Interference reflection microscope and method for analysing a sample

Abstract

The invention relates to an interference reflection microscope which is operated with a light source which is spatially coherent and temporally incoherent. As a result, the formation of speckle patterns can be avoided and filters for filtering out such speckle patterns are no longer required. The invention also relates to an associated method.

Description

[0001] Interference reflection microscope and method for analyzing a sample

[0002] The invention relates to an interference reflection microscope and a method for analyzing a sample using such an interference reflection microscope.

[0003] Microscopes are generally used for various types of sample examination or observation. One possible type of microscope is an interference reflection microscope, which typically illuminates a sample with excitation light and uses the reflected light for analysis. Due to interference effects, information content can arise in the light, allowing, for example, photometric mass spectrometry of molecules.

[0004] An example implementation is disclosed in Young et al., Science 360, 423–427 (2018) and the accompanying supplementary material. This implementation uses a central obstruction filter to suppress speckle patterns.

[0005] It is an object of the invention to provide an interference reflection microscope that is alternative to or better than known designs. It is also an object of the invention to provide a corresponding method for analyzing a sample.

[0006] This is achieved according to the invention by an interference reflection microscope and a method according to the respective main claims. Advantageous embodiments can be found, for example, in the respective subclaims. The content of the claims is incorporated into the content of the description by express reference.

[0007] The invention relates to an interference reflection microscope. The interference reflection microscope comprises a light source, illumination optics, a sample arrangement, detection optics, and a detector. The illumination optics direct light from the light source as excitation light onto the sample arrangement. The detection optics direct light reflected from the sample arrangement onto the detector. It is expedient for the light source to be spatially coherent. It is also expedient for the light source to be temporally incoherent.

[0008] The design of such an interference reflection microscope offers the advantage that the temporal incoherence of the light source prevents the formation of speckle patterns in the reflected light. Such patterns typically arise as interference phenomena due to unavoidable roughness, contamination, or chemical heterogeneity of a surface of the sample arrangement and / or the illumination or detection optics. In known designs, they typically significantly interfere with the analysis, which is why they are filtered out using strong filters in the beam path. While such filters allow for better analysis without significant interference from the speckle patterns, they also result in a significant portion of the light reflected back from the sample being lost.This, in turn, means that with conventional designs, considerably higher radiance must be used to maintain sufficient light intensity for analysis even after passing through a filter. Such high light outputs can lead to bleaching or destruction, particularly when examining organic molecules. With the interference reflection microscope design presented here, however, it is possible to significantly reduce the power density on a sample, which in turn allows for a longer exposure time without damaging the molecules.

[0009] An interference reflection microscope is basically a microscope in which a sample is illuminated and in which reflected and / or scattered light is collected and evaluated, in particular in a direction that is at least approximately opposite to the illuminating light. This light generally contains information that arises due to interference effects. Such interference effects can occur in particular due to interface transitions, for example between glass and liquid or between liquid and sample, or due to distances between sample and glass. Instead of glass, a transparent plastic can also be used, for example. The sample can be a molecule, in particular an organic molecule and in particular a molecule with a mass of, for example, several kDa (kilodaltons). Several such molecules can also be used.

[0010] A characteristic of an interference reflection microscope is that the interference occurs at the sample or at least in the immediate vicinity of the sample, for example, at a distance of at most 100 nm, at most 1 m, at most 10 pm, at most 100 pm, or at most 1 mm from the sample, for example, at an interface between glass and liquid or between liquid and sample. This distinguishes an interference reflection microscope from microscope designs that have a separate path into which light from a main light beam is coupled, which then interferes with light reflected from the sample. Such separate paths are typically not present in interference reflection microscopes.

[0011] The light source generates the excitation light. The illumination optics direct the excitation light onto the sample arrangement. A Köhler arrangement, for example, can be used for this. The sample arrangement can, in particular, be an arrangement that can hold and / or provide the sample. For example, the sample arrangement can be a vessel in which a liquid can be absorbed, whereby one or more molecules can be present in the liquid. Such molecules can be referred to as a sample. They can, in particular, be examined using an interference reflection microscope. Alternatively, a sample arrangement can, for example, also represent the sample itself. In this case, such a sample, such as a solid object, can be examined directly.

[0012] The detection optics direct the light reflected and / or scattered by the sample arrangement to the detector. This typically generates a signal indicative of the intensity or intensities of the light incident on the detector.

[0013] For example, the detector can be a two-dimensional detector divided into pixels. This allows the acquisition of a two-dimensional image, which can be evaluated appropriately. Regarding the structure of the optics and other components of the interference reflection microscope, reference is made in particular to the description of an exemplary embodiment below with reference to the accompanying drawing.

