Interference reflection microscope and method for analyzing sample

Abstract

The invention relates to an interference reflection microscope which operates with a spatially coherent, temporally incoherent light source. Thus, the formation of speckle patterns can be avoided, and an optical filter for filtering out such speckle patterns is no longer required. The invention also relates to a related method.

Description

Technical Field

[0001] The present invention relates to an interference reflection microscope and a method for analyzing a sample using the interference reflection microscope. Background Art

[0002] Microscopes are commonly used to examine or observe various types of samples. One possible microscope design is the interference reflection microscope, which typically illuminates a sample with an excitation light beam and uses the reflected light for evaluation. Due to interference effects, information can be generated from the light, enabling, for example, photometric mass spectrometry of molecules.

[0003] Young et al. disclose an exemplary embodiment in Science 360, 423-427 (2018) and its accompanying supplementary materials. In this embodiment, a central blocking filter is used to suppress speckle patterns. Summary of the Invention

[0004] An object of the present invention is to provide an interference reflection microscope that can replace or be superior to existing designs. Another object of the present invention is to provide a corresponding method for analyzing a sample.

[0005] According to the invention, this object is achieved by an interference reflection microscope and a method thereof according to the respective independent claims. Preferred embodiments can be found, for example, in the respective dependent claims. The contents of the claims are expressly incorporated by reference into the description.

[0006] The present invention relates to an interference reflection microscope. The interference reflection microscope comprises a light source, an illumination optical system, a sample arrangement, a detection optical system, and a detector. The illumination optical system directs light from the light source as excitation light to the sample arrangement. The detection optical system directs reflected light from the sample arrangement to the detector. A spatially coherent light source is advantageous. A temporally incoherent light source is also advantageous.

[0007] The advantage of the light source design in this interference reflection microscope is that, due to its temporal incoherence, it prevents the formation of speckle patterns in the reflected light. Such patterns often appear as interference phenomena due to unavoidable roughness, impurities, or chemical heterogeneity in the surface where the sample is arranged and / or in the illumination or detection optics. In known designs, these patterns often interfere with the evaluation to a considerable extent, necessitating the use of strong filters in the beam path to remove them. While such filters enable better evaluation without significant interference from the speckle pattern, they also result in significant loss of light reflected from the sample. This, in turn, means that in known designs, higher radiation intensities must be used to achieve sufficient light intensity after the filters for evaluation. Such high light output can particularly cause organic molecules to fade or be destroyed during detection. However, in the present interference reflection microscope design, the power density at the sample can be significantly reduced, allowing for longer exposure times without damaging the molecules.

[0008] An interference reflection microscope is essentially a microscope in which a sample is illuminated and the reflected and / or scattered light, in particular in a direction at least approximately opposite to the illumination light, is collected and evaluated. This light essentially contains information generated by interference effects. Such interference effects can occur, in particular, due to interface transitions, for example, between glass and liquid, between liquid and sample, or due to the distance between sample and glass. For example, transparent plastic can also be used instead of glass. For example, molecules can be used as samples, in particular organic molecules, and in particular molecules with a mass of, for example, several kDa (kilodaltons). A plurality of such molecules can also be used.

[0009] A characteristic of interference reflection microscopy 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 nanometers (nm), at most 1 micrometer (μm), at most 10 micrometers, at most 100 micrometers, or at most 1 millimeter (mm) from the sample, such as at the interface between glass and liquid or between liquid and sample. This distinguishes interference reflection microscopy from microscope designs that have a separate path into which the light of the main beam is coupled and then interferes with the light reflected from the sample. Interference reflection microscopy generally does not have such a separate path.

[0010] The light source is used to generate excitation light. The illumination optical component guides the excitation light to the sample arrangement position. For example, a Kohler device ( For this purpose, a sample arrangement can be used. The sample arrangement can in particular be an arrangement capable of holding and / or providing a sample. For example, the sample arrangement can be a container for collecting a liquid, wherein the liquid can contain one or more molecules. These molecules can be referred to as a sample. They can in particular be examined using the interference reflection microscope. Alternatively, a sample arrangement can also represent the sample itself. In this case, a sample, such as a solid object, can be examined directly.

[0011] The detection optics direct light reflected and / or scattered by the sample arrangement to the detector. This typically generates a signal indicating the intensity of the light incident on the detector. For example, the detector can be a two-dimensional detector divided into a plurality of pixels. This allows the recording of a two-dimensional image and its evaluation in a suitable manner.

