Total internal reflection fluorescence (TIRF) microscope and TIR prism for use in a TIRF microscope

Abstract

Total internal reflection fluorescence (TIRF) microscope (100) comprising: an excitation light source (101) and an excitation light path (108), the excitation light source (101) being configured to emit an excitation light (110) propagating along the excitation light path (108) towards a sample (106) with at least one fluorophore and to generate an evanescent wave that is configured to permeate the sample and that is configured to excite an emission of fluorescence emission light (107) by the sample (106); an optical microscope setup (300) configured to project an image of at least a portion of the sample (106) positioned in an object plane (OP) onto an image plane (IP) of the optical microscope setup (300); a sensor (304) configured to be positioned in the image plane (IP) and to record the fluorescence emission light (107) emitted by the sample (106); and a total internal reflection (TIR) prism (201; 501; 600; 700) configured to support the sample (106), the total internal reflection (TIR) prism (201; 501; 600; 700) comprising: a base surface (A) facing away from the sensor; a sample-supporting surface (B) opposite to the base surface (A) and facing the sensor; an excitation light incidence surface (C) oriented at an angle 0° < ∝ < 90° with respect to the sample-supporting surface (B) and the excitation light path (108) being configured such that at least a portion of the excitation light (110) hits the excitation light incidence surface (C) and propagates via the TIR prism (200; 500; 600; 700) to the sample-supporting surface (B); an excitation light exit surface (D) opposite the excitation light incidence surface (C); and at least one light trap element (202; 502; 602; 703) matching the refractive index of the TIR prism (200; 500; 600; 700) and being optically coupled to the excitation light exit surface (D) of the TIR prism (200; 500; 600; 700).

Description

[0001] Total internal reflection fluorescence (TIRF) microscope and TIR prism for use in a TIRF microscope

[0002] The invention relates to a total internal reflection fluorescence (TIRF) microscope and a TIR prism for use in a TIRF microscope, specifically in the field of In Vitro diagnostics (IVD), where TIRF microscopes are used together with analyzers (for example, without limitations, optical and / or digital analyzers) to analyze samples, specifically medical samples, for example, in electroluminescence analyzers.

[0003] Widefield fluorescence microscopy is often applied in IVD including applications such as cell and tissue imaging, quantification of biomarkers, pathogen detection, gene expression studies and drug testing. By providing high-resolution images and the ability to label specific molecular targets, widefield fluorescence microscopy is very useful in diagnosing and understanding a variety of medical conditions.

[0004] Common widefield fluorescence microscopy typically excites and detects fluorescence in the entire volume of a specimen. Signal contribution from outside the focal plane causes higher background signal, blurring and low contrast, which makes image analysis difficult or even impossible. In this respect, US 2005 / 0092934 Al describes a fluorescence microscope and method of observing samples using the microscope.

[0005] TIRF microscopy is a method to excite fluorescence in only a very narrow layer of the specimen. This technique is often used in cell biology, where the use of large volumes is compelling for the study of living cells. TIRF microscopy may further be used to study (functionalized) surfaces, where only dyes bound to the surface generate a signal and dyes in the supernatant remain dark. In more detail, TIRF microscopes are powerful tools which are often used in various biomedical applications, including IVD. For example, TIRF microscopy can be applied in Single-Molecule Detection to detect individual molecules with high sensitivity due to its ability to selectively illuminate and excite fluorophores at or near the cell membrane. This is particularly useful in diagnostics for detecting low-abundance biomarkers. Further, TIRF microscopy may be used for studying cell membrane dynamics, specifically for observing processes occurring at the cell membrane, such as receptor-ligand interactions, endocytosis, and exocytosis. This can lead to a better understanding and diagnosis of cellular responses and diseases. Moreover, TIRF microscopy can be used in highly sensitive immunoassays for the detection of specific proteins or antigens. This high sensitivity is crucial for early disease detection and monitoring. TIRF microscopy may also allow real-time imaging of live cells with minimal photo damage, which is beneficial for studying dynamic biological processes relevant to disease progression. Besides the fact, that TIRF microscopy can provide superior resolution, also the contrast is improved making the principle useful for studying the spatial distribution of molecules on the cell surface, which is critical for understanding cellular mechanisms and pathology. Overall, TIRF microscopy has the ability to provide detailed and sensitive measurements at the cellular and molecular levels making it a valuable tool in the field of IVD.

[0006] TIRF microscopy is often used in two ways:

[0007] On the one hand, TIRF microscopy may be used in an objective-based approach in which the sample is illuminated through a detection objective. This setup can typically only be realized with lasers, which results in a limited number of wavelengths in one instrument and high costs. Further, only specific objective lenses with a high numerical aperture and magnification can be used, which limits the range of magnifications and entails high costs. Moreover, adjustment for every sample is complex and time consuming and has to be done by highly trained operators.

[0008] On the other hand, TIRF microscopy may be realized in / by a prism based approach, for which the sample is usually prepared on a surface of a prism. In this respect, B.P. Olveczky et al.: “Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy", BIOPHYSICAL JOURNAL, vol. 73, no. 5, November 1997 (1997-11), pages 2836-2847, XP055077202,ISSN: 0006-3495, DOI: 10.1016 / S0006- 3495(97)78312-7 describes a prism-based approach.

[0009] When using the prism-based approach, reflections may occur at the surfaces of the prism and may lead to signal loss and a high background. The sample usually cannot be examined using transmitted light. In addition, for this approach, illumination is usually realized with lasers, which limits the number of wavelengths in the instrument and which can become expensive. The known principles of TIRF microscopes therefore require improvement with respect to the limitation in the total number of fluorescent dyes, the limitation in the range of magnification, complex and time consuming operations and cost efficiency.

[0010] Summary of the Invention

[0011] It is therefore desirable to develop and improve TIRF microscopes with respect to the limitation in the total number of fluorescent dyes, the limitation in the range of magnification, the number of complex and / or time consuming operations and / or the cost efficiency.

[0012] At least one of these desired improvements are realized by the aspects of this disclosure, i.e. the subject matter defined by the independent claims. Other advantageous effects and / or improvements are achieved by the specific embodiments, i.e. the corresponding subject matter covered by the dependent claims.

[0013] According to an aspect, which may be considered a first aspect, a total internal reflection fluorescence (TIRF) microscope comprises: an excitation light source and an excitation light path, the excitation light source being configured to emit an excitation light propagating along the excitation light path towards a sample with at least one fluorophore and to generate an evanescent wave that is configured to permeate the sample, specifically with a depth in the nm-range, more specifically about 50nm to 200nm, and that is configured to excite an emission of fluorescence emission light by the sample; an optical microscope setup configured to project an image of at least a portion of the sample positioned in an object plane onto an image plane of the optical microscope setup; a sensor configured to be positioned in the image plane and to record the fluorescence emission light emitted by the sample upon excitation; and a total internal reflection (TIR) prism configured to support the sample, the TIR prism comprising: a base surface facing away from the sensor; a sample-supporting surface opposite (for example, without limitation, parallel to) the base surface and facing the sensor; an excitation light incidence surface oriented at an angle of about 0° < oc < 90° with respect to the sample-supporting surface (and / or about 90° < oc < 180° with respect to the base surface) and the excitation light path being configured such that at least a portion of the excitation light hits the excitation light incidence surface and propagates via the TIR prism to the sample-supporting surface; an excitation light exit surface opposite the excitation light incidence surface; and at least one light trap element matching the refractive index of the TIR prism and being optically coupled to the excitation light exit surface of the TIR prism.

[0014] It is an advantage to provide a light trap element matching the refractive index of the TIR prism and being optically coupled to the excitation light exit surface of the TIR prism as light leaving the prism at the second leg is trapped to suppress the background signal. Therefore, the TIRF microscope has an improved sensitivity compared to known TIRF microscopes. Further, using the TIRF microscope according to the first aspect, the sample can be examined in transmitted light, widefield fluorescence and TIRF at a wide range of magnification by the use of standard objective lenses. The handling and / or operating of the TIRF microscope is comparable to usual laboratory microscopes or even simplified. The number of adjustments can be kept low and the system is robust and stable. In other words, not many re-adjustments are required to operate the TIRF microscope.