[0014] In particular, the light source can be temporally incoherent in that a value of a coherence length is at most 100 pm, at most 75 pm or at most 50 pm. The coherence length can be defined in particular on the basis of the spectrum of the light source. In particular, the coherence length can be defined as the maximum wavelength squared divided by the half-width in the spectrum of the light source. Such a definition has proven to be a useful definition of a coherence length for assessing temporal coherence or incoherence. With the specified values, the light source can be referred to as temporally incoherent. The values ​​have proven to be particularly advantageous for the application at hand. Even with temporally incoherent light sources, the coherence length is typically not exactly zero, but is, for example, at least one wavelength.

[0015] In particular, a half-width can be defined as the width in the spectrum that connects two points at which the intensity, seen from a maximum, is half that of the maximum.

[0016] In particular, the light source can be a superluminescent light-emitting diode (SULED). Such SLEDs generate spatially coherent and temporally incoherent light, which has proven advantageous for the present application for the reasons already mentioned. In principle, however, the use of other light sources is also possible, provided they meet the required properties.

[0017] Typically, the light source is not a laser light source. Laser light sources are typically resonant feedback optical amplifiers that generate light based on stimulated emission. Laser light sources are typically not suitable for the application relevant here. In particular, the light source can be spatially coherent if a value of an area over which the emitted light has a fixed phase relationship is at least 1 pm. 2 , at least 5 pm 2 , at least 10 pm 2 , at least 100 pm 2 , at least 250 pm 2and / or the total radiating area of ​​the light source. This has proven to be a useful definition of a coherence surface for defining a spatially coherent light source. The specified values ​​can be assumed to be a spatially coherent light source that delivers the required performance and enables the necessary light power density to illuminate the sample.

[0018] It should be understood that the given definitions of temporal incoherence and spatial coherence can also be understood as alternatives to the formulations used in claim 1 of the application.

[0019] Preferably, no attenuating filter is present in the beam path from the sample arrangement through the detection optics to the detector. An attenuating filter is understood to be an element whose task is to attenuate the light beam, in particular to attenuate it significantly, for example by at least one, two, or three orders of magnitude. Other optical elements such as lenses, which for technical reasons never have 100% transmission, or beam splitters, which serve to split a beam and thus naturally only allow a portion of the incident light to pass through in a specific direction, are not considered attenuating filters.By eliminating the need for an attenuating filter, a significantly higher proportion of the reflected light reaches the detector, allowing a lower power density to be used for excitation. This allows for longer exposure times without damaging samples.

[0020] In particular, in the beam path from the sample arrangement through the detection optics to the detector, at least 10%, at least 25%, at least 50%, at least 80% or at least 90% of the light reflected by the sample arrangement can reach the detector. Such values ​​can typically be achieved if, for example, an attenuating filter is omitted from this beam path. The positive effects described above can be achieved through the high transmission from the sample arrangement to the detector. The values ​​mentioned are also realistic, for example, if there is a beam splitter in the beam path from the sample arrangement to the detector, which inevitably does not allow part of the light to pass through to the detector but redirects it in another direction.

[0021] If a proportion of light is mentioned here, this may in particular refer to the power of the light which is transmitted, for example, at least to the stated proportion.

[0022] According to one embodiment, the interference reflection microscope has a beam splitter. The illumination optics can, in particular, direct the light from the light source onto the beam splitter, and the beam splitter can, in particular, direct the light at least partially onto the sample arrangement. The beam splitter can, in particular, direct the light reflected from the sample arrangement at least partially onto the detection optics. Such a beam splitter enables a simple and practical design of an interference reflection microscope, since the beam splitter enables superposition of the excitation light beam and the reflected light.

[0023] The light source and / or the illumination optics can, in particular, direct the light from the light source onto the beam splitter in a polarized manner. In particular, this can be a linear polarization. The beam splitter can, in particular, be polarization-sensitive such that it directs at least 70%, at least 80%, or at least 90% of the polarized light from the light source onto the sample arrangement. By using a polarization-sensitive beam splitter and polarized light from the light source, it can be achieved, in particular, that a higher proportion of the light source hits the sample arrangement than when using a 50% reflecting and transmitting beam splitter. As a result, less light from the light source is lost due to the splitting in the beam splitter.

[0024] In particular, the reflected light from the sample arrangement can be polarized and impinge on the beam splitter. The beam splitter can in particular be polarization-sensitive in such a way that it directs at least 70%, at least 80%, or at least 90% of the polarized light reflected from the sample arrangement onto the detection optics. As a result, a polarization-sensitive design of the beam splitter in the beam path from the sample arrangement to the detector can also ensure that less light is directed by the beam splitter in a direction in which it cannot be used, and a higher proportion of the light is passed through to the detector. The polarization of the light reflected from the sample arrangement and impinging on the beam splitter can in particular be oriented transversely, for example at an angle of 90° or at an angle between 85° and 95°, to the polarization of the light impinging on the beam splitter from the light source.This can be achieved, for example, by using a 4x plate between the beam splitter and the sample arrangement.