[0012] Regarding the structure of the optical elements and other components of the interference reflection microscope, reference may be made in particular to the description of the following embodiments in conjunction with the accompanying drawings.

[0013] In particular, the light source may be temporally incoherent, with a coherence length having a value of at most 100 microns, at most 75 microns or at most 50 microns. The coherence length can be defined in particular based on 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. It has been shown that this definition is a useful definition of a coherence length for assessing temporal coherence or incoherence. A light source that is temporally incoherent can be called a temporally incoherent light source with a given numerical value. These numerical values have been shown to be particularly advantageous for current applications. Even for a temporally incoherent light source, the coherence length is usually not completely zero, but for example at least one wavelength.

[0014] In particular, the half-width can be defined as the width connecting two points in the spectrum where, viewed from a maximum, the intensity of the two points is half of the maximum.

[0015] In particular, the light source can be a superluminescent diode or a superluminescent diode. Such superluminescent diodes generate spatially coherent and temporally incoherent light, which has proven advantageous for this application for the reasons mentioned above. However, in principle, it is also possible to use other light sources as long as they have the required properties.

[0016] Typically, the light source is not a laser light source. A laser light source is typically a resonant feedback optical amplifier that generates light based on stimulated emission of radiation. Laser light sources are generally not suitable for the applications described here.

[0017] In particular, the light source may be spatially coherent, wherein the light emitted over an area of at least 1 square micrometer (μm) has a fixed phase relationship. 2 ), at least 5 square microns, at least 10 square microns, at least 100 square microns, at least 250 square microns, and / or a total radiant area of the light source (LI). This has proven to be a useful definition of a coherence surface for a spatially coherent light source. Based on the given values, it can be assumed that a spatially coherent light source provides the required performance and is capable of illuminating the sample with the necessary light power density.

[0018] It should be understood that the given definitions of temporal incoherence and spatial coherence may also be understood as replacing the formula used in claim 1 of the present application.

[0019] Preferably, there is no attenuation filter in the light beam path from the sample arrangement through the detection optics to the detector. An attenuation filter is understood in particular to be an element whose task is to attenuate the light beam, in particular to attenuate the light beam significantly, for example by at least one, two or three orders of magnitude. Other optical elements, such as lenses which cannot achieve 100% penetration for technical reasons, or beam splitters which serve the purpose of splitting the light beam and therefore naturally only allow part of the incident light to pass in a specific direction, are not considered to be attenuation filters. By omitting an attenuation filter, it can advantageously be achieved that a significantly higher proportion of the reflected light reaches the detector, so that a lower power density can be used for excitation. This allows longer exposure times without damaging the sample.

[0020] In particular, in the beam path from the sample arrangement position 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 from the sample arrangement position reaches the detector. Such values are typically achieved, for example, if no attenuation filter is used in this beam path. This positive effect is achieved due to the high penetration from the sample arrangement position to the detector. This is also true, for example, if a beam splitter is present in the beam path from the sample arrangement position to the detector, which inevitably prevents some light from passing through the detector and redirects it in another direction.

[0021] If a proportion of light is mentioned in this context, this may particularly mean, for example, a power of light that transmits at least the stated proportion.

[0022] According to one embodiment, the interference reflection microscope includes a beam splitter. The illumination optical system can specifically direct light from the light source to the beam splitter, while the beam splitter can specifically direct the light at least partially to the sample arrangement location. In particular, the beam splitter can direct at least partially light reflected from the sample arrangement location to the detection optical system. Because the beam splitter can superimpose the excitation light beam and the reflected light, such a beam splitter enables a simple and practical design of an interference reflection microscope.

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

[0024] In particular, it can be provided that the reflected light from the sample arrangement position is polarized when it impinges on the beam splitter. The beam splitter can in particular be polarization-sensitive in such a way that it is able to split the polarized light reflected from the sample arrangement position in such a way that at least 70%, at least 80% or at least 90% is directed towards the detection optics. This means that, in the beam path from the sample arrangement position to the detector, a polarization-sensitive design of the beam splitter can ensure that less light is directed from the beam splitter in an unusable direction and a higher proportion of light passes through the detector. The polarization of the light reflected by the sample arrangement position and incident on the beam splitter can in particular be transversely aligned, for example at an angle of 90° or an angle between 85° and 95° to the polarization of the light incident on the beam splitter from the light source. This can be achieved, for example, by using a λ / 4 plate between the beam splitter and the sample arrangement position.