[0015] TIR allows light to be reflected entirely (no transmission via the reflective interface / surface / transition of refractive indexes) within a medium, making it a valuable phenomenon for various optical applications, especially where high-efficiency light transmission or precise imaging is required. Understanding TIR enables the design and optimization of systems that utilize this effect for enhanced performance in scientific, medical, and communication technologies.

[0016] More precisely, TIR may be considered an optical phenomenon that generally occurs when a light wave traveling through a medium with a higher refractive index (such as glass or water) hits the boundary with a medium of lower refractive index (such as air) at an angle greater than a specific critical angle. When this happens, instead of passing through the boundary and refracting into the second medium, the light wave is entirely reflected back into the first medium.

[0017] The refractive index may be considered a measure of how much the speed of light is reduced inside a medium compared to its speed in a vacuum, being denoted as n. The critical angle, which is the minimum angle of incidence at which total internal reflection occurs can be calculated using the formula:

[0018] 6C= arcsin (tr / t^) where:

[0019] 0Cis the critical angle. ni is the refractive index of the denser medium and = c / v and c is the speed of light in vacuum. n2 is the refractive index of the less dense medium and n2= c / v2and c is the speed of light in vacuum.

[0020] The excitation light path may be configured such that at least a portion of the excitation light hits the sample-supporting surface (and / or the excitation light incidence surface) at an angle of incidence, which corresponds to an angle between the incident light ray and the normal (perpendicular) to the boundary surface, i.e. the sample-supporting surface (and / or the excitation light incidence surface). The conditions for total internal reflection are the following:

[0021] 1. High to Low Refractive Index Transition: TIR occurs only when light travels from a medium with a higher refractive index m to a medium with a lower refractive index n2.

[0022] 2. Angle of Incidence Greater than the Critical Angle: For TIR to occur, the angle of incidence must be greater than the critical angle.

[0023] TIR is essential to TIRF Microscopy, where TIR can be used for example to selectively illuminate and excite fluorophores at or near the surface of a cell membrane, providing high-contrast images of cellular processes occurring at the membrane.

[0024] In other typical applications, TIR is applied in optical fibers, where TIR is the fundamental principle behind the functioning of optical fibers, which are used extensively in telecommunications and medical instruments like endoscopes. Light signals can travel long distances with minimal loss due to repeated internal reflections.

[0025] In general, TIR may use the prism-based approach. Prisms are used in various optical instruments that use TIR to reflect light internally, enhancing the performance of devices such as binoculars and periscopes. In addition, certain types of sensors use TIR to detect changes in the refractive index near the surface, useful in chemical and biological sensing applications.

[0026] The TIR prism is configured for at least one total internal reflection at the sample-supporting surface of the excitation light emitted by the excitation light source (for example, without limitation, broadband and / or narrow band light source) when being incident on the samplesupporting surface at an angle that fulfills the TIR condition. At least one total internal reflection at the sample-supporting surface means that one or more reflections either occur directly at the interface between the sample-supporting surface and the medium on top of the sample-supporting surface, such as the sample or indirectly at the interface between a slide and a sample on top of the slide. In the latter case, the slide may be index matched with the TIR prism and an index matching oil may be provided between the slide and the sample-supporting surface of the TIR prism. The TIRF microscope only requires the occurrence of one single total internal reflection. However, two, three, four, five or more total internal reflections may occur in the TIR prism.

[0027] At the very position at which the light undergoes a total internal reflection, an evanescent wave propagates into the medium with an effective depth of the evanescent wave of about 50-200 nm, depending from the refractive indexes, the angles, the wavelength etc. Generating an evanescent wave that is configured to permeate the sample, specifically with a depth in the nm-range, more specifically about 50nm to 200nm, and that is configured to excite an emission of fluorescence emission light by the sample has the advantage of reducing underground signals and / or scattering that might arise when passing a sample instead of only marginally penetrating with the evanescent wave.

[0028] In general, an evanescent wave is a near-field standing wave that decays exponentially with distance from the interface at which it is formed. These waves commonly arise in the context of total internal reflection, where light is reflected off the boundary between two mediums with different refractive indices. Although the light does not pass into the second medium, an evanescent wave extends into it but quickly diminishes in amplitude - in fact, the evanescent wave in TIRF microscopy excites the fluorescence in the sample. Evanescent waves are significant in various applications, including optical fibers, surface Plasmon resonance (SPR) being utilized in sensors to detect molecular interactions and microscopy techniques, being the key in total internal reflection fluorescence microscopy (TIRFM) for visualizing cell membranes and other thin biological samples.

[0029] It is an advantage in view of light losses due to reflection and / or scattering to provide the excitation light incidence surface having an orientation with respect to the sample-supporting surface that allows introducing the excitation light easily under a rectangular incidence or under the Brewster condition. This is specifically the case, when the excitation light incidence surface is oriented at an angle of about 0° < oc < 90° with respect to the sample-supporting surface (and / or about 90° < oc < 180° with respect to the base surface) and specifically when the excitation light path being configured such that at least a portion of the excitation light hits the excitation light incidence surface and propagates via the TIR prism to the sample-supporting surface.

[0030] The base surface of the TIR prism is oriented in the TIRF microscope facing away from the sensor. The sample-supporting surface is opposite (e.g. in some cases parallel to) the base surface and faces the sensor. The sample-supporting surface is therefore closer to the sensor than the base surface. The base surface may in some cases act like an illumination incidence surface where an illumination light hits the surface of the TIR prism first. The excitation light exit surface is oriented opposite to the excitation light incidence surface, which may be also denoted “entrance surface”. The excitation light exit surface may, without limitation, be oriented at around 90°+ / -20° with respect to the sample-supporting surface and / or the base surface. All mentioned surfaces may have a rectangular and / or a square and / or an irregular shape. At least one light trap element which is matching the refractive index of the TIR prism is optically coupled to, specifically affixed to and / or arranged at the excitation light exit surface of the TIR prism. In other words, one, two, three, four, five, six, or more light trap elements may be optically and specifically physically coupled in a permanent manner to the excitation light exit surface of the TIR prism.

[0031] The light trap element is configured to trap light that reaches the excitation light exit surface, which will mostly be reflected excitation light after TIR and potentially some scattered ambient light and / or fluorescence. The light trap element may comprise a black body. The light trap element may directly or indirectly be attached to the excitation light exit surface of the TIR prism. The light trap element may absorb more than 95%, specifically more than 97% and more specifically more than 99% of the scattered and / or reflected light that reaches the excitation light exit surface.

[0032] The TIRF microscope may be operable with different excitation light sources (an excitation narrowband light source, specifically comprising an LED and / or a Laser medium; an excitation broadband light source, specifically an excitation white light source configured to emit the excitation light which comprises a white excitation light, specifically the white excitation light ranges between 100 nm and 1000 nm, more specifically between 380 nm and 780 nm) without the need for major adjustments and / or changes in the setup of the TIRF microscope. The setup of the TIRF microscope already allows using different types of excitation light sources and possibly exchange them if required. The TIRF microscope may be operable with excitation light sources emitting divergent light such as broadband light sources, specifically an excitation white light sources. The advantage of being able of using a broadband, non-collimated light source (like an arc lamp) provides a significant practical and cost advantage over complex, single-wavelength laser systems which are often used, and it is directly enabled by the inclined-surface prism design.

[0033] Exciting a fluorescence may be realized by taking into account some or all of the (optional) steps as outlined below as a specific embodiment without limitation:

[0034] Select an appropriate fluorophore for providing to a sample: Choose a fluorescent molecule that absorbs light at one wavelength and emits light at another, typically longer wavelength. A sample may already be provided and / or naturally comprise a fluorophore.