[0025] In particular, it can be provided that the illumination optics between the light source and the beam splitter and the detection optics between the beam splitter and the detector are designed completely separately from each other. A completely separate design can be understood, in particular, to mean that the respective optical components such as mirrors, lenses, or apertures are used only for one of the two optics. Joint mounting on an optical table or in a holder does not preclude this. It can also be provided that the illumination optics are designed completely separately from the detection optics.

[0026] In particular, it can be provided that a beam path in the illumination optics between the light source and the beam splitter and a beam path in the detection optics between the beam splitter and the detector are completely separate from one another. This can mean, in particular, that the beams do not overlap in the said area and that a beam in one optic is not generated from a beam of the other optic. Typically, the beam splitter is the first optical element after the light source or the last optical element before the detector, which is struck by both the beam of the excitation optics and the beam of the detection optics. It can also be provided that a beam path in the illumination optics and a beam path in the detection optics are completely separate from one another.

[0027] The interference reflection microscope can, in particular, have an objective lens. This objective lens can be positioned optically directly in front of the sample arrangement. In particular, it can be positioned between the beam splitter and the sample arrangement. Using such an objective lens, the light can be specifically directed onto the sample arrangement and structured. Reflected light can also be specifically captured.

[0028] For example, the objective lens can collimate the excitation light toward the sample array. In this case, the sample array is illuminated with an excitation light whose diameter remains constant, at least over the relevant range. Alternatively, the objective lens can also focus the excitation light onto the sample array.

[0029] In particular, the illumination optics can focus the excitation light. A focus can be located in the beam path between the illumination optics and the objective. In particular, the focus can be located in a rear conjugate plane of the objective. Typically, after this focus, the beam then expands again before hitting the objective and being processed in the objective in the manner intended for the sample arrangement.

[0030] The illumination optics and / or the beam splitter can, in particular, direct the excitation light onto the lens in such a way that it is incident parallel to an optical axis of the lens. The optical axis can, in particular, be an axis of symmetry of the lens. It can, in particular, be arranged centrally in a cross-section in the lens. By parallel incidence of the light onto the lens, it can, in particular, be achieved that the beam also exits the lens parallel to this optical axis. It should be understood that “parallel” here also includes “identical”. The excitation light can therefore also be incident on the lens in such a way that a center point of the excitation light lies on the optical axis of the lens.

[0031] According to one embodiment, the illumination optics and / or the beam splitter can direct the excitation light onto the objective lens in such a way that a center point of the excitation light is incident adjacent to the optical axis of the objective lens. It does not coincide with the optical axis. It can also be said that the center point of the excitation light is spaced from the optical axis of the objective lens, i.e., has a non-zero distance. This allows asymmetric illumination of the objective lens. The excitation light can, in particular, exit the objective lens at an angle to the optical axis of the objective lens. This enables illumination of the sample arrangement at an angle to the optical axis. In particular, the excitation light can strike a surface of the sample arrangement at an angle, be reflected by the sample arrangement, and be reflected back to the objective lens.Such a reflected beam is then typically oblique to the surface of the sample arrangement. It is also typically oblique to the optical axis of the objective.

[0032] The excitation light can be arranged to strike the surface of the sample arrangement at an angle of at least 0°, greater than 0°, at least 1°, at least 5°, at least 10°, at least 20°, or at least 40°. The excitation light can be arranged to strike the surface of the sample arrangement at an angle of at most 1°, at most 5°, at most 10°, at most 20°, at most 40°, or at most 80°. A suitable interval can be formed from each lower value using each larger upper value. A normal to the surface typically serves as a reference for defining the angle. The angle is therefore typically specified relative to the surface normal. An angle of 0° therefore corresponds to a non-oblique incidence. The excitation light can also strike the surface of the sample arrangement at the Brewster angle. This allows unwanted reflections to be avoided as best as possible.The Brewster angle depends on the materials used, especially the material of the sample assembly, which is typically glass. However, it can be easily calculated using the corresponding formula.

[0033] According to one embodiment, it can be provided that the light reflected at the sample arrangement strikes the objective at a different point on the objective than the point at which the excitation light exits the objective towards the sample arrangement.

[0034] The designs just described allow for oblique illumination of the sample arrangement. This ensures that the light reflected from the sample arrangement contains less directly reflected excitation light and more light that has interacted with the sample. This further increases the information content of the detected light.