[0025] In particular, it is possible to design the illumination optics between the light source and the beam splitter, and the detection optics between the beam splitter and the detector, completely independently of one another. A completely independent design can be understood to mean, in particular, that the respective optical component, such as a reflector, lens, or aperture, is only used in one of the two optical components. This does not preclude their joint installation on an optical platform or in a bracket. It is also possible to design the illumination optics completely independently of 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 independent of one another. In particular, this can mean that the beams do not overlap in the aforementioned area and that the beam in one optical element is not generated by the beam of another optical element. Typically, the beam splitter is the first optical element after the light source or the last optical element before the detector, and both the beam of the illumination optics and the beam of the detection optics hit the beam splitter. 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 arranged optically directly in front of the sample arrangement location. In particular, the objective lens can be arranged between the beam splitter and the sample arrangement location. Using such a lens, the light can be directed and specifically structured onto the sample arrangement location. Reflected light can also be specifically captured.

[0028] For example, the objective lens may collimate the excitation light toward the sample arrangement location. In this case, the sample arrangement location is illuminated by an excitation light whose diameter remains constant at least within the relevant range. Alternatively, the objective lens may focus the excitation light onto the sample arrangement location.

[0029] In particular, the illumination optics can focus the excitation light. In particular, a focal point can be arranged in the beam path between the illumination optics and the objective lens. In particular, the focal point can be arranged in a back-conjugate plane of the objective lens. Typically, after this focal point and before hitting the objective lens, the light beam broadens again and is processed in the objective lens so that it hits the sample arrangement in the desired manner.

[0030] The illumination optics and / or the beam splitter can in particular direct the excitation light onto the objective lens in such a way that the excitation light is incident parallel to an optical axis of the objective lens. The optical axis can in particular be an axis of symmetry of the objective lens. In particular, it can be arranged at the center of the cross section of the objective lens. By ensuring that the light is incident on the objective lens in parallel, it can be ensured that the light beam leaves the objective lens parallel to this optical axis. It should be understood that "parallel" here also includes "identical". The excitation light can also be incident on the objective lens in such a way that a center of the excitation light is located on the optical axis of the objective 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 near the optical axis of the objective lens. Therefore, it does not coincide with the optical axis. It can also be said that the center of the excitation light is spaced apart from the optical axis of the objective lens, i.e. has a non-vanishing distance. This allows an asymmetric illumination of the objective lens to be achieved. The excitation light can in particular leave the objective lens at an angle to the optical axis of the objective lens. This allows the sample arrangement position to be illuminated at an angle to the optical axis. In particular, the excitation light can impinge obliquely on a surface of the sample arrangement position, be reflected at the sample arrangement position and be reflected back to the objective lens. This reflected light beam is typically also inclined to the surface of the sample arrangement position. It is then typically also inclined to an optical axis of the objective lens.

[0032] It can also be provided that the excitation light impinges on the surface of the sample arrangement location 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°. It can also be provided that the excitation light impinges on the surface of the sample arrangement location 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 by any lower value and any larger higher value. Usually, a normal on the surface serves as a reference for defining the angle. Therefore, the angle is usually given relative to the surface normal. Therefore, an angle of 0° corresponds to a non-oblique incidence. The excitation light can also be incident on the surface of the sample arrangement location at the Brewster angle. This allows unwanted reflections to be avoided as much as possible. The Brewster angle depends on the material used, in particular the material of the sample arrangement location, which is usually glass. However, it can be easily calculated once the corresponding formula is known.

[0033] According to one embodiment, it may be provided that the light reflected at the sample arrangement position hits the objective lens at a different point than the point at which the excitation light leaves the objective lens towards the sample arrangement position.

[0034] The above design allows for oblique illumination of the sample arrangement location. This can, in particular, result in less directly reflected excitation light from the sample arrangement location, and a greater proportion of light interacting with the sample. This further increases the information content of the detected light.

[0035] According to one embodiment, the excitation light may exit the objective lens parallel to the optical axis and / or may strike the surface transversely to the surface where the sample is arranged. For example, the light may be reflected back in the opposite direction to the excitation light, i.e. offset by exactly or at least approximately 180°, for example at an angle of at least 170° and / or at most 190°.

[0036] The objective lens can in particular 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 to be advantageous for typical interference reflection microscopes.