[0035] Choose the excitation source: Use an excitation light source that can provide the specific wavelength of light that matches the absorption spectrum of the fluorophore in the sample. Excitation light sources may include LED lights, Laser diodes, and / or Arc lamps (e.g., mercury or xenon). In more detail, the excitation light source may comprise at least one of: an excitation narrowband light source, specifically comprising an LED and / or a Laser medium; an excitation broadband light source, specifically an excitation white light source configured to emit the excitation light which comprises a white excitation light, specifically the white excitation light ranges between 100 nm and 1000 nm, more specifically between 380 nm and 780 nm. Filter the excitation light: Use optical (excitation) filters to narrow down the light to the desired wavelength range that efficiently excites the fluorophore.

[0036] Align the sample and excitation light: Direct the filtered excitation light onto the sample containing the fluorophore. This can be realized by operating and / or aligning elements of the optical microscope setup, a cuvette, or other suitable platforms depending on the experimental setup.

[0037] Collect the emitted fluorescence: The excited fluorophores will emit light at a different wavelength (e.g. without limitation a longer wavelength), which can be captured by using appropriate emission filters and detected using sensors such as Photomultiplier tubes (PMTs), imaging sensors (for example CCD, CMOS), and / or Avalanche photodiodes (APDs)

[0038] Analyze the data: The detected fluorescence can be quantified and analyzed using suitable software to interpret the intensity and other properties of the fluorescence signal. The TIRF microscope may further comprise an illumination light source and an illumination light path, the illumination light source being configured to emit an illumination light along the illumination light path and to illuminate the sample and the illumination light path being configured such that at least a portion of the illumination light hits the base surface and propagates via the TIR prism to the sample-supporting surface, specifically, the illumination light source comprising: an illumination brightfield light source, specifically, wherein the illumination brightfield light source comprises an illumination white light source configured to emit the illumination light which comprises a white illumination light, specifically the white illumination light ranges between 300 nm and 800 nm, more specifically between 380 nm and 780 nm; and / or a narrow band illumination light source, specifically ranging between 300 nm and 800 nm, more specifically between 380 nm and 780 nm.

[0039] The illumination light emitted by the illumination light source allows a user to view the sample while focusing, the light passing through the optical microscope setup, i.e. bringing the sample into the position of the object plane of the optical microscope setup, i.e. adjusting the height / the z- position of the sample. Further, the illumination light allows adjusting the position of the sample in the x / y-plane.

[0040] In general, the term “light path” as used herein, such as the excitation light path and the illumination light path, is defined by the emission direction of the respective light source and optionally by optical elements which can process, form, change and / or deviate the emitted light such that the emitted light once it has passed the respective optical element is somewhat different from before passing the optical element, for example in terms of propagation direction, size and / or shape of light spot, intensity, focus etc.. The light path may be considered the path that at least a portion of the light that is emitted onto the light path propagates / travels along.

[0041] The illumination light path may be considered the path of the illumination light between the illumination light source and the sample. The excitation light path may be considered the path of the excitation light between the excitation light source and the sample or even the excitation light exit surface of the TIR prism. The path between the sample and the sensor that is passed by the fluorescence emission light may be considered a fluorescence emission light path.

[0042] The illumination may, for example, be realized by a broadband light source in combination with one or more different filters, and therefore a broad variety of dyes can be used by simply exchanging the filters and / or using a multipass filter.

[0043] The optical microscope setup may comprise an emission filter for filtering the fluorescence emission light, specifically wherein the emission filter comprises at least one of: a bandpass filter, specifically comprising a bandpass range between 20 to 30 nm, a notch filter, a multipass filter, a longpass filter, specifically wherein the filter is removably and / or exchangeably coupled to the TIRF microscope (100); and / or wherein the excitation light path comprises an excitation filter for filtering the excitation light, specifically wherein the excitation filter comprises at least one of: a bandpass filter, specifically comprising a bandpass range between 20 to 30 nm, a notch filter, a multipass filter, a shortpass pass filter, specifically wherein the filter is removably and / or exchangeably coupled to the light source and / or the TIRF microscope.

[0044] In general, an optical filter is a device used to selectively transmit or block specific wavelengths of light, while allowing others to pass through. The emission filter may for example selectively transmit the wavelength or the wavelength range of the fluorescence emission while other wavelengths, specifically the wavelength of the excitation light are blocked. Whereas, an excitation filter may selectively transmit the wavelength or the wavelength range of the excitation light. In the following, different types of filters which may be used in the context of this disclosure are described in more detail.

[0045] The emission filter may be positioned between the sample that emits the fluorescence emission light and the sensor that should detect the fluorescence emission light. The excitation filter may be positioned between the excitation light source and the TIR prism.

[0046] Bandpass Filters: These filters allow light within a specific wavelength range (the passband) to pass through and block light outside that range. They may specifically be useful in fluorescence microscopy, where it is necessary to isolate particular wavelengths.

[0047] Longpass and Shortpass Filters: Longpass filters transmit wavelengths longer than a specific cutoff wavelength and block shorter wavelengths, whereas shortpass filters do the opposite. Such filters may specifically be used in imaging and sensing applications, including fluorescence microscopy, to control the spectral composition of light.

[0048] The TIR prism may comprise at least two pieces including the at least one light trap element and a single-piece prism or a multi-piece prism, specifically, the TIR prism may comprise two pieces including the at least one light trap element and a monolithic prism defining the base surface, the sample-supporting surface, the excitation light incidence surface and the excitation light exit surface. Alternatively, the TIR prism may comprise three pieces including the at least one light trap element and a two-piece prism comprising a rectangular cuboid prism or cube prism defining the base surface, at least a portion of the sample-supporting surface and the excitation light exit surface combined with a wedge being optically coupled to the rectangular cuboid prism or cube prism and defining the excitation light incidence surface. At least some of the three pieces may be individually commercially available and relatively cheap in production. In other words, at least some of the three pieces may not require being tailor-made for the application. This provides simplicity and / or cost-efficiency when building a TIRF microscope according to this embodiment.

[0049] Alternatively, the TIR prism may comprise four pieces including the at least one light trap element and a three-piece prism comprising a first triangular prism defining the base surface and the excitation light exit surface combined with a second triangular prism being optically coupled to the first triangular prism and defining at least a portion of the sample-supporting surface and combined with a wedge being optically coupled to the second triangular prism and defining the excitation light incidence surface. At least some of the four pieces may be individually commercially available and relatively cheap in production. In other words, at least some of the four pieces may not require being tailor-made for the application. This provides simplicity and / or cost-efficiency when building a TIRF microscope according to this embodiment.

[0050] The TIR prism may for example comprise one, two, three, four, five, six or more light trap elements and a single-piece prism. The TIR prism may for example comprise a single light trap element and a single-piece prism. The TIR prism may for example comprise a single light trap and a multi-piece prism that comprises two, three, four, five six, seven or more components. The TIR prism may for example comprise one, two, three, four, five, six or more light trap elements and a multi-piece prism that comprises two, three, four, five six, seven or more components.

[0051] A cuboid prism or cube prism, as used herein, may be considered as having the form of a dice with all sides having the substantially the same length. Alternatively, the prism may have a shape of a brick with non-parallel planes having different lengths. Brick-shapes and / or dice-shaped prisms have 6 planes / surfaces, comprising three couples of planes which are parallel to each other and perpendicular to the remaining planes / surfaces. In other words: A brick-shaped prism (also known as a rectangular prism) and a dice-shaped prism (a cube) both have 6 faces. These 6 faces comprise three pairs of planes where each pair is parallel to each other and perpendicular to the remaining planes. This means: There are three sets of parallel faces. Each face is perpendicular to the other faces that it is not parallel with. For example, in a rectangular prism: The front and back faces are parallel to each other. The top and bottom faces are parallel to each other and perpendicular to the front, back, and side faces. The left and right faces are parallel to each other and perpendicular to the front, back, top, and bottom faces.

[0052] A wedge, as used herein, has a triangular shape in cross-section. It consists of two inclined planes that come together to form a sharp edge or point at one end. When viewed from the side, the wedge appears as a narrow triangle or an elongated triangle, with a broad base and a narrow tip. The TIR prism may also have a shape, where the base surface and the sample-supporting surface are not parallel, for example, where their planes may enclose a sharp angle larger than about 0° and smaller than about 45°, without limitation.