[0035] According to one embodiment, the excitation light can exit the objective parallel to the optical axis and / or can impinge on a surface of the sample arrangement perpendicular to this surface. The light can be reflected back, for example, opposite to the excitation light, i.e., with an offset of exactly or at least approximately 180°, for example, at an angle of at least 170° and / or at most 190°.

[0036] In particular, the objective lens may have a numerical aperture of at least 1.1, at least 1.2, at least 1.3, or at least 1.4. Such numerical apertures have proven advantageous for typical interference reflection microscopes.

[0037] The sample arrangement can, for example, comprise a vessel for a liquid, wherein the vessel is optically transparent at least to the extent that the light directed onto the sample arrangement can penetrate and light reflected within the liquid can escape. As a result, a sample in the form of one or more molecules can be present within such a liquid, for example, and such molecules can be observed. The vessel can, for example, be made of glass or a transparent plastic. The liquid can, for example, be present statically or it can be conducted through the vessel as part of a flow.

[0038] According to one embodiment, the light source comprises a substrate and a waveguide that is inclined relative to the substrate. The inclined waveguide can, for example, have two parallel outer surfaces that are inclined relative to a surface of the substrate. This allows spatially incoherent light to be generated.

[0039] For example, the waveguide can be coated with an antireflective coating on two opposite sides. This prevents the formation of modes in the waveguide that would lead to temporally coherent light. In particular, the light source can be designed without a resonator. This can also prevent the formation of modes that would lead to temporally coherent light.

[0040] In particular, the light source cannot be a laser light source. Laser light is typically temporally and spatially coherent, making it unsuitable for the intended application.

[0041] For example, the light source's light output can be a maximum of 10 mW, a maximum of 20 mW, a maximum of 100 mW, or a maximum of 1,000 mW. These light outputs can be used for typical applications, but other light outputs are also possible.

[0042] According to an advantageous embodiment, the light source has a radiating surface of at most 1 pm 2 , maximum 5 pm 2 , maximum 10 pm 2 , maximum 50 pm 2 , maximum 100 pm 2 , maximum 500 pm 2 or a maximum of 1,000 pm 2 Such sizes of radiating surfaces of the light source have proven advantageous for the present design.

[0043] In particular, the light source can illuminate the sample with a power density of not more than 1,000 kW / cm 2 , maximum 500 kW / cm 2 , maximum 100 kW / cm 2 , maximum 50 kW / cm 2 , maximum 10 kW / cm 2 , maximum 2 kW / cm 2 , maximum 1 kWcm 2 or a maximum of 0.5 kW / cm 2 illuminate. Such low power densities at the sample can be used with the design provided here, since, as already mentioned, a filter in the beam path to the detector can be omitted. In other words, a particularly high transmission from the sample arrangement to the detector can be used, which enables the use of such low power densities. The low power densities, in turn, create the advantage that a longer exposure time is possible. The power density is specified specifically at the sample.

[0044] In particular, the detector can be a 2D camera or a 1D camera. It can be implemented, for example, as a CCD detector and / or a CMOS camera.

[0045] Such designs have proven advantageous for typical applications. Typically, the detector is two-dimensional, allowing a two-dimensional pattern or interference pattern to be advantageously detected. This can be used, for example, for photographic mass spectrometry. However, depending on the application, a one-dimensional detector design may also be sufficient. Typically, the detector has several pixels, each measuring a light intensity.

[0046] The invention further relates to a method for analyzing a sample using an interference reflection microscope. In particular, this can be an interference reflection microscope as described herein. With regard to the interference reflection microscope, all embodiments and variants described herein can be used. The method comprises, in particular, the following steps:

[0047] Introducing the sample into the sample arrangement or as a sample arrangement, and illuminating the sample using the light source, while recording at least one image using the detector.

[0048] Such a method enables advantageous analysis of a sample using the interference reflection microscope described herein. The advantages already described can also be achieved with the method.

[0049] In particular, multiple images can be recorded using the detector. After recording each image, a difference image can be generated between this image and a previously recorded image or an averaged image. An averaged image can be generated, for example, as a mean or median across multiple previously recorded images. With an average, an average is typically generated for each pixel using the intensity values ​​of the images to be averaged, particularly for the same pixels. In other words, an average can be calculated for each pixel using the respective intensity values ​​or other measured values. This can be an arithmetic mean or a geometric mean.With a median, a mathematical mean is not typically generated; rather, a value is determined at which the respective intensity value for the respective pixel is below this value in half of the images to be averaged and above this value in the other half. Averaged images of this type can be used in particular to provide a basis for comparison with a previously recorded image. Noise effects are averaged out in images averaged in this way. This can be achieved by either averaging or median calculation. By generating difference images, it is particularly possible to detect a change compared to previous images. This can, for example, make it possible to detect molecules that have recently landed on a surface. These can be measured, and then, for example, it can be observed when they move away from the surface again.

