.

[0040] Also note that the conoscopy imaging beam path 120.1 is shown. While the orthoimaging beam path 120.2 represents the beam for the objective lens 106 that images the sample 128 at infinity, the conoscopy beam path 120.1, as described above, represents the beam path that images the exit pupil 108 into the intermediate image 104.

[0041] Furthermore, the microscope 100 includes modulation elements 110 positioned on the optical axis 120, i.e., along the z-direction, at the positions of each conjugate plane 124 or within a predetermined range around the conjugate plane 124. The modulation elements 110 can be moved in a direction parallel to the optical axis 120 (z-direction) and in a direction lateral to the optical axis 120 (x-direction). The modulation elements are positioned on a modulation element positioning device 130, which will be described in more detail with reference to Figure 2.

[0042] Furthermore, the microscope 100 includes a control device 118 equipped with a processor (which may be a PC or a control unit built into the microscope) configured to automatically position the modulation element 110 on the optical axis 120 at the above-mentioned position. In particular, the control device 118 is electrically connectable to the element positioning device 130 to move and position the modulation element 110 (automatically) as needed.

[0043] In addition, the control device 118 is electrically connected to the focus drive device 115 to receive information about the axial position of each objective lens 106. This makes it possible to determine the required axial position of the modulation element 110. Furthermore, the control device 118 is electrically connected to the focusing lens aperture changer 126 to illuminate the sample with a predetermined spatial illumination spectrum. This may include controlling the focusing lens aperture changer 126 or its focusing aperture diaphragm changer 116 to change the aperture diameter (by opening and closing the aperture diaphragm).

[0044] Figure 2 shows a modulation element positioning device 130 for a microscope according to a preferred embodiment of the present invention in more detail than that shown in Figure 1. The modulation element positioning device 130 comprises a z-rail 200 and a z-slider 202. The z-slider 202 is positioned on the z-rail 200 so that it can move in the z-direction. A motor 204 and a drive shaft 212 are provided to move the z-slider and position it with extremely high precision in the z-direction.

[0045] Furthermore, the z-slider 202 includes an x-rail 206 and an x-slider 208. The x-slider 208 is positioned on the x-rail 206 so that it can move in the x-direction. A motor 210 and a drive shaft (not shown) are provided to move the x-slider and position it with very high precision in the x-direction. A modulation element 110 is positioned on the x-slider 208 (which may be tilted as described above). Both motors 204 and 210 can be connected to and controlled by the control device 118. The z-rail 200 can be fixed inside or positioned in the microscope 100.

[0046] The modulation element positioning device 130 is positioned relative to the optical axis 120 of the microscope 100 such that its z-direction coincides with or is parallel to the optical axis 120 (the z-direction shown in Figure 1). Therefore, the modulation element 110 is movable in the z-direction, i.e., parallel to the optical axis 120, and in the x-direction, i.e., laterally relative to the optical axis 120. Note that the modulation element 110 is particularly plate-shaped or embedded in a planar parallel plate having, for example, an anti-reflective coating. Such a plate is then positioned perpendicular to or slightly inclined to the optical axis 120 in order to reduce reflections.

[0047] Figures 3a and 3b show different views of the modulation element positioning device 130 of Figure 2, viewed from above along the optical axis 120 shown in Figure 2. In particular, Figure 3a shows the modulation element 110 positioned on the optical axis 120. This is visualized by image 300.1 of the exit pupil 108. As can be seen from the figure, a portion of the opaque or partially transparent section or region of the modulation element 110 (indicated by two parallel lines; see Figure 4a for a more detailed view) is located within image 300.1.

[0048] Figure 3b specifically shows the situation where the modulation element 110 is positioned outside the optical axis 120. As can be seen from the figure, all opaque and partially transparent sections or regions of the modulation element 110 (indicated by two parallel lines) are located outside the image, which is shown as 300.2. To move the modulation element 110 (or its opaque region) away from the imaging beam path (image 300.1 in Figure 3a), the x-slider is moved in the x-direction (as the lateral direction). Note that the modulation element 110 includes a (fully) transparent section, which is positioned so that the imaging beam path passes through the transparent section of the modulation element 110, based on a predetermined lateral displacement of the modulation element 110 away from its position on the optical axis 120.