[0037] The sample arrangement location can, for example, comprise a container for a liquid, wherein the container is optically transparent, at least to the extent that light directed to the sample arrangement location can penetrate and light reflected in the liquid can escape. This means, for example, that one or more molecules of a sample can be present in this liquid and that these molecules can be observed. The container can be made of, for example, glass or transparent plastic. For example, the liquid can be static or can also flow through the container as part of a fluid.

[0038] According to one embodiment, the light source includes a substrate and a waveguide tilted relative to the substrate. The tilted waveguide may, for example, have two parallel outer surfaces that are tilted relative to a surface of the substrate. This can generate spatially incoherent light.

[0039] For example, the waveguide can be anti-reflective coated on both opposite sides. This prevents the formation of modes in the waveguide that would lead to temporal coherence of the light. In particular, it can be provided that the light source does not have a resonator. This also prevents the formation of modes that would lead to temporal coherence of the light.

[0040] In particular, it can be provided that the light source is not a laser light source. Lasers are generally temporally and spatially coherent and are therefore unsuitable for the applications envisaged herein.

[0041] For example, the light output of the light source may be at most 10 mW, at most 20 mW, at most 100 mW, or at most 1000 mW. Such light outputs may be useful for typical applications, but other light outputs are also possible.

[0042] According to an advantageous embodiment, the light source has a radiation surface of at most 1 square micrometer, at most 5 square micrometers, at most 10 square micrometers, at most 50 square micrometers, at most 100 square micrometers, at most 500 square micrometers or at most 1000 square micrometers. Such a size of the radiation surface of the light source has proven to be advantageous for the present design.

[0043] In particular, the light source may be at a power of not more than 1000 kW / cm 2 , not exceeding 500kW / cm 2 , not exceeding 100kW / cm 2 , not exceeding 50kW / cm 2 , not exceeding 10kW / cm 2 , not exceeding 2kW / cm 2 , not exceeding 1kW / cm 2 or not more than 0.5kW / cm 2 The sample is illuminated with a power density of . Such a low power density at the sample can be used in the design provided herein because, as previously mentioned, a filter in the beam path to the detector can be omitted. In other words, a particularly high transmittance from the sample arrangement to the detector can be used, making it possible to use such a low power density. In turn, the advantage of a low power density is that a longer exposure time is possible. This refers in particular to the power density at the sample.

[0044] In particular, the detector can be a two-dimensional (2D) camera or a one-dimensional (1D) camera. For example, it can be designed as a CCD detector and / or a CMOS camera. Such designs have proven advantageous for typical applications. Typically, the detector is of two-dimensional design so that two-dimensional patterns or interference patterns can be easily detected. This can be used, for example, for photographic mass spectrometry. However, depending on the application, a one-dimensional design of the detector may also be sufficient. Typically, the detector has a plurality of pixels, each of which measures a light intensity.

[0045] The present invention also 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 respect to the interference reflection microscope, all designs and variants described herein can be used. The procedure of the method specifically comprises the following steps:

[0046] - introducing said sample into said sample arrangement location or as a sample arrangement location, and

[0047] - illuminating the sample with the light source while recording at least one image with the detector.

[0048] Such a method enables advantageous analysis of a sample using the interference reflection microscope described herein. Said method also achieves the advantages described above.

[0049] In particular, the detector can be used to capture multiple images. After each image is captured, a difference image can be generated between that image and the previously captured image or an average image. For example, an average image can be created as the average or median of the previously captured images. Averaging typically involves using the intensity values of the images to be averaged, particularly the intensity values of identical pixels, to generate a mean for each pixel. In other words, a mean value can be calculated for each pixel based on the respective intensity values or other measured values. The mean value can be an arithmetic mean or a geometric mean. For median values, a mathematical mean is not typically generated; instead, a value is determined at which the intensity values of the corresponding pixels in one half of the image to be averaged are below the mean value and the intensity values of the corresponding pixels in the other half are above the mean value. Such average images are particularly useful as a basis for comparison with a correspondingly recorded image. In such average images, the effects of noise are averaged out. This can be accomplished by calculating the mean or median value. Generating a difference image makes it possible to detect changes compared to the previous image. This makes it possible, for example, to detect new molecules that have landed on a surface. For example, these molecules can be measured and observed as they leave the surface again.