[0050] The sample can, for example, be one molecule or can comprise multiple molecules. In particular, these can be organic molecules that can be dissolved in a solution. The molecule or molecules can, for example, each have a mass of at least 2 kDa, at least 3 kDa, or at least 10 kDa. Such molecules can be analyzed particularly advantageously using the method described herein and / or using the interference reflection microscope described herein.

[0051] In particular, the mass of one or more molecules can be determined based on one or more images or difference images. Automated image analysis can be used for this purpose. This can be based, for example, on artificial intelligence, neural networks, and / or machine learning. The advantages of the design described herein enable longer and non-destructive illumination.

[0052] In particular, several images can be captured consecutively using the detector. These images can then be used to create a video. Such a video is typically a sequence of several consecutively captured images. This allows, for example, molecules to be observed landing on and / or moving away from a surface. A change in molecular orientation can also be observed.

[0053] The illumination of the sample and / or the acquisition of one or more images can, for example, take place over a continuous period of at least one minute, at least two minutes, at least five minutes, at least ten minutes, or at least twenty minutes. Such periods are typically possible using the embodiment described herein, since the power density of the sample illumination can be significantly reduced compared to known embodiments. In particular, the previously described power densities or other data of the light source or the interference reflection microscope can be used for this purpose.

[0054] In particular, the embodiments described herein can be used for simultaneous localization measurements and / or mass photometry. For example, they can be used to investigate biomolecules such as proteins or nanoparticles. For example, the embodiments described herein can dispense with a marker that is otherwise typically used for nanoparticles or proteins. For example, single-molecule detection, molecular weight determination, or other evaluation can be performed.

[0055] The light source can, in particular, be linearly polarized. This can be used, as already explained above, especially in conjunction with a polarization-sensitive beam splitter.

[0056] Spatial coherence typically allows light to be collimated and focused like a laser. Temporal incoherence prevents speckle patterns. Polarization enables efficient use of the light power. For this purpose, a polarization-sensitive beam splitter can be used, for example, as described above. The excitation light can have a wavelength of 450 nm, for example. Any other wavelength that is suitable for the respective purpose can also be used. The light can be collimated, focused or expanded using several lenses, for example, and cut off at two irises, for example, before it falls on a sample behind an objective. Appropriate use of waveplates (λ / 2 or λ / 4) and a polarization beam splitter can reduce the loss of light power.The incident light can be reflected by sample glass surfaces and / or biomolecules or nanoparticles, interfere with the image, and travel via the detection path to the detector or camera. For example, frame rates of more than 100 frames per second, more than 500 frames per second, more than 1,000 frames per second, more than 2,000 frames per second, and / or up to 2,000 frames per second, up to 5,000 frames per second, or up to 10,000 frames per second can be recorded. Such images can then be partially averaged to create differential images. The image contrast is typically proportional to the molecular weight of the sample. Thus, for example, molecules or particles with a molecular weight of a few MDa up to the specified values ​​can be detected and localized.If the molecules are moving, they can be tracked for several minutes, as only very low light power is required, which does not cause photodamage. Due to the measured background noise, the designs described here can measure down to a molecular weight of a few kDa.

[0057] For example, protein analysis, molecular weight determination, protein complex formation, oligomerization, biomolecular interaction or interaction, macromolecule assembly, single-particle tracking, or single-molecule localization can be realized with the embodiments described herein.

[0058] It is also possible to define a temporally incoherent light source by the coherence length, which can be determined, for example, as described above, being smaller than the distance between the light source and the nearest optical interface. Such an optical interface can be provided, for example, by a lens. Typical coherence lengths of lasers used in microscopy, in contrast, are on the order of magnitude of meters. The temporally incoherent light sources used here therefore typically have a coherence length that is, for example, at least four to six orders of magnitude smaller. In particular, the light source used here is not a laser.

[0059] A normal light-emitting diode typically has an emitting area on the order of a few square millimeters or larger. A superluminescent light-emitting diode, or more generally a light source used in this field, typically has emitting areas on the order of a few square micrometers, i.e., about six orders of magnitude smaller. For a similar beam angle, the emitted light output of an LED is therefore about 100 times greater than that of a superluminescent light-emitting diode—for example, 500 mW for LEDs compared to 5 mW for superluminescent light-emitting diodes. Accordingly, the power density of a superluminescent light-emitting diode is typically about four orders of magnitude higher than that of a simple light-emitting diode. At the same time, however, it is significantly lower than that of a laser.

[0060] Spatial coherence can also be defined, for example, by the fact that a light source has the same phase regardless of its position on a radiating surface.

[0061] Further features and advantages will become apparent to those skilled in the art from the embodiment described below with reference to the accompanying drawings. Fig. 1 shows an interference reflection microscope, and

[0062] Fig. 2: a representation of a contrast depending on a molecular mass.