[0049] Figures 4a and 4b schematically illustrate different types of modulation elements for a microscope according to different preferred embodiments of the present invention. These modulation elements are intended for use in the modulation contrast method. Figure 4a shows in more detail the modulation element 110, which is basically shown in Figures 3a and 3b. The modulation element 110 has the form of a rectangular plate and comprises an opaque section or region 400 (e.g., 100% absorption coefficient or 0% transparency), a partially transparent section or region 402 (e.g., 80% absorption coefficient or 20% transparency), and a (completely) transparent section or region 404 (e.g., 0% absorption coefficient or 100% transparency).

[0050] Image 300.1 shows an image of the exit pupil when the modulation element 110 is positioned on the optical axis, and Image 300.2 shows an image of the exit pupil when the modulation element 110 is positioned off the optical axis. In the latter case, the complete imaging beam path passes through the completely transparent section 404 (only). This means that the modulation element 110 (or its effect) is deactivated. To achieve this, the modulation element 110 is moved laterally off the optical axis (vertical direction in Figure 4a, x-direction in Figures 3a and 3b) by a predetermined lateral displacement. In the example shown in Figure 4a, such a predetermined lateral displacement corresponds to the distance between the centers of the two images 300.1 and 300.2, indicated by reference numeral d.

[0051] Figure 4b shows the modulation element 110 and an additional second or further modulation element 410. As an example, the two modulation elements 110 and 410 are provided as a single or combined modulation element 412, providing the modulation effect of modulation element 110 having regions or sections 400, 402 and 404. In addition, the modulation element also provides another modulation effect through a partially transparent section or region 406 (e.g., 80% absorption coefficient or 20% transparency) and an opaque section or region 408 (e.g., 100% absorption coefficient or 0% transparency).

[0052] The absorption coefficient may be the same as that of the modulation element 110, but these regions or sections 406, 408 are rotated 90° with respect to each section or region 400, 402 and arranged in reverse order. In this way, the modulation contrast provided by the modulation element 410 is rotated 90° with respect to the modulation element 110.

[0053] To position the modulation element 410 on the optical axis, the combined modulation element 412 is further moved laterally (vertically in Figure 4b) so that the exit pupil image 300.3 is achieved. The modulation element positioning device is then able to achieve the required lateral displacement. These two modulation elements 110 and 410 can also be provided as two separate elements or plates. The difference between the two modulation elements can include, for example, the strength of the contrast with an appropriate aperture.

[0054] Figures 5a and 5b show the amplitude transfer function 500 (Figure 5a) and phase transfer function 502 (Figure 5b) for modulated contrast microscopy and imaging. In both cases, the contrast C (in arbitrary units) is the vertical versus the horizontal (k x ,k y ) direction (μm -1 These functions are shown in ). These functions can be determined or calculated by the control unit from each data and detected image of the entire system, making it possible to reconstruct a quantitative phase image of the captured sample. The frequency space range of the phase transfer function for quantitative phase reconstruction can be improved by using a modulator arrangement such as the one shown as 410 (or the combined element 412) in Figure 4b.

[0055] Figure 6 shows the amplitude transfer function 600 in bright-field microscopy. The contrast C (in arbitrary units) is the vertical-to-horizontal ratio (k). x ,k y ) direction (μm -1 This is shown in ). Similar to the transfer function for modulation contrast, this function can be determined or calculated by the control unit from each data and detected image of the entire system, making it possible to reconstruct a quantitative phase image of the acquired sample.

[0056] The transfer functions in FIGS. 5a, 5b, and 6 have the role of the transfer function H that correlates the complex refractive index distribution of the object with the intensity image detected by the system (see also the above interpretation regarding Michael Chen, Lei Tian, and Laura Waller, 3D differential phase contrast microscopy, Biomed. Opt. Express 7, 3940 - 3950 (2016)). Since forward image formation is usually formulated as a convolution operation, it is common to show the Fourier amplitude of each transfer function as in each figure.

[0057] The modulation contrast phase transfer function 502 (FIG. 5b) converts the phase change of the object (k y > 0 or k y <0) into positive or negative intensity modulation and converts the amplitude (absorption rate) of the object into negative intensity modulation of the image. It can be easily seen that the phase transfer function for transmitted bright - field imaging is zero everywhere and is thus omitted. As described above, the range of the modulation element, for example, the overall phase transfer function of the system, can be improved by using element 410. With the help of these transfer functions, a forward model of the complete imaging system can be formulated and finally inverted as described above.