[0050] For example, the sample can be a single molecule or can contain multiple molecules. In particular, these molecules can be organic molecules that are soluble in solution. For example, one or more molecules can each have a mass of at least 2 kDa, at least 3 kDa, or at least 10 kDa. Such molecules can be particularly advantageously analyzed using the methods and / or interference reflection microscopy described herein.

[0051] In particular, the mass of one or more molecules can be determined based on one or more images or difference images. This can be achieved, for example, using automated image analysis. This can be based on, for example, artificial intelligence, neural networks, and / or machine learning. The design described herein has the advantage of enabling longer illumination times and lossless illumination.

[0052] In particular, the detector can be used to capture multiple images one after another. These images can be used to create a video. This type of video is typically a series of consecutively captured images. This allows, for example, observing the landing and / or departure of molecules on a surface. It can also be used to observe changes in the molecular orientation.

[0053] The illumination of the sample and / or the acquisition of one or more images can be performed, for example, 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 generally possible using the designs described herein because the power density of the sample illumination can be significantly reduced compared to known designs. In particular, the power density or other data of the light source or the interference reflection microscope described above 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 study biomolecules such as proteins or nanoparticles. For example, in the embodiments described herein, a label typically used for nanoparticles or proteins can be omitted. For example, single molecule detection, molecular weight determination, or other assessments can be performed.

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

[0056] Spatial coherence generally allows the light to be collimated and focused like a laser. Temporal incoherence prevents the generation of speckle patterns. Polarization enables efficient use of the light output. For this purpose, for example, a polarization-sensitive beam splitter as described above can be used. The excitation light can, for example, have a wavelength of 450 nanometers (nm). Any other wavelength suitable for the respective purpose can also be used. For example, multiple lenses can be used to collimate, focus, or expand the light and cut off at, for example, two apertures before it falls on a sample behind an objective lens. Appropriate use of retarders (λ / 2 or λ / 4) and a polarizing beam splitter can reduce light output losses. The incident light can be reflected by the sample glass surface and / or biomolecules or nanoparticles, which interfere and pass through the detection path to the detector or camera. For example, frame rates exceeding 100 frames per second, exceeding 500 frames per second, exceeding 1000 frames per second, exceeding 2000 frames per second, and / or up to 2000 frames per second, up to 5000 frames per second, or up to 10,000 frames per second can be recorded. These images can then be partially averaged to create differential images. The image contrast is generally proportional to the molecular weight of the sample. Thus, for example, molecules or particles weighing several MDa up to a specified value can be detected and located. As molecules move, they can be tracked for several minutes without causing photodamage, since only very low light output is required. Due to the background noise of the measurement, the design described here can be used to measure molecular weights as low as a few kDa.

[0057] For example, embodiments described herein may be utilized to perform protein analysis, molecular weight determination, protein complex formation, oligomerization, biomolecular interaction or mutual influence, macromolecular assembly, single particle tracking, or single molecule localization.

[0058] A temporally incoherent light source can also be defined by the coherence length, which can be determined, for example, as described above, to be less than the distance between the light source and the nearest optical interface. Such an optical interface can be provided, for example, by a lens. On the other hand, the typical coherence length of lasers used in microscopes is on the order of meters. Therefore, the temporally incoherent light source used herein has a coherence length that is typically, for example, at least four to six orders of magnitude smaller. In particular, the light source used herein is not a laser.

[0059] A typical light-emitting diode typically has an emitting area of a few square millimeters or more. For a superluminescent diode, or more generally, a light source as used herein, the typical radiating surface is a few square micrometers, or about six orders of magnitude smaller. At a similar beam angle, the radiant light output of an LED is approximately 100 times greater than that of a superluminescent diode. For example, an LED has a radiant light output of 500 mW, while a superluminescent diode has a radiant light output of 5 mW. Therefore, the power density of a superluminescent diode is typically about four orders of magnitude higher than that of a simple LED. However, at the same time, its power density is significantly lower than that of a laser.

[0060] Spatial coherence can also be defined, for example, as having the same phase regardless of the position of a light source on the radiating surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Further features and advantages will become apparent to those skilled in the art from the embodiments described below with reference to the accompanying drawings.