[0063] Fig. 1 shows an interference reflection microscope IRM according to an embodiment of the invention. The interference reflection microscope IRM has a light source LI. This emits light which is used as excitation light and is shown in dash-dotted lines. This light initially strikes a first lens L1 and a subsequent second lens L2. The first lens L1 collimates the light, and the second lens L2 focuses it onto an iris diaphragm, also known as the aperture iris AI. This filters out interfering components of the light. Downstream in the beam path is a third lens L3, which in turn collimates the light. Downstream is an X / 2 plate, by means of which the polarization of the light can be adjusted so that the polarization is optimized with respect to the beam splitter described below.After the ½ x-ray plate there is a field iris Fl for shaping the light beam as well as a first mirror M1 and a second mirror M2, which redirect the light to a fourth lens L4. This fourth lens L4 focuses the light, which then strikes a beam splitter ST. The transmitted part then passes through a ¼ x-ray plate, whereby the initially linear polarization becomes circular polarization. A third mirror M3 redirects the light to an objective 0, with a focus of the light focused by the fourth lens L4 lying in front of the objective 0. The focus lies in a rear conjugate plane of the objective 0. The objective 0 collimates the light and directs it to a sample arrangement PA. The sample arrangement PA is implemented here in the form of a transparent plate with a sample applied to it. Other solutions are also possible, for example a reservoir for a liquid.The excitation light is reflected at the sample arrangement PA, and a component of the excitation light is further reflected by interaction with interfaces, particularly with a sample such as an organic molecule. This creates interference patterns, which are contained in the back-reflected light. The back-reflected light is shown in dashed lines by a surrounding beam in Fig. 1. This is first guided via the third mirror M3 to the 4-wavelength plate and then impinges on the beam splitter ST.

[0064] The beam splitter ST is designed to allow a very high proportion of the incoming light with the polarization of the excitation light to pass through toward the objective 0. It is also designed to direct light with the polarization that the reflected light has after passing through the 4-wavelength plate twice, predominantly to a fourth mirror M4. This allows the polarization dependence of the beam splitter ST to be exploited to direct as much of the desired light as possible to the desired elements. Any unused light components are directed to a beam blocker SB.

[0065] The fourth mirror M4 directs the light through a Bertrand lens LB, which can be inserted into the beam path for control purposes but is normally not in the beam path for image acquisition, and then through a fifth lens L5. The light is refocused and then strikes a sixth lens L6, which in turn collimates it and directs it to a seventh lens L7. This lens L7 focuses the light onto a detector D. Detector D records a two-dimensional image of the incoming light, thus enabling analysis. A two-dimensional image can, for example, contain an interference pattern, which provides an indication of the size of a molecule in the sample arrangement PA. In particular, several images can be recorded in succession to create a video, or, for example, to create respective differences between images, which allow for better tracking of changes.Please refer to the explanations given above in this regard. The first to fourth lenses L1, L2, L3, L4, the aperture iris AI, the half-wave plate, the field iris Fl, and the first and second mirrors M1, M2 together form an illumination optics BO. The fourth mirror M4, the Bertrand lens LB, and the fifth to seventh lenses L5 to L7 together form a detection optics DO.

[0066] The light source LI in this case is a spatially coherent, temporally non-coherent light source in the form of a superluminescent light-emitting diode. This temporal non-coherence makes it possible to effectively prevent the formation of speckle patterns on the sample arrangement PA. This allows light to reach the detector D without such interfering speckle patterns, thus eliminating the need for filters known in the art that attenuate the light in front of the detector D to suppress this speckle pattern. This, in turn, allows the use of a light source LI with a significantly reduced power density compared to known designs, allowing longer irradiation even of sensitive samples without damaging them.

[0067] Fig. 2 shows a diagram illustrating the molecular mass in kDa for different molecules and the contrast achievable using the experimentally implemented interference reflection microscope described here. Different molecules are shown, and it is clear that the contrast increases with molecular mass. However, compared to existing designs, generally better contrasts can be achieved.

[0068] The contrasts were measured with a light power density of less than 0.5 kW / cm 2 This power density results in a contrast of 1.1% for a molecular mass of 100 kDa, for example. In comparison, the implementation disclosed in Young et al., Science 360, 423-427 (2018) achieves contrasts of only 0.7% for a molecular mass of 100 kDa with a power density of 420 kW / cm 2The implementation described here thus enables significantly lower power densities and, at the same time, even better contrast.

[0069] The steps mentioned in the method according to the invention can be carried out in the specified order. However, they can also be carried out in a different order, as long as this is technically reasonable. The method according to the invention can be carried out in one of its embodiments, for example, with a specific combination of steps, in such a way that no further steps are carried out. However, in principle, further steps can also be carried out, even those not mentioned.