[0058] As can be seen from FIG. 5b, there is a line where the magnitude of the phase transfer function disappears at k y = 0. That is, the phase structure in this direction does not generate any contrast in the image. This is not attractive for reconstruction. With the help of the second position, the modulator 410 can rotate this line (by the opaque and partially transparent regions rotated by 90°) to k x = 0, so that all frequency components except k x = k y = 0 are covered. This makes it possible to generate a significantly better reconstruction.

[0059] Figure 7 shows the steps performed by a microscope control device according to a preferred embodiment of the present invention, for example, the control device 118 shown in Figure 1, to position or move a modulation element.

[0060] In step 700, the control unit receives information I1 from the objective lens changer indicating that the objective lens is currently positioned on the optical axis. In step 702, the control unit receives information I2 from the focus drive unit regarding the axial position of the objective lens currently positioned on the optical axis. In step 704, the control unit combines the received information with further data D C For example, based on data from the optical components of a microscope, calibration data, etc., the axial displacement d of each conjugate surface is calculated. z To decide.

[0061] In step 706, the control device determines the axial displacement d z is a predetermined threshold d' z Check if it exceeds the limit, and if it does, in step 708, the axial displacement d z The modulation element is moved in the z-direction. This specifically involves controlling the modulation element positioning device and its motor.

[0062] In a further step 710, if deactivation of the modulation element is required, the control device may move the modulation element out of the optical axis by moving it by a predetermined lateral displacement d (as described above).

[0063] As used herein, the term "and / or" includes all possible combinations of one or more of the items listed herein and may be abbreviated as " / ".

[0064] While several embodiments have been described in the context of the apparatus, it is clear that these embodiments also represent descriptions of the corresponding methods, where blocks or apparatus correspond to steps or features of steps. Similarly, embodiments described in the context of steps also represent descriptions of the corresponding blocks, items, or features of the corresponding apparatus.

[0065] Some embodiments relate to microscopes equipped with control devices as described in relation to one or more of Figures 1 to 7. Alternatively, the microscope may be part of a system as described in relation to one or more of Figures 1 to 7, or may be connected to a system as described in relation to one or more of Figures 1 to 7. Figure 1 shows a schematic diagram of a microscope 100 having a control device (system) 118 configured to carry out the methods described herein. The microscope 100 comprises a control device (computer system) 118. The microscope 100 is configured to take images and is connected to the computer system 118. The computer system 118 is configured to carry out at least a portion of the methods described herein. The computer system 118 may be configured to run machine learning algorithms. The computer system 118 and the microscope 100 may be separate entities, or they may be integrated within a single common housing. The computer system 118 may be part of the central processing system of the microscope 100, and / or the computer system 118 may be part of the dependent components of the microscope 100, such as sensors, actors, cameras, or lighting units.

[0066] The computer system 118 may be a local computer device (e.g., a personal computer, laptop, tablet computer, or mobile phone) comprising one or more processors and one or more storage devices, or it may be a distributed computer system (e.g., a cloud computing system comprising one or more processors and one or more storage devices distributed to various locations such as local clients and / or one or more remote server farms and / or data centers). The computer system 118 may include any circuit or combination of circuits. In one embodiment, the computer system 118 may include one or more processors, which may be of any kind. As used herein, the processor may be intended to be any kind of computing circuit, such as a microprocessor for a microscope or microscopic component (e.g., a camera), a microcontroller, a composite instruction set computing (CISC) microprocessor, a reduced instruction set computing (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, a graphics processor, a digital signal processor (DSP), a multicore processor, a field-programmable gate array (FPGA), or any other kind of processor or processing circuit. Other types of circuits that may be included in the computer system 118 may be custom circuits, application-specific integrated circuits (ASICs), etc., such as one or more circuits (communication circuits, etc.) used in wireless devices such as mobile phones, tablet computers, laptop computers, two-way radios, and similar electronic systems. The computer system 118 may also include one or more storage devices that may include one or more memory elements suitable for a particular application, such as main memory in the form of random access memory (RAM), one or more hard drives and / or one or more drives that handle removable media such as compact discs (CDs), flash memory cards, digital video discs (DVDs), etc.The computer system 118 may also include a display device, one or more speakers and a controller which may include a keyboard and / or mouse, trackball, touchscreen, voice recognition device, or any other device which enables a user of the system to input information into and receive information from the computer system 118.