[0053] An optical coupling, as used herein, refers to a coupling that allows the transmission of light signals from one optical device or medium to another with minimal loss of light. Two media that are optically coupled with each other may preferably be coupled physically via an index matching material, such as an index matching oil that fluidly fills the gap(s) between the two media or an index matching cement that couples the two media not only optically but also couples them physically in a permanent manner, i.e. establishing a bonding between the two media, while the media and the index matching cement are index matched.

[0054] An optical cement may be considered a type of adhesive material specifically designed for bonding optical components, such as lenses and prisms, together. This cement may have certain properties to ensure the optimal performance of the optical system. Key properties often include:

[0055] • Transparency: It needs to be highly transparent to not interfere with the passage of light.

[0056] • Refractive Index: The refractive index of the cement should be similar to that of the optical components to minimize refraction and reflection losses at the interfaces.

[0057] • Stability: The cement must remain stable over time, resisting yellowing or degradation when exposed to light, temperature changes, and environmental factors.

[0058] • Mechanical Strength: It should provide strong adhesion to maintain the alignment and integrity of the optical elements under mechanical stress.

[0059] • Low Shrinkage: During curing, minimal shrinkage is required to avoid inducing stress or distortion in the optical system.

[0060] Optical cements can be cured using different methods, such as UV light, heat, or even a two-part chemical reaction, depending on the specific application and the type of optical cement used.

[0061] Index matching, as used herein refers to the practice of minimizing reflections and maximizing the transmission of light between two optical interfaces of two media by using materials that have the same or similar refractive indexes (similar may refer to a difference of about + / - 0,3, specifically about + / - 0,1 and more specifically about + / - 0,05 in the refractive index). This technique is commonly employed in various photonic applications to enhance the efficiency of light transmission. Each material / medium has a characteristic refractive index, which measures the speed of light in the medium compared to the speed of light in vacuum. When light passes from one material to another with a different refractive index, e.g. from glass to air without limitation, some of the light is reflected at the interface, which can lead to signal loss or image degradation. When joining two optical components, reflections can arise at the air gap between adjacent surfaces. By using an intermediate material with a refractive index close to that of the materials / media being joined, reflections at the interface can be minimized, ensuring that more light passes through. These materials can be in the form of liquids, gels, or solid adhesives. Moreover, anti-reflective coatings may be used as layers with varying refractive indexes to minimize reflections. In addition to using the concept of index matching, the prism may therefore also be coated at the surfaces with anti-reflective coatings to reduce reflections. By carefully matching the refractive indexes of the materials involved, optical systems can achieve higher efficiency and better overall performance.

[0062] The at least one light trap element may cover at least about 10%, specifically at least about 30%, more specifically at least about 50% and even more specifically at least about 90% of the excitation light exit surface; and / or the length of the light trap element may correspond to at least about 25%, specifically at least about 50%, more specifically at least about 75% and even more specifically at least about 90% of the length of the excitation light exit surface.

[0063] The TIRF microscope may further comprise a fiber defining at least a portion of the excitation light path between the excitation light source and the excitation light incidence surface of the TIR prism; and / or a collimating lens or a collimating lens system defining at least a portion of the excitation light path between the excitation light source and the excitation light incidence surface of the TIR prism, specifically between the fiber and the excitation light incidence surface of the TIR prism and configured for collimating at least a portion of the excitation light.

[0064] The fiber may be configured and positioned to transmit at least a portion of the excitation light (for example being emitted by a broadband light source) towards the excitation light incidence surface of the TIR prism. The fiber may comprise a filter and / or filter properties to transmit only a wavelength or a range of wavelengths and block others. The fiber may comprise a single optical fiber or a bundle of optical fibers.

[0065] The collimating lens or the collimating lens system may be configured and positioned to collimate at least a portion of the excitation light. The collimating lens or system may be positioned between the fiber and the excitation light incidence surface of the TIR prism.

[0066] The sensor may comprise a detector and / or a camera; and / or the optical microscope setup may comprise a microscope objective and / or a tube lens, specifically wherein the microscope objective may be removably and / or exchangeably coupled to the TIRF microscope.

[0067] The microscope objective may be removably and / or exchangeably coupled to the TIRF microscope, such that objectives with different magnifications may be swapped in the optical microscope setup without the requirement for substantial rearrangement of the optical microscope setup. This makes it easy and / or efficient for a user to operate the TIRF microscope. Further, the TIRF microscope is very versatile allowing a user to analyze a sample at different magnifications.

[0068] The TIRF microscope may further comprise a collimator defining at least a portion of the illumination light path between the illumination light source and the base surface of the TIR prism and being configured to collimate at least a portion of the illumination light. The TIR prism may comprise at least one of the following materials: an optical glass, such as a crown glass, a flint glass, an optical filter glass, or a colored glass; a technical glass, such as a borosilicate glass, or a white glass; a quartz glass, fused Silica, SiCh, Sapphire, Silicon, MgF?, BaF?, LiF, Ge, Si, GaAs, ZnSe, ZnS, CaF2, Amtir; an optical polymer, such as Polycarbonate, Acrylic (PMMA), Polyester; and / or the at least one light trap element may comprise at least one of the following materials: dyed optical glass, dyed polymer; and / or the optical index matching medium, that establishes the optical coupling between the TIR prism and the at least one light trap element, may comprise at least one of the following materials: an index matching oil, an adhesive, a composite, a gel, an elastomer.

[0069] The excitation light incidence surface may be oriented at an angle about 5° < oc < 90°, specifically at an angle about 10° < oc < 87° and more specifically at an angle about 55° < oc < 85° or 45° < oc < 75° with respect to the sample-supporting surface.

[0070] Alternatively or in addition, the excitation light incidence surface may be oriented at an angle about 95° < oc < 150°, specifically at an angle about 100° < oc < 120° with respect to the illumination light incident surface (A).

[0071] In all cases, the excitation light incidence surface is oriented at and / or forms an angle about 0° < oc < 90° with respect to the sample-supporting surface. The inclination of the excitation light incidence surface allows the excitation light that is directed towards the sample-supporting surface from the side to hit the excitation light incidence surface at a substantially right angle. At least, there is no sharp angle between the incident light and the excitation light incidence surface required to direct the incident light towards the sample-supporting surface as it would be the case if the excitation light incidence surface was not inclined with respect to the sample-supporting surface.

[0072] The sample-supporting surface may be configured to receive the sample directly and / or indirectly, specifically wherein the sample-supporting surface may be configured to receive a slide that is configured to receive the sample, specifically wherein the sample-supporting surface may be configured to receive an immersion oil to match the refractive index between the TIR prism and the slide.

[0073] The samples may be prepared on standard slides and be examined with the TIRF microscope, resulting in stable preparations as well as fluidic cells can be used.

[0074] The slide or a set of slides may or may not be considered as a component of the TIRF microscope. Often slides for supporting samples (object slides) are disposables and comprise a transparent glass that allows the transmission of light in a broad range, specifically in the visible range. The slide may be supported by the sample-supporting surface or may at least be in physical contact with the sample-supporting surface. The physical contact may be a direct contact in which the two surfaces are touching each other or an index matching material such as an index matching oil may be provided in a gap between the slide and the sample-supporting surface. The TIR prism, the slide and / or the index matching material may be index matched to reduce and / or avoid large differences in the refractive indexes and therefore reflections and / or scattering. In all cases, the TIR prism and the slide are optically connected such that at least a portion of an incident light can pass through the TIR prism, the index matching material and the slide to reach the sample.

[0075] The excitation light path may be configured such that at least a portion of the excitation light hits the excitation light incidence surface at an inclined angle of at least about 0° to 60° with respect to the surface normal of the excitation light incidence surface, specifically of at least about 0° to 45° with respect to the surface normal of the excitation light incidence surface, and more specifically of at least about 0° to 20° with respect to the surface normal of the excitation light incidence surface. In other words, the excitation light path or a beam of light propagating along the excitation light path is directed and / or configured such that at least a portion of the excitation light perpendicularly hits the excitation light incidence surface with a maximum allowed deviation from the perpendicular incidence of about + / - 60°, specifically about + / -45° and more specifically about + / -200or oven more specifically about + / -100.