[0070] It should be noted that features may be described in combination in the claims and the description, for example, to facilitate understanding, although they may also be used separately. Those skilled in the art will recognize that such features may also be combined independently with other features or combinations of features.

[0071] References in subclaims may indicate preferred combinations of the respective features, but do not exclude other combinations of features.

[0072] List of reference symbols

[0073] LI light source

[0074] L lenses

[0075] AI Aperture Iris

[0076] Fl Field Iris

[0077] BO lighting optics

[0078] DO detection optics

[0079] M Mirror

[0080] ST beam splitter

[0081] 0 lens

[0082] PA sample arrangement

[0083] SB beam blocker

[0084] LB Bertrand lens

[0085] D detector

[0086] IRM interference reflection microscope

Claims

Patent claims 1. Interference reflection microscope (IRM), comprising a light source (LI), an illumination optics (BO), a sample arrangement (PA), a detection optics (DO), and a detector (D), wherein the illumination optics (BO) directs light from the light source (LI) as excitation light onto the sample arrangement (PA), wherein the detection optics (DO) directs light reflected from the sample arrangement (PA) onto the detector (D), and wherein the light source (LI) is spatially coherent and temporally incoherent.

2. Interference reflection microscope (IRM) according to claim 1, wherein the light source (LI) is temporally incoherent in that a value of a coherence length, which is defined by the maximum wavelength squared divided by the half-width in the spectrum of the light source (LI), is at most 100 pm, at most 75 pm or at most 50 pm.

3. Interference reflection microscope (IRM) according to one of the preceding claims, wherein the light source (LI) is a superluminescent light-emitting diode.

4. Interference reflection microscope (IRM) according to one of the preceding claims, wherein the light source is not a laser light source.