[0067] Some or all of the steps may be performed by a hardware device (or by using a hardware device), such as a processor, microprocessor, programmable computer, or electronic circuit. In some embodiments, one or more of the most important steps may be performed by such a device.

[0068] Depending on certain implementation requirements, embodiments of the present invention may be implemented in hardware or software. This implementation is feasible using a non-transient recording medium, which is a digital recording medium, etc., that stores electronically readable control signals and cooperates (or can cooperate) with a programmable computer system to carry out each method. Examples include floppy disks, DVDs, Blu-rays, CDs, ROMs, PROMs and EPROMs, EEPROMs, or FLASH memory. Thus, the digital recording medium may be computer-readable.

[0069] Some embodiments of the present invention include a data carrier having electronically readable control signals that can cooperate with a programmable computer system so as to carry out any of the methods described herein.

[0070] Generally, embodiments of the present invention can be implemented as a computer program product comprising program code, which operates to perform one of the methods when the computer program product is executed on a computer. This program code may be stored, for example, on a machine-readable carrier.

[0071] Another embodiment includes a computer program stored in a machine-readable carrier for carrying out any of the methods described herein.

[0072] Therefore, in other words, embodiments of the present invention are computer programs having program code for carrying out any of the methods described herein when the computer program is executed on a computer.

[0073] Accordingly, another embodiment of the present invention is a recording medium (or data carrier or computer-readable medium) containing a stored computer program for carrying out any of the methods described herein when executed by a processor. The data carrier, digital recording medium, or recording medium is typically tangible and / or non-transient. Another embodiment of the present invention is an apparatus, such as those described herein, comprising a processor and a recording medium.

[0074] Therefore, another embodiment of the present invention is a data stream or signal sequence representing a computer program for carrying out any of the methods described herein. The data stream or signal sequence may be configured to be transmitted, for example, over a data communication connection, such as the Internet.

[0075] Another embodiment includes processing means, for example, a computer or programmable logic device configured or adapted to carry out any of the methods described herein.

[0076] Another embodiment includes a computer having an installed computer program for carrying out any of the methods described herein.

[0077] Another embodiment of the present invention includes an apparatus or system configured to transfer (e.g., electronically or optically) a computer program for carrying out any of the methods described herein to a receiver. The receiver may be, for example, a computer, a mobile device, a storage device, etc. The apparatus or system may include, for example, a file server for transferring the computer program to the receiver.

[0078] In some embodiments, a programmable logic device (e.g., a field-programmable gate array) may be used to perform some or all of the functions of the methods described herein. In some embodiments, the field-programmable gate array may cooperate with a microprocessor to carry out any of the methods described herein. Generally, the methods are advantageously carried out by any hardware device. [Explanation of symbols]

[0079] 100 Microscopes 102 Lens System 104 Intermediate image 106 Objective lens 108 Exit pupil 110 modulation elements 112 Digital Image Detection Module 114 Objective Lens Changer 115 Focusing mechanism 116. Light-gathering aperture changer 117 Focusing lens 118 Control device 120 Optical axis 120.1 Conoscope imaging path 120.2 Orthorectal imaging path 120.3 Lighting beam path 122 Lighting Unit 124 Conjugate surfaces 126 Focusing Lens Aperture Changer 128 samples 130 Modulation element positioning device 200 z-rail 202 z-slider 204,210 motors 206 x-rail 208 x-slider 212 Drive shaft 300.1, 300.2, 300.3 Images of the exit pupil 400,408 Opaque sections 402,406 Partially transparent sections 404 Transparent Section 500, 502, 600 transfer functions d. predetermined lateral displacement d z Displacement of axial position d' z Threshold of displacement at a predetermined axial position C Contrast D c data I1, I2 Information x, y, z, k x ,k y direction 700-708 steps

Claims

1. A microscope (100) for imaging a sample at least by a transmitted light contrast method, wherein the microscope (100) comprises: an objective lens holder or changer (114) configured to dispose an objective lens (106) among objective lenses or a plurality of objective lenses on an optical axis (120) of the microscope (100); a lens system (102) for forming an intermediate image (104) formed on each conjugate plane (124) conjugate to an exit pupil (108) of any one of the objective lenses (106) disposed on the optical axis (120); a control device (118) configured to automatically position a modulation element (110) at a position of each conjugate plane (124) or on the optical axis (120) associated with each conjugate plane (124); The microscope (100) comprising the above.