[0062] Figure 1 : shows an interference reflection microscope, and

[0063] Figure 2 : Displays a contrast ratio depending on the mass of a molecule. DETAILED DESCRIPTION

[0064] Figure 1An interference reflection microscope IRM according to an embodiment of the present invention is shown. The interference reflection microscope IRM has a light source LI. The light emitted by the light source LI serves as excitation light and is indicated by a dotted line. The light first strikes a first lens L1 and then a second lens L2. The first lens L1 collimates the light, while the second lens L2 focuses it onto a variable aperture, also called an aperture AI. This eliminates interfering components in the light. Following the beam path is a third lens L3, which further collimates the light. Below this is a λ / 2 plate, by which the polarization of the light can be adjusted to optimize it with respect to the beam splitter described below. Following the λ / 2 plate is a field aperture F1 for shaping the 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 portion that penetrates then passes through a λ / 4 plate, which converts the originally linear polarization into circular polarization. The light is redirected to an objective lens O by a third reflector M3, and after being focused by the fourth lens L4, the focus of the light is located in front of the objective lens O. The focus is particularly located in a rear conjugate plane of the objective lens O. The objective lens O collimates the light and directs it to a sample arrangement position PA. The sample arrangement position PA is in the form of a transparent plate on which a sample is placed. Other solutions are also applicable, such as a reservoir of a liquid. The excitation light is reflected at the sample arrangement position PA, and a component of the excitation light is further reflected by interacting with an interface, in particular with a sample such as an organic molecule. This produces an interference pattern contained in the reflected light. The reflected light is Figure 1 This is first directed by the third mirror M3 to the λ / 4 plate and then hits the beam splitter ST.

[0065] The beam splitter ST is designed to allow a very high proportion of incident light having the polarization of the excitation light to pass in the direction of the objective lens O. It is further designed to direct the polarized light reflected after two passes through a λ / 4 reflector primarily to a fourth mirror M4. This allows the polarization dependence of the beam splitter ST to be exploited, forcing as much of the desired light as possible to the desired element. Any unused light components are directed to a beam blocker SB.

[0066] 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 typically not in the beam path for image acquisition. The light then passes through a fifth lens L5. After being refocused, the light then strikes a sixth lens L6, which collimates the light and directs it to a seventh lens L7. The seventh lens L7 focuses the light onto a detector D. The detector D records a two-dimensional image of the incoming light, which can then be evaluated. For example, the two-dimensional image can include an interference pattern that provides an indication of the size of a molecule in the sample arrangement PA. In particular, multiple images can be captured consecutively to create a video, or, for example, to generate differences between images in order to better track changes. For comments on this aspect, please see above. The first to fourth lenses L1, L2, L3, L4, the aperture AI, the λ / 2 plate, the field stop F1, and the first and second mirrors M1 and M2 together form the illumination optics BO. The fourth reflector M4, the Bertrand lens LB, and the fifth to seventh lenses L5 to L7 together constitute a detection optical element DO.

[0067] Light source LI is a spatially coherent, temporally incoherent light source in the form of a superluminescent diode (SLLD). Due to its temporal incoherence, the sample arrangement is effectively prevented from forming a speckle pattern when positioned on the PA. Consequently, the light reaching detector D does not contain such an interfering speckle pattern, eliminating the need for filters known in the prior art to attenuate light in front of detector D to shield it from such speckle patterns. This, in turn, allows the use of light source LI with a significantly lower power density than known designs, enabling longer illumination of sensitive samples without damaging them.

[0068] Figure 2 A graph is shown illustrating the molecular weight (kDa) of various molecules and the contrast achievable using the design described herein for implementing the interference reflection microscopy experiment. Different molecules are shown, and it can be observed that the contrast increases with increasing molecular weight. However, generally, better contrast can be achieved compared to known designs.

[0069] The contrast ratio is obtained when the optical power density is less than 0.5kW / cm 2 For example, at this power density, a molecule with a molecular weight of 100 kDa causes a contrast of 1.1%. In contrast, the embodiment disclosed by Young et al. in Science 360, 423-427 (2018) achieves a contrast of 1.1% at a molecular weight of 100 kDa and a power density of 420 kW / cm 2In the case of , the contrast ratio is only 0.7%. Therefore, the embodiments described herein can significantly reduce the power density while having a better contrast ratio.

[0070] The steps mentioned in the method according to the present invention may be performed in the order specified. However, they may also be performed in a different order, as long as it is technically reasonable. The method according to the present invention may be performed according to one of its specific embodiments, for example, using a particular combination of steps so that other steps do not need to be performed. However, in principle, other steps may also be performed even if not mentioned.

[0071] It should be noted that the claims and the specification may describe features in combination, for example, to facilitate understanding, but these features can also be used alone. Those skilled in the art will recognize that these features can also be independently combined with another feature or feature combination.