5. Interference reflection microscope (IRM) according to one of the preceding claims, wherein the light source (LI) is spatially coherent in that a value of an area over which the emitted light has a fixed phase relationship is at least 1 pm 2 , at least 5 pm 2 , at least 10 pm 2 , at least 100 pm 2 , at least 250 m 2and / or a total radiating area of ​​the light source (LI). Interference reflection microscope (IRM) according to one of the preceding claims, wherein no attenuating filter is present in the beam path from the sample arrangement (PA) through the detection optics (DO) to the detector (D). Interference reflection microscope (IRM) according to one of the preceding claims, wherein in the beam path from the sample arrangement (PA) through the detection optics (DO) to the detector (D), at least 10%, at least 25%, at least 50%, at least 80%, or at least 90% of the light reflected by the sample arrangement (PA) reaches the detector (D).Interference reflection microscope (IRM) according to one of the preceding claims, which has a beam splitter (ST), wherein the illumination optics (BO) directs the light from the light source (LI) onto the beam splitter (ST), and the beam splitter (ST) directs the light at least partially onto the sample arrangement (PA), and wherein the beam splitter (ST) directs the light reflected from the sample arrangement (PA) at least partially onto the detection optics (DO). Interference reflection microscope (IRM) according to claim 8, wherein the light source (LI) and / or the illumination optics (BO) direct the light from the light source (LI) in a polarized manner onto the beam splitter (ST), wherein the beam splitter (ST) is polarization-sensitive such that it directs at least 70%, at least 80%, or at least 90% of the polarized light from the light source (LI) onto the sample arrangement (PA).Interference reflection microscope (IRM) according to claim 9, wherein reflected light from the sample arrangement (PA) impinges on the beam splitter (ST) in a polarized manner, and. wherein the beam splitter (ST) is polarization-sensitive such that it directs at least 70%, at least 80%, or at least 90% of the polarized light reflected from the sample arrangement (PA) onto the detection optics (DO). Interference reflection microscope (IRM) according to one of claims 8 to 10, wherein the illumination optics (BO) between the light source (LI) and the beam splitter (ST) and the detection optics (DO) between the beam splitter (ST) and the detector (D) are completely separate from one another. Interference reflection microscope (IRM) according to one of claims 8 to 11, wherein a beam path in the illumination optics (BO) between the light source (LI) and the beam splitter (ST) and a beam path in the detection optics (DO) between the beam splitter (ST) and the detector (D) are completely separate from one another. Interference reflection microscope (IRM) according to one of the preceding claims, wherein the illumination optics (BO) are designed completely separately from the detection optics (DO).Interference reflection microscope (IRM) according to one of the preceding claims, wherein a beam path in the illumination optics (BO) and a beam path in the detection optics (DO) are completely separate from one another. Interference reflection microscope (IRM) according to one of the preceding claims, which has an objective (0) arranged optically directly in front of the sample arrangement (PA). Interference reflection microscope (IRM) according to claim 15, wherein the objective (0) collimates the excitation light toward the sample arrangement (PA). The interference reflection microscope (IRM) according to claim 15, wherein the objective (0) focuses the excitation light onto the sample arrangement (PA). The interference reflection microscope (IRM) according to one of claims 15 to 17, wherein the illumination optics (BO) focuses the excitation light, wherein a focus is arranged in the beam path between the illumination optics (BO) and the objective (0), and / or wherein a focus is arranged in a rear conjugate plane of the objective (0). The interference reflection microscope (IRM) according to one of claims 15 to 18, wherein the illumination optics (BO) and / or the beam splitter (ST) direct the excitation light onto the objective (0) such that it is incident parallel to an optical axis of the objective (0).Interference reflection microscope (IRM) according to one of claims 15 to 19, wherein the illumination optics (BO) and / or the beam splitter (ST) direct the excitation light onto the objective (0) such that a center point of the excitation light is incident adjacent to the optical axis of the objective (0). Interference reflection microscope (IRM) according to one of claims 15 to 20, wherein the excitation light exits the objective (0) obliquely to the optical axis of the objective (0). Interference reflection microscope (IRM) according to one of claims 15 to 21, wherein the excitation light impinges obliquely on a surface of the sample arrangement (PA), is reflected by the sample arrangement (PA), and is reflected back to the objective (0). Interference reflection microscope (IRM) according to claim 22, wherein the excitation light is at an angle of at least 0°, greater than 0°, at least 1°, at least 5°, at least 10°, at least 20° or at least. 40° to the surface normal, and / or wherein the excitation light impinges on the surface of the sample arrangement (PA) at an angle of at most 1°, at most 5°, at most 10°, at most 20°, at most 40°, or at most 80° to the surface normal, and / or wherein the excitation light impinges on the surface of the sample arrangement (PA) at the Brewster angle. Interference reflection microscope (IRM) according to one of claims 22 or 23, wherein the light reflected by the sample arrangement (PA) impinges on the objective (O) at a different point on the objective (O) than the point at which the excitation light exits the objective (O) toward the sample arrangement (PA).Interference reflection microscope (IRM) according to one of the preceding claims, wherein the sample arrangement (PA) comprises a vessel for a liquid, the vessel being optically transparent at least to the extent that the light directed onto the sample arrangement (PA) can penetrate and light reflected within the liquid can escape. Interference reflection microscope (IRM) according to one of the preceding claims, wherein the light source (LI) comprises a substrate and a waveguide that is inclined relative to the substrate. Interference reflection microscope (IRM) according to claim 26, wherein the waveguide is antireflectively coated on two opposite sides. Interference reflection microscope (IRM) according to one of the preceding claims, wherein the light source (LI) does not comprise a resonator. Interference reflection microscope (IRM) according to one of the preceding claims, wherein the light source (LI) has a radiating area of ​​at most 1 pm 2 , maximum 5 pm2 , maximum 10 pm 2 , maximum 50 pm 2 , maximum 100 pm 2 , maximum 500 pm 2 or a maximum of 1,000 pm 2 Interference reflection microscope (IRM) according to one of the preceding claims, wherein the light source (LI) illuminates the sample with a power density of at most 1,000 kW / cm 2 , maximum 500 kW / cm 2 , maximum 100 kW / cm 2 , maximum 50 kW / cm 2 , maximum 10 kW / cm 2 , maximum 2 kW / cm 2 , maximum 1 kW / cm 2 or a maximum of 0.5 kW / cm 2 illuminated. Interference reflection microscope (IRM) according to one of the preceding claims, wherein the detector (D) is a 2D camera or a 1D camera, and / or a CCD detector and / or a CMOS camera. Method for analyzing a sample using an interference reflection microscope (IRM) according to one of the preceding claims, wherein the method comprises the following steps: Placing the sample in the sample assembly (PA) or as a sample assembly (PA), and Illuminating the sample using the light source (LI), while capturing at least one image using the detector (D). The method according to claim 32, wherein multiple images are captured using the detector (D), and wherein, after capturing each image, a difference image is generated between this image and a previously captured image or an averaged image, wherein an averaged image is generated as the mean or median across multiple previously captured images. The method according to claim 32 or 33, wherein the sample is one or more molecules. The method according to claim 34, wherein the molecule or molecules have a mass of at least 2 kDa, at least 3 kDa, or at least 10 kDa. The method according to claim 34 or 35, further comprising the following step: Determining a mass of one or more molecules based on one or more images or difference images. The method according to any one of claims 32 to 36, wherein multiple images are acquired consecutively by the detector (D), and a video is created from these images. The method according to any one of claims 32 to 37, wherein the illumination of the sample and / or the acquisition of one or more images occurs over a continuous period of at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 20 minutes.

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