[0076] The excitation light path may be configured such that at least a portion of the excitation light hits the excitation light incidence surface at the Brewster angle. The excitation light path may be configured such that at least a portion of the excitation light hits the excitation light incidence surface at an inclined angle of at least about 0° to 20° with respect to the Brewster angle, more specifically at least about 0° to 5° with respect to the Brewster angle.

[0077] The Brewster angle, also known as the polarization angle, is the angle of incidence at which light with a particular polarization is perfectly transmitted through a transparent dielectric surface, with no reflection. When unpolarized light strikes a surface at this angle, the reflected light will be completely polarized perpendicular to the plane of incidence.

[0078] The said angles or ranges of angles allow at least one total internal reflection to occur at the interface of the slide and the sample (if a slide is provided) or at the interface of the samplesupporting surface of the TIR prism and the sample (if no slide is provided).

[0079] An optical microscope setup may without limitation comprise the following components that may work together to magnify and visualize structures in a sample:

[0080] An optical microscope setup may, without limitation, comprise an illumination system having an illumination light source and a condenser. The illumination light source may provide the necessary illumination to view the sample. Illumination light sources may for example include LEDs, halogen lamps, or lasers as already described. The condenser may focus the illumination light onto the sample. It generally comprises lenses beneath sample-supporting plane and / or the stage and / or the object plane and concentrates and directs the illumination light.

[0081] An optical microscope setup may, without limitation, comprise a sample-supporting element such as a sample-supporting surface, plane and / or stage. If a stage is provided, it serves as a platform, where the sample or specimen slide is placed and / or the TIR prism is received by the platform. The stage may be adjustable in the x, y, and sometimes z directions to accurately position the sample (in the object plane) under the objective lens. The stage may be provided with stage clips and / or the stage may correspond to a mechanical stage that is configured to receive, affix and / or hold the sample slide and / or the sample and / or the TIR prism in place. A mechanical stage allows precise movement of the sample.

[0082] An optical microscope setup may, without limitation, comprise optical components, such as objective lenses corresponding to a set of lenses located close to the sample. They provide primary magnification and are often part of a rotating turret to switch between different magnifications (e.g., about 4x, lOx, 40x, lOOx). Optical components may, without limitation, comprise an eyepiece (also denoted ocular lens) through which a user may look through and which usually provides additional magnification (typically lOx). Some microscopes have binocular or tri-nocular eyepieces for dual or camera-assisted viewing. Optical components may, without limitation, comprise a body tube, corresponding to a housing that connects the eyepiece to the objective lenses and aligns them for proper magnification and focusing.

[0083] An optical microscope setup may, without limitation, comprise a focusing mechanism, which may comprise a coarse focus knob for substantial adjustments to the focus, allowing the user to bring the sample into general focus and / or a fine focus knob for making fine adjustments to the focus, enabling the user to finely tune the image clarity.

[0084] An optical microscope setup may further, without limitation, comprise other components, such as a diaphragm and / or an iris for controlling the amount of light reaching the sample. Adjusting the diaphragm can enhance contrast and resolution. Other components may comprise one or more mirrors, which are sometimes used in place of a built-in light source, particularly in older or simpler microscopes, to reflect external light towards the sample.

[0085] An optical microscope setup may be operated by using some basic usage steps, which may comprise without limitation:

[0086] Preparation: Place the sample on a microscope slide and cover it with a cover slip.

[0087] Illumination: Turn on the light source and adjust the condenser and diaphragm for optimal light and contrast.

[0088] Placement: Position the sample slide on the stage and secure it with stage clips.

[0089] Magnification: Start with the lowest magnification objective lens, usually 4x or lOx.

[0090] Focusing: Use the coarse focus knob to bring the sample into general focus, then refine the focus using the fine focus knob.

[0091] Observation: Look through the eyepiece and adjust the light intensity and diaphragm as needed for the best image quality. Switch to higher magnification objectives if necessary.

[0092] In addition to the fluorescence which uses specific wavelengths of light to excite fluorescent dyes in the specimen, and which allows visualization of specific structures or molecules, other advanced features may be combined, such as a phase contrast for enhancement of the contrast of transparent samples without staining. Further, a camera used as the sensor may be coupled and / or attached to the microscope for capturing images or videos of the sample.

[0093] Specifically, in terms of one or more embodiments described in the present disclosure, an illumination source was developed to upgrade a usual commercial widefield microscope to a TIRF microscope. An important component may be considered a robust (monolithic) prism element, which combines the coupling of the excitation light, an optional illumination of the sample and a light trap. All optical surfaces may be optimized to reduce stray light. The samples may be prepared on standard slides and be examined with the TIRF microscope, meaning stable preparations as well as fluidic cells can be used. Illumination may for example be done by a broadband light source and filters, meaning a broad variety of dyes can be used by easy change of filters. The sample can be examined in transmitted light, widefield fluorescence and TIRF at a wide range of magnification by the use of standard objective lenses. Operating the microscope is comparable to usual laboratory microscopes.

[0094] According to an aspect, which may be considered a second aspect, a TIR prism for use in a TIRF microscope to support the sample comprises: a base surface; a sample-supporting surface opposite to the base surface; an excitation light incidence surface oriented at an angle about 0° < oc < 90° with respect to the sample-supporting surface; an excitation light exit surface opposite the excitation light incidence surface; and at least one light trap element matching the refractive index of the TIR prism and being optically coupled to the excitation light exit surface of the TIR prism via an optical index matching medium.

[0095] The TIR prism may comprise at least two pieces including the at least one light trap element and a single-piece prism or a multi-piece prism, specifically, the TIR prism may comprise two pieces including the at least one light trap element and a monolithic prism defining the base surface, the sample-supporting surface, the excitation light incidence surface and the excitation light exit surface; or the TIR prism may comprise three pieces including the at least one light trap element and a two-piece prism comprising a rectangular cuboid prism or cube prism defining the base surface, at least a portion of the sample-supporting surface and the excitation light exit surface combined with a wedge being optically coupled to the rectangular cuboid prism or cube prism and defining the excitation light incidence surface; or the TIR prism may comprise four pieces including the at least one light trap element and a three-piece prism comprising a first triangular prism defining the base surface and the excitation light exit surface combined with a second triangular prism being optically coupled to the first triangular prism and defining at least a portion of the sample-supporting surface and combined with a wedge being optically coupled to the second triangular prism and defining the excitation light incidence surface. Detailed of the Invention

[0096] In the following, some example embodiments will be described in detail, wherein the invention should not be understood to be limited to the examples and embodiments described. The following examples, embodiments and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. Single features being described in a particular embodiment may be arbitrarily combined, given that they are not excluding each other. In addition, different features, which are provided together in the example embodiments are not to be considered restrictive to the invention.

[0097] Skilled artisans appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements whereas other elements may have been left out or represented in a reduced number in order to enhance clarity and improve understanding of the aspects of the present disclosure.

[0098] The same reference numerals are used among different embodiments and examples for the same or similar elements or elements that have similar or the same effects.

[0099] Fig- 1 is a schematic drawing of a TIRF microscope according to an embodiment;

[0100] Fig. 2a is a schematic drawing of a two-piece TIR prism according to an embodiment;

[0101] Fig. 2b is a schematic drawing of a three-piece TIR prism according to an embodiment; and

[0102] Fig. 2c is a schematic drawing of a four-piece TIR prism according to an embodiment.

[0103] Fig. 1 is a schematic drawing of a TIRF microscope 100 according to an embodiment. The TIRF microscope 100 comprises an excitation light source 101 emitting an excitation light 110 that is guided and therefore propagates at least partially along an excitation light path 108 via a (first) collimating lens(es) 102, a fiber 104, a (second) collimating lens or collimating lens system 105 and a TIR prism 200 towards a sample 106. The sample 106 comprises at least one fluorophore for being excited by the excitation light 110 to emit a fluorescence emission light 107.