[0072] The references in the dependent claims may indicate preferred combinations of individual features, but do not exclude other combinations of a plurality of features.

[0073] Reference Symbols List

[0074] LI light source

[0075] L lens

[0076] AI aperture (aperture Iris)

[0077] Fl field aperture (fieldIris)

[0078] BO lighting optics

[0079] DO detection optics

[0080] M reflector

[0081] ST Beam Splitter

[0082] OObjective lens

[0083] PA Sample Arrangement Location

[0084] SB beam blocker

[0085] LB Bertrand lens

[0086] D detector

[0087] IRM Interference Reflection Microscope

Claims

1. An interference reflection microscope (IRM), characterized in that: The interference reflection microscope comprises: a light source (LI), an illumination optic (BO), A sample arrangement position (PA), a detection optic (DO), and A detector (D), wherein the illumination optics (BO) directs light from the light source (LI) onto the sample arrangement position (PA) as excitation light, wherein the detection optics (DO) directs the light reflected by the sample arrangement position (PA) from the sample arrangement position (PA) to the detector (D), and Wherein the light source (LI) is spatially coherent and temporally incoherent.

2. The interference reflection microscope (IRM) according to claim 1, characterized in that: The light source (LI) is temporally incoherent, wherein a coherence length defined by the square of the maximum wavelength in the spectrum of the light element (L1) divided by the half width has a value of at most 100 microns, at most 75 microns or at most 50 microns.

3. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The light source (LI) is a superluminescent diode.

4. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The light source is not a laser light source.

5. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The light source (LI) is spatially coherent, wherein the light emitted over an area has a fixed phase relationship, wherein the area is at least 1 square micron, at least 5 square microns, at least 10 square microns, at least 100 square microns, at least 250 square microns, and / or a total radiation area of the light source (LI).

6. Interference reflection microscope (IRM) according to any one of the preceding claims, characterized in that: No attenuation filter is present in the beam path from the sample arrangement position (PA) through the detection optics (DO) to the detector (D).

7. Interference reflection microscope (IRM) according to any one of the preceding claims, characterized in that: In the beam path from the sample arrangement position (PA) through the detection optical element (DO) to the detector (D), at least 10%, at least 25%, at least 50%, at least 80% or at least 90% of the light is reflected by the sample arrangement position (PA) to the detector (D).

8. Interference reflection microscope (IRM) according to any one of the preceding claims, characterized in that: The interference reflection microscope has a beam splitter (ST), wherein the illumination optics (BO) directs the light of the light source (LI) onto the beam splitter (ST), and the beam splitter (ST) directs the light at least partially onto the sample arrangement position (PA), and The beam splitter (ST) directs at least part of the light reflected by the sample arrangement (PA) onto the detection optics (DO).

9. The interference reflection microscope (IRM) according to claim 8, characterized in that: The light source (LI) and / or the illumination optics (BO) direct the light of the light source (LI) onto the beam splitter (ST) in a polarized manner, The beam splitter (ST) is polarization-sensitive, thereby directing at least 70%, at least 80% or at least 90% of the polarized light of the light source (LI) to the sample arrangement position (PA).

10. The interference reflection microscope (IRM) according to claim 9, characterized in that: The reflected light from the sample arrangement position (PA) is incident on the beam splitter (ST) in a polarized manner, and Wherein the beam splitter (ST) is polarization sensitive, thereby directing 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).

11. The interference reflection microscope (IRM) according to any one of claims 8 to 10, characterized in that: The illumination optics (BO) between the light source (LI) and the beam splitter (ST) as well as the detection optics (DO) between the beam splitter (ST) and the detector (D) are designed to be completely independent of each other.

12. The interference reflection microscope (IRM) according to any one of claims 8 to 11, characterized in that: 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 independent of each other.

13. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The illumination optics (BO) are designed to be completely independent from the detection optics (DO).

14. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: A beam path in the illumination optics (BO) and a beam path in the detection optics (DO) are completely independent of one another.

15. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The interference reflection microscope has an objective (O) which is arranged optically directly in front of the sample arrangement (PA).

16. The interference reflection microscope (IRM) according to claim 15, characterized in that: The objective lens (O) collimates the excitation light toward the sample arrangement position (PA).

17. The interference reflection microscope (IRM) according to claim 15, characterized in that: The objective lens (O) focuses the excitation light onto the sample arrangement position (PA).