2. The control device (118) is configured to automatically move the modulation element (110) in an axial direction (z) parallel to the optical axis (120). The microscope (100) according to Claim 1.

3. The control device (118) is connected to the objective lens holder or changer (114) to receive information (I １ ) about each objective lens (106) arranged on the optical axis (120). The microscope (100) according to Claim 1.

4. The control device (118) is connected to the focus driving device (115) of the microscope (100) in order to receive information (I ２ ) about the axial position of each of the objective lenses (106), and the focus driving device (115) is configured to move each of the objective lenses (106) disposed on the optical axis (120) in the axial direction (z) parallel to the optical axis (120). The microscope (100) according to Claim 1.

5. The control device (118) moves the modulation element (110) in the axial direction only when the displacement (d ｚ ) of the axial position of each conjugate plane (124) exceeds a predetermined threshold value (d' ｚ ). The microscope (100) according to Claim 1.

6. The control device (118) is configured to automatically move the modulation element (110) laterally with respect to the optical axis (120). The microscope (100) according to Claim 1.

7. The control device (118) is configured to determine a lateral displacement of the modulation element (110) according to quantitative image quality analysis. The microscope (100) according to Claim 6.

8. The quantitative image quality analysis includes at least one of quantitative reproducibility of image impression and quantitative reproducibility of contrast transfer function. The microscope (100) according to Claim 7.

9. The control device (118) is configured to replace a first modulation element (110) with another second modulation element (410) by a lateral movement of the first modulation element (110) outside the optical axis (120) and a lateral movement of a second modulation element (410) onto the optical axis (120). The microscope (100) according to Claim 1.

10. The control device (118) is further configured to adapt illumination of the microscope (100) according to the second modulation element. The microscope (100) according to Claim 9.

11. The microscope (100) includes an illumination unit (122) that generates an illumination beam path for illuminating the sample to be imaged. Each objective lens (106) disposed on the optical axis (120) generates an imaging beam path for imaging the sample. The microscope (100) according to claim 1.

12. The modulation element (110) includes a transparent section arranged such that when a predetermined lateral displacement (d) of the modulation element (110) deviates from its position on the optical axis, the imaging beam path passes through the transparent section of the modulation element (110). The microscope (100) according to claim 11.

13. The control device (118) is configured to laterally move the modulation element (110) by the predetermined lateral displacement (d) to deactivate the modulation element (110). The microscope (100) according to claim 12.

14. The modulation element (110) is embedded in a plane-parallel plate having an anti-reflection coating. The microscope (100) according to claim 1.

15. The modulation element (110) is inclined with respect to the optical axis (120). The microscope (100) according to claim 1.

16. The illumination unit includes a condenser lens aperture changer (126) for generating a collimated illumination beam path for each aperture. To illuminate the sample with a predetermined spatial illumination spectrum, the control device (118) is connected to the condenser lens aperture changer (126). The microscope (100) according to claim 11.

17. The condenser lens aperture changer (126) includes a condenser lens (117) and a condenser aperture diaphragm changer (116). The microscope (100) according to claim 16.

18. The microscope (100) further includes a digital image detection module (112) for detecting an image of the sample to be imaged. The microscope (100) according to claim 1.

19. The control device (118) is connected to the digital image detection module (112) at least for detecting intensity images for the display, documentation, quantitative reconstruction of the standard phase modulation, and quantitative reconstruction of the sample amplitude modulation. The microscope (100) according to claim 18.

20. To reconstruct the quantitative phase image of the sample, the control device (118) is configured to determine at least one of the phase transfer function (502) and the amplitude transfer functions (500, 600) of the optical system of the microscope (100). The microscope (100) according to claim 1.

21. The microscope (100) further includes a modulation element positioning device (130) including at least one slider (202, 208) configured to carry the modulation element (110), and the at least one slider is configured to move in at least one of a direction parallel and transverse to the optical axis (120). The microscope (100) according to claim 1.