[0104] In this embodiment, the (first) collimating lenses 102 comprise two lens elements. An excitation filter 103 is positioned between the two lens elements of the (first) collimating lenses 102. Thus the excitation light path 108 comprises an excitation filter 103 for filtering the excitation light 110, i.e. transmitting light at a wavelength that comprises at least some of the wavelengths of the excitation light 110 and blocking light with other wavelength. The excitation filter 103 may comprise at least one of: a bandpass filter, specifically comprising a bandpass range between 20 to 30 nm, a notch filter, a multipass filter, a shortpass pass filter, specifically wherein the filter is removably and / or exchangeably coupled to the light source and / or the TIRF microscope 100. The fiber 104 defines at least a portion of the excitation light path 108 between the excitation light source 101 and the collimating lens or the collimating lens system 105. Due to the total internal reflections of light inside fibers, the fiber 104 provided in this embodiment of Fig. 1 allows bending the excitation light path 108 around curves without having to add optical components such as mirrors and without losing substantial amounts of light.

[0105] The collimating lens system 105 also defines at least a portion of the excitation light path 108 between the excitation light source 101 and the TIR prism 200. The collimating lens system 105 is configured for collimating at least a portion of the excitation light 110 before hitting the TIR prism 200.

[0106] The TIR prism 200 in Fig. 1 comprises two pieces, namely at least one light trap element 202 and a monolithic prism 201. The monolithic prism 201 defines a base surface A, a sample-supporting surface B, an excitation light incidence surface C and an excitation light exit surface D.

[0107] The sample 106 is positioned in Fig. 1 on a slide 204 that may preferably be index matched with the TIR prism 200. An immersion oil / index matching oil 203 is provided between the TIR prism 200 and the slide 204, namely on the sample-supporting surface B. Since the TIR prism 200, the index matching oil 203 and the slide 204 are index matched, the excitation light 110 experiences a (here one single) total internal reflection at the very transition / interface between the slide surface and the sample 106 or medium above the slide 204. The excitation light 110 creates an evanescent wave with a depth of several nm to several hundred nm, specifically about 50-200 nm. This means that an evanescent wave permeates the sample in a very thin layer and excites the fluorescence needed to perform TIRF microscopy.

[0108] The reflected excitation light 110 travels along the monolithic prism 201 towards the light trap element 202 that is coupled to and index matched with the monolithic prism 201 (forming together a two-piece prism). The light trap element 202 is coupled to the excitation light exit surface D of the monolithic prism 201 by an optical cement that matches the refractive indexes of the monolithic prism 201 and the light trap element 202. Therefore, the optical cement functions as a physical coupling agent and an optical coupling agent. The reflected excitation light 110 is substantially not at all reflected at the interface between the monolithic prism 201 and the light trap element 202 and substantially completely absorbed by the light trap element 202.

[0109] The TIRF microscope 100 comprises an optical microscope setup 300 configured to project an image of at least a portion of the sample 106 that is positioned in an object plane OP onto an image plane IP of the optical microscope setup 300. The sample 106 is therefore actively positioned in the object plane OP. An x-y-z-stage (not shown) may be provided configured for supporting the TIR prism 200 and configured for moving the sample in the x-y plane and along the z axis (tentatively indicated as coordinate system in Fig. 1) to position the sample 106 in the object plane OP. A sensor 304, such as a camera being positioned and / or being configured to be positioned in the image plane IP records the fluorescence emission light 107 that is emitted by the sample 106 when the excitation takes place.

[0110] The sensor 304 may comprises a detector and / or a camera 304. The optical microscope setup 300 further comprises a microscope objective 301 and a tube lens 303, specifically the microscope objective may be removably and / or exchangeably coupled to the TIRF microscope 100 to make it possible to swap the objective.

[0111] The optical microscope setup 300 further comprises an emission filter 302 for filtering the fluorescence emission light 107. The emission filter 302 may comprise at least one of a bandpass filter, specifically comprising a bandpass range between 20 to 30 nm, a notch filter, a multipass filter, a longpass pass filter, specifically wherein the filter is removably and / or exchangeably coupled to the TIRF microscope 100.

[0112] When being correctly positioned in the TIRF microscope 100, the TIR prism that is configured to support the sample 106 has the base surface A facing away from the sensor, the sample-supporting surface B which is opposite (specifically parallel to) the base surface A faces the sensor and the excitation light incidence surface C being oriented at an angle about 0° < oc < 90° with respect to the sample-supporting surface A. The excitation light incidence surface may be oriented at an angle about 5° < oc < 90°, specifically at an angle about 10° < oc < 87° and more specifically at an angle about 55° < oc < 85° or 45° < oc < 75° with respect to the sample-supporting surface.

[0113] The excitation light path 108 is configured such that at least a portion of the excitation light 110 hits the excitation light incidence surface C, preferably at a substantially right angle, i.e. around 0° with respect to the normal of the excitation light incidence surface C, and propagates via the TIR prism 200 to the sample-supporting surface B, as can be seen in Fig. 1.

[0114] In general, the excitation light path 108 may be configured such that at least a portion of the excitation light 110 hits the excitation light incidence surface C at an inclined angle of at least about 0° to 60° (0° + / - 60°) with respect to the surface normal of the excitation light incidence surface C, specifically of at least about 0° to 45° (0° + / - 45°) with respect to the surface normal of the excitation light incidence surface C, and more specifically of at least about 0° to 20° (0° + / - 20°) with respect to the surface normal of the excitation light incidence surface C. The excitation light path 108 may be configured such that at least a portion of the excitation light 110 hits the excitation light incidence surface C at the Brewster angle. The excitation light path 108 may be configured such that at least a portion of the excitation light 110 hits the excitation light incidence surface C at an inclined angle of at least about 0° to 20° with respect to the Brewster angle, more specifically at least about 0° to 5° with respect to the Brewster angle.

[0115] The excitation light exit surface D is opposite the excitation light incidence surface C but is not required to be parallel to the excitation light exit surface D. In Fig. 1, the excitation light exit surface D is perpendicular to the sample-supporting surface B and the base surface A. The excitation light source 101 may comprise at least one of: an excitation narrowband light source, specifically comprising an LED and / or a Laser medium; an excitation broadband light source, specifically an excitation white light source configured to emit the excitation light 110 which comprises a white excitation light, specifically the white excitation light ranges between 100 nm and 1000 nm, more specifically between 380 nm and 780 nm.

[0116] The TIRF microscope 100 further comprises an illumination light source 401 and an illumination light path 404. The illumination light source 401 is configured to emit an illumination light 403 along the illumination light path 404 and to illuminate the sample 106. The illumination light path 404 is configured such that at least a portion of the illumination light 403 hits the base surface A and propagates via the TIR prism to the sample-supporting surface B. The illumination light source 401 may comprise: an illumination brightfield light source, specifically, wherein the illumination brightfield light source comprises an illumination white light source configured to emit the illumination light 403 which comprises a white illumination light, specifically the white illumination light ranges between 300 nm and 800 nm, more specifically between 380 nm and 780 nm, and / or a narrow band illumination light source. The illumination light source 401 may alternatively or in addition comprise an illumination narrowband light source, specifically comprising an LED and / or a Laser medium and / or a filter.

[0117] The TIRF microscope 100 further comprises a collimator 402 defining at least a portion of the illumination light path 404 between the illumination light source 401 and the base surface A of the TIR prism 200 and being configured to collimate at least a portion of the illumination light 403.

[0118] Referring to Fig. 2a-2c, the respective TIR prism 200; 500; 600; 700 of the shown embodiments comprises at least two pieces including the at least one light trap element 202; 502; 602; 703 and a single-piece prism 201; 501 or a multi -piece prism 601, 604; 701, 702, 705. The at least one light trap element 202; 502; 602; 703 is optically and mechanically coupled to the single-piece prism 201; 501 or multi-piece prism 601, 604; 701, 702, 705 via an optical cement 503; 603; 704. In general, the optical cement may be considered an index matching agent that is transparent for at least a portion of the light that is transmitted and mechanically bonds the prism elements with each other.