18. The interference reflection microscope (IRM) according to any one of claims 15 to 17, characterized in that: 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 (O), and / or One of the focal points is arranged in a back conjugate plane of the objective lens (O).

19. The interference reflection microscope (IRM) according to any one of claims 15 to 18, characterized in that: The illumination optical element (BO) and / or the beam splitter (ST) guide the excitation light to the objective lens (O) in a manner such that the excitation light is incident on the objective lens (O) in parallel with an optical axis of the objective lens (O).

20. The interference reflection microscope (IRM) according to any one of claims 15 to 19, characterized in that: The illumination optical element (BO) and / or the beam splitter (ST) guide the excitation light to the objective lens (O) in such a manner that a center point of the excitation light is incident near the optical axis of the objective lens (O).

21. The interference reflection microscope (IRM) according to any one of claims 15 to 20, characterized in that: The excitation light is emitted from the objective lens (O) in a manner inclined with respect to the optical axis of the objective lens (O).

22. The interference reflection microscope (IRM) according to any one of claims 15 to 21, characterized in that: The excitation light obliquely impinges on a surface of the sample arrangement position (PA), is reflected at the sample arrangement position (PA), and is reflected back to the objective lens (O).

23. The interference reflection microscope (IRM) according to claim 22, characterized in that: The excitation light is irradiated 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° relative to the surface normal, and / or the excitation light impinges on the surface at the sample arrangement position (PA) at an angle of at most 1°, at most 5°, at most 10°, at most 20°, at most 40° or at most 80° relative to the surface normal, and / or The excitation light impinges on the surface at the sample arrangement position (PA) at a Brewster angle.

24. The interference reflection microscope (IRM) according to any one of claims 22 to 23, characterized in that: The light reflected at the sample arrangement position (PA) impinges on the objective lens (O) at a different point than the point at which the excitation light leaves the objective lens (O) towards the sample arrangement position (PA).

25. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The sample arrangement position (PA) comprises a container for a liquid, which container is optically transparent at least to a certain extent, so that light directed onto the sample arrangement position (PA) can penetrate and light reflected in the liquid can escape.

26. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The light source (LI) has a substrate and a waveguide tilted relative to the substrate.

27. The interference reflection microscope (IRM) according to claim 26, characterized in that: Two opposing sides of the waveguide are coated with an anti-reflection coating.

28. Interference reflection microscope (IRM) according to any one of the preceding claims, characterized in that: The light source (LI) does not have a resonator.

29. Interference reflection microscope (IRM) according to any one of the preceding claims, characterized in that: The light source (LI) has a radiation area of at most 1 square micron, at most 5 square microns, at most 10 square microns, at most 50 square microns, at most 100 square microns, at most 500 square microns or at most 1000 square microns.

30. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The light source (LI) has a power of at most 1000 kW / cm 2 , up to 500kW / cm 2 , up to 100kW / cm 2 , up to 50kW / cm 2 , up to 10kW / cm 2 , up to 2kW / cm 2 , up to 1kW / cm 2 or up to 0.5kW / cm 2 The sample is irradiated with a power density of .

31. Interference reflection microscope (IRM) according to any of the preceding claims, characterized in that: The detector (D) is a 2D camera or a 1D camera, and / or a CCD detector and / or a CMOS camera.

32. A method for analyzing a sample using an interference reflection microscope (IRM) according to any one of the preceding claims, characterized in that: The method comprises the following steps: placing a sample in the sample arrangement position (PA) or as a sample arrangement position (PA), and The sample is illuminated using the light source (LI) while at least one image is recorded using the detector (D).

33. The method according to claim 32, wherein: recording a plurality of images by means of said detector (D), and After recording a corresponding image, a difference image is generated between the corresponding image and a previously recorded image or an average image, wherein the average image is generated by an average value or a median value of a plurality of previously recorded images.

34. The method according to any one of claims 32 or 33, characterized in that: The sample is one molecule or contains multiple molecules.

35. 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.

36. The method according to any one of claims 34 or 35, characterized in that: The method further comprises the following steps: The mass of the one or more molecules is determined based on the one or more images or difference images.

37. The method according to any one of claims 32 to 36, characterized in that: a plurality of images are recorded one by one by the detector (D), and Therein, a video is created from these images.

38. The method according to any one of claims 32 to 37, characterized in that: Illuminating the sample and / or capturing one or more images occurs over a continuous period of time that is at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 20 minutes.

Images