[0119] Fig. 2a is a schematic drawing of a two-piece TIR prism 500 according to the embodiment shown in Fig- 1 The TIR prism 200; 500 comprises two pieces including the light trap element 502 and a monolithic prism 501 defining the base surface A, the sample-supporting surface B, the excitation light incidence surface C and the excitation light exit surface D. In this embodiment, the light trap element 502 has the same length as the excitation light exit surface D and may therefore fully cover the excitation light exit surface D, i.e. about 100%.

[0120] Fig. 2b is a schematic drawing of a three-piece TIR prism 600 according to an embodiment. The TIR prism 600 comprises three pieces including the at least one light trap element 602 and a two- piece prism 601, 604 comprising a rectangular cuboid prism or cube prism 601 and a wedge 604. The rectangular cuboid prism or cube prism 601 defines the base surface A, at least a portion of the sample-supporting surface B and the excitation light exit surface D. The wedge 604 being mechanically and optically coupled to the rectangular cuboid prism or cube prism 601 via an optical cement 605 and defines the excitation light incidence surface C. In this embodiment, the light trap element 602 is of approximately two thirds of the length of the excitation light exit surface D and may therefore cover the excitation light exit surface D to about 66,66%.

[0121] Fig. 2c is a schematic drawing of a four-piece TIR prism 700 according to an embodiment. The TIR prism 700 comprises four pieces including the at least one light trap element 703 and a three- piece prism 701, 702, 705 comprising a first triangular prism 701, a second triangular prism 702, and a wedge 705. The first triangular prism 701 defines the base surface A and the excitation light exit surface D. The second triangular prism 702 is mechanically and optically coupled to the first triangular prism 701 and defines at least a portion of the sample-supporting surface B. The wedge 705 is mechanically and optically coupled to the second triangular prism 702 and defines the excitation light incidence surface C. In this embodiment, the light trap element 602 is of approximately a third of the length of the excitation light exit surface D and may therefore cover the excitation light exit surface D to about 33,33%.

[0122] In general, the at least one light trap element may cover at least about 10%, specifically at least about 30%, more specifically at least about 50% and even more specifically at least about 90% of the excitation light exit surface D. The length of the light trap element may correspond to at least about 25%, specifically at least about 50%, more specifically at least about 75% and even more specifically at least about 90% of the length of the excitation light exit surface D.

[0123] The TIR prism 200; 500; 600; 700 may comprise at least one of the following materials: an optical glass, such as a crown glass, a flint glass, an optical filter glass, or a colored glass; a technical glass, such as a borosilicate glass, or a white glass; a crystal, such as a quartz glass, fused Silica, SiCh, Sapphire, Silicon, MgF?, BaF?, LiF, Ge, Si, GaAs, ZnSe, ZnS, CaF2, Amtir; an optical polymer, such as Polycarbonate, Acrylic (PMMA), Polyester. The at least one light trap element 202; 502; 602; 703 may comprises at least one of the following materials: dyed optical glass, dyed polymer. The optical index matching medium and / or optical cement that establishes the mechanical and optical coupling between the TIR prism 200; 500; 600; 700 and the at least one light trap element 202; 502; 602; 703 may comprises at least one of the following materials: an index matching oil, an adhesive, a composite, a gel, an elastomer.

[0124] The TIR prism may have, without limitation, dimensions of about 1mm < side length < 10cm, specifically about 3mm < side length < 5cm and / or a volume of about 27cmm < volume < 125ccm.

[0125] Reference list

[0126] 100 Total internal reflection fluorescence (TIRF) microscope

[0127] 101 Excitation light source, e.g. white light source Collimating lens

[0128] Excitation filter

[0129] Fiber

[0130] Collimating lens (-system)

[0131] Sample

[0132] Fluorescence emission light

[0133] Excitation light path

[0134] Fluorescence emission light path

[0135] Excitation light

[0136] TIR prism (two-pieces TIR prism)

[0137] Monolithic prism

[0138] Light trap element

[0139] Immersion oil

[0140] Slide

[0141] Microscope objective

[0142] Emission filter

[0143] Tube lens

[0144] Sensor

[0145] Illumination light source

[0146] Collimator

[0147] Illumination light

[0148] Illumination light path

[0149] TIR prism (two-pieces TIR prism)

[0150] Monolithic prism

[0151] Light trap element, specifically OD filter

[0152] Optical index matching medium, specifically an optical cement

[0153] TIR prism (three-pieces TIR prism)

[0154] Cuboid prism or cube prism

[0155] Light trap element

[0156] Optical index matching medium, specifically an optical cement

[0157] Wedge

[0158] Optical index matching medium, specifically an optical cement

[0159] TIR prism (four-pieces TIR prism)

[0160] First triangular prism

[0161] Second triangular prism

[0162] Light trap element

[0163] Optical index matching medium, specifically an optical cement

[0164] Wedge

[0165] Optical index matching medium, specifically an optical cement

[0166] Optical index matching medium, specifically an optical cement A Base surface oc Angle between Base surface and excitation light incidence surface

[0167] B Sample-supporting surface

[0168] C Excitation light incidence surface

[0169] D Excitation light exit surface

[0170] IP Image plane

[0171] OP Object plane

Claims

- 23 -Patent Claims1. Total internal reflection fluorescence (TIRF) microscope (100) comprising: an excitation light source (101) and an excitation light path (108), the excitation light source (101) being configured to emit an excitation light (110) propagating along the excitation light path (108) towards a sample (106) with at least one fluorophore and to generate an evanescent wave that is configured to permeate the sample and that is configured to excite an emission of fluorescence emission light (107) by the sample (106); an optical microscope setup (300) configured to project an image of at least a portion of the sample (106) positioned in an object plane (OP) onto an image plane (IP) of the optical microscope setup (300); a sensor (304) configured to be positioned in the image plane (IP) and to record the fluorescence emission light (107) emitted by the sample (106); and a total internal reflection (TIR) prism (201; 501; 600; 700) configured to support the sample (106), the TIR prism (201; 501; 600; 700) comprising: a base surface (A) facing away from the sensor; a sample-supporting surface (B) opposite to the base surface (A) and facing the sensor; an excitation light incidence surface (C) oriented at an angle 0° < oc < 90° with respect to the sample-supporting surface (B) and the excitation light path (108) being configured such that at least a portion of the excitation light (110) hits the excitation light incidence surface (C) and propagates via the TIR prism (200; 500; 600; 700) to the samplesupporting surface (B); an excitation light exit surface (D) opposite the excitation light incidence surface (C); and at least one light trap element (202; 502; 602; 703) matching the refractive index of the TIR prism (200; 500; 600; 700) and being optically coupled to the excitation light exit surface (D) of the TIR prism (201; 501; 600; 700).

2. The TIRF microscope (100) of claim 1, wherein the excitation light source (101) comprises at least one of: an excitation narrowband light source, specifically comprising an LED and / or a Laser medium; an excitation broadband light source, specifically an excitation white light source configured to emit the excitation light (110) which comprises a white excitation light, specifically the white excitation light ranges between 100 nm and 1000 nm, more specifically between 380 nm and 780 nm.

3. The TIRF microscope (100) of claim 1 or 2, further comprising an illumination light source (401) and an illumination light path (404), the illumination light source (401) being configured to emit an illumination light (403) along the illumination light path (404) and to illuminate the sample (106) and the illumination light path (404) being configured such that at least a portion of the illumination light (403) hits the base surface (A) and propagates via the TIR prism to the sample-supporting surface (B), specifically, the illumination light source (401) comprising: an illumination brightfield light source, specifically, wherein the illumination brightfield light source comprises an illumination white light source configured to emit the illumination light (403) which comprises a white illumination light, specifically the white illumination light ranges between 300 nm and 800 nm, more specifically between 380 nm and 780 nm; and / or a narrow band illumination light source.

4. The TIRF microscope (100) of any one of the preceding claims, wherein the optical microscope setup (300) comprises an emission filter (302) for filtering the fluorescence emission light (107), specifically wherein the emission filter (302) comprises at least one of: a bandpass filter, specifically comprising a bandpass range between 20 to 30 nm, a notch filter, a multipass filter, a longpass filter, specifically wherein the filter is removably and / or exchangeably coupled to the TIRF microscope (100); and / or wherein the excitation light path (108) comprises an excitation filter (103) for filtering the excitation light (110), specifically wherein the excitation filter (103) comprises at least one of: a bandpass filter, specifically comprising a bandpass range between 20 to 30 nm, a notch filter, a multipass filter, a shortpass pass filter, specifically wherein the filter is removably and / or exchangeably coupled to the light source and / or the TIRF microscope (100).

5. The TIRF microscope (100) of any one of the preceding claims, wherein the TIR prism (200; 500; 600; 700) comprises at least two pieces including the at least one light trap element (202; 502; 602; 703) and a single-piece prism (201; 501) or a multi-piece prism (601, 604; 701, 702, 705), specifically, wherein the TIR prism (200; 500) comprises two pieces including the at least one light trap element (202; 502) and a monolithic prism (201; 501) defining the base surface (A), the samplesupporting surface (B), the excitation light incidence surface (C) and the excitation light exit surface (D); or wherein the TIR prism (600) comprises three pieces including the at least one light trap element (602) and a two-piece prism (601, 604) comprising a rectangular cuboid prism or cube prism (601) defining the base surface (A), at least a portion of the sample-supporting surface (B) and the excitation light exit surface (D) combined with a wedge (604) being optically coupled to the rectangular cuboid prism or cube prism (601) and defining the excitation light incidence surface (C); orwherein the TIR prism (700) comprises four pieces including the at least one light trap element (703) and a three-piece prism (701, 702, 705) comprising a first triangular prism (701) defining the base surface (A) and the excitation light exit surface (D) combined with a second triangular prism (702) being optically coupled to the first triangular prism (701) and defining at least a portion of the sample-supporting surface (B) and combined with a wedge (705) being optically coupled to the second triangular prism (702) and defining the excitation light incidence surface (C).

6. The TIRF microscope (100) of any one of the preceding claims, wherein the at least one light trap element (202; 502; 602; 703) covers at least 10%, specifically at least 30%, more specifically at least 50% and even more specifically at least 90% of the excitation light exit surface (D); and / or wherein the length of the light trap element (202; 502; 602; 703) corresponds to at least a 25%, specifically at least 50%, more specifically at least 75% and even more specifically at least 90% of the length of the excitation light exit surface (D).

7. The TIRF microscope (100) of any one of the preceding claims, further comprising: a fiber (104) defining at least a portion of the excitation light path (108) between the excitation light source (101) and the excitation light incidence surface (C) of the TIR prism (200; 500; 600; 700); and / or a collimating lens or a collimating lens system (105) defining at least a portion of the excitation light path (108) between the excitation light source (101) and the excitation light incidence surface (C) of the TIR prism (200; 500; 600; 700), specifically between the fiber (104) and the excitation light incidence surface (C) of the TIR prism (200; 500; 600; 700) and configured for collimating at least a portion of the excitation light (110).

8. The TIRF microscope (100) of any one of the preceding claims, wherein the sensor (304) comprises a detector and / or a camera (304); and / or wherein the optical microscope setup (300) comprises a microscope objective (301) and / or a tube lens (303), specifically wherein the microscope objective is removably and / or exchangeably coupled to the TIRF microscope (100).

9. The TIRF microscope (100) of any one of claims 3 to 8, further comprising a collimator (402) defining at least a portion of the illumination light path (404) between the illumination light source (401) and the base surface (A) of the TIR prism (200; 500; 600; 700) and being configured to collimate at least a portion of the illumination light (403).

10. The TIRF microscope (100) of any one of the preceding claims,- 26 - wherein the TIR prism (200; 500; 600; 700) comprises at least one of the following materials: an optical glass, such as a crown glass, a flint glass, an optical filter glass, or a colored glass; a technical glass, such as a borosilicate glass, or a white glass; a quartz glass, fused Silica, SiCh, Sapphire, Silicon, MgF?, BaF?, LiF, Ge, Si, GaAs, ZnSe, ZnS, CaF2, Amtir; an optical polymer, such as Polycarbonate, Acrylic (PMMA), Polyester; and / or wherein the at least one light trap element (202; 502; 602; 703) comprises at least one of the following materials: dyed optical glass, dyed polymer; and / or wherein the optical index matching medium that establishes the optical coupling between the TIR prism (200; 500; 600; 700) and the at least one light trap element (202; 502; 602; 703) comprises at least one of the following materials: an index matching oil, an adhesive, a composite, a gel, an elastomer.

11. The TIRF microscope (100) of any one of the preceding claims, wherein the excitation light incidence surface (C) is oriented at an angle of:5° < oc < 90°, specifically at an angle 10° < oc < 87° and more specifically at an angle 55° < oc < 85° with respect to the sample-supporting surface (B); and / or95° < oc < 150°, specifically at an angle 100° < oc < 120° with respect to the illumination light incident surface (A).

12. The TIRF microscope (100) of any one of the preceding claims, wherein the samplesupporting surface (B) is configured to receive the sample (106) directly and / or indirectly, specifically wherein the sample-supporting surface (B) is configured to receive a slide (204) that is configured to receive the sample (106), specifically wherein the sample-supporting surface (B) is configured to receive an immersion oil (203) to match the refractive index between the TIR prism (200; 500; 600; 700) and the slide (204).

13. The TIRF microscope (100) of any one of the preceding claims, wherein the excitation light path (108) is configured such that at least a portion of the excitation light (110) hits the excitation light incidence surface (C) at an inclined angle of at least 0° to 60° with respect to the surface normal of the excitation light incidence surface (C), specifically of at least 0° to 45° with respect to the surface normal of the excitation light incidence surface (C), and more specifically of at least 0° to 20° with respect to the surface normal of the excitation light incidence surface (C); and / or wherein the excitation light path (108) is configured such that at least a portion of the excitation light (110) hits the excitation light incidence surface (C) at the Brewster angle; and / or wherein the excitation light path (108) is configured such that at least a portion of the excitation light (110) hits the excitation light incidence surface (C) at an inclined angle of at least 0° to 20c- 27 - with respect to the Brewster angle, more specifically at least 0° to 5° with respect to the Brewster angle.

14. A TIR prism (200; 500; 600; 700) for use in a TTRF microscope (100) to support the sample (106), the TIR prism comprising: a base surface (A); a sample-supporting surface (B) opposite to the base surface (A); an excitation light incidence surface (C) oriented at an angle 0° < oc < 90° with respect to the sample-supporting surface (B); an excitation light exit surface (D) opposite the excitation light incidence surface (C) ; and at least one light trap element (202; 502; 602; 703) matching the refractive index of the TIR prism (200; 500; 600; 700) and being optically coupled to the excitation light exit surface (D) of the TIR prism (200; 500; 600; 700) via an optical index matching medium, wherein the TIR prism (600) comprises three pieces including the at least one light trap element (602) and a two-piece prism (601, 604) comprising a rectangular cuboid prism or cube prism (601) defining the base surface (A), at least a portion of the sample-supporting surface (B) and the excitation light exit surface (D) combined with a wedge (604) being optically coupled to the rectangular cuboid prism or cube prism (601) and defining the excitation light incidence surface (C); or wherein the TIR prism (700) comprises four pieces including the at least one light trap element (703) and a three-piece prism (701, 702, 705) comprising a first triangular prism (701) defining the base surface (A) and the excitation light exit surface (D) combined with a second triangular prism (702) being optically coupled to the first triangular prism (701) and defining at least a portion of the sample-supporting surface (B) and combined with a wedge (705) being optically coupled to the second triangular prism (702) and defining the excitation light incidence surface (C).

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