Microscope system with oblique illumination

Abstract

SOLUTION: A microscope system has an illumination optical system comprising a multi-mode optical fiber having an egress for emitting a laser beam. The egress is located in a plane that is conjugate to a microscope sample plane. The illumination optical system is configured such that the laser beam is incident on the objective lens laterally displaced from the principal optical axis of the objective lens in order that the objective lens delivers the laser beam to the sample plane obliquely to the principal optical axis.EFFECT: Utilization of a multi-mode optical fiber for laser delivery in an oblique illumination microscope, such as a TIRF microscope, solves problems associated with using single-mode optical fibers such as alignment and uniformity of illumination.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an oblique illumination microscope, particularly, but not limited to, a total internal reflection (TIR) microscope.

Background Art

[0002] In an optical microscope, illuminating a sample at an oblique angle with respect to the optical axis of the microscope is called oblique illumination. Oblique illumination can observe a sample with higher contrast than that obtained using bright-field illumination parallel to the optical axis. Thereby, the visibility of the sample can be improved. For example, oblique illumination is particularly suitable for use with transparent or colorless samples.

[0003] TIR microscopes, including total internal reflection fluorescence (TIRF) microscopes, are widely recognized as one of the best surface inspection imaging methods and are routinely used to observe biological interactions at the cell membrane interface. Many commercially available TIRF microscopes utilize single-mode optical fibers to irradiate laser light at an oblique angle through a high numerical aperture objective lens. However, single-mode optical fibers are difficult to adjust and are subject to wavelength and power limitations. Also, when used as a light source for TIRF illumination, single-mode optical fibers show non-uniform (Gaussian) illumination analysis results, and the coherence of the light emitted from the fiber tends to lead to the generation of interference fringes that distort and degrade the quality of the TIRF image. Many applications of TIR microscopes, such as quantitative TIRF imaging and related super-resolution imaging applications, can benefit from eliminating or reducing the disadvantages associated with single-mode optical fiber laser irradiation.

Summary of the Invention

[0004] In a first aspect, the present invention provides the following: A microscope system for imaging a sample placed on a sample surface, comprising: an objective lens of a microscope; and an illumination optical system configured to illuminate the sample surface with a light beam through the objective lens, the illumination optical system having a multimode optical fiber having an outlet for emitting the light beam, wherein the outlet of the illumination optical system is located at a beam outlet surface conjugate to the sample surface, and the illumination optical system is configured such that the light beam is displaced laterally from the principal optical axis of the objective lens and incident on the objective lens so that the objective lens irradiates the sample surface with the light beam at an oblique angle with respect to the principal optical axis of the objective lens.

[0005] Preferably, the illumination optical system is configured such that the light beam is incident on the peripheral portion of the objective lens.

[0006] Preferably, the illumination optical system is configured to image the beam exit surface to an equivalent sample surface that is intermediate between the beam exit surface and the sample surface and conjugate to the sample surface. A preferred illumination optical system includes at least one lens configured to image the beam exit surface to the equivalent sample surface.

[0007] In a preferred embodiment, the illumination optical system comprises at least one collimating lens having a front focal plane and a back focal plane that coincide with the beam exit plane, and the illumination optical system is configured to relay the back focal plane of the at least one collimating lens to the back focal plane of the objective lens. Preferably, the at least one lens comprises the at least one collimating lens and at least one imaging lens, and the at least one imaging lens is configured to form an image of the beam exit on the equivalent sample plane.

[0008] Preferably, the illumination optical system comprises at least one lens positioned between the equivalent sample plane and the objective lens, configured to collimate light from the sample plane to the back focal plane of the objective lens. Optionally, the at least one lens includes a microscope tube lens.

[0009] In a preferred embodiment, the illumination optical system includes positioning means for positioning the light beam such that the light beam is displaced laterally from the principal optical axis of the objective lens and incident on the objective lens. The positioning means may include one or more optical components configured to move the light beam relative to the principal optical axis of the microscope system, optionally tilt the light beam, and / or move the light beam laterally with respect to the principal axis. The at least one collimating lens is optionally positioned such that its optical axis is displaced laterally from the principal optical axis of the microscope system.

[0010] In a preferred embodiment, the multimode optical fiber is connected to a light source for generating the light beam. The light source is typically a laser light source, and the light beam is a laser beam, preferably a homogenized laser beam.

[0011] In some embodiments, the illumination optical system is configured such that the angle causes total internal reflection at the sample interface.

[0012] Advantageously, the illumination optical system is configured to focus the beam exit onto the sample surface.

[0013] In a preferred embodiment, the illumination optical system comprises at least one collimating lens arranged to collimate light from the beam exit, and at least one relay lens arranged to relay the light collimated by the at least one collimating lens to the back focal plane of the objective lens. The at least one relay lens is preferably configured to relay light from the back focal plane of the at least one collimating lens to the back focal plane of the objective lens.

[0014] Advantageously, the illumination optical system is configured to image the beam outlet onto the sample surface.

[0015] Preferably, the multimode optical fiber has a square or rectangular cross-sectional shape.

[0016] Typically, the illumination optical system includes means for homogenizing the light beam.

[0017] In a second aspect, the present invention provides: an illumination optical system for a microscope system for imaging a sample placed on a sample surface, comprising a multimode optical fiber having an outlet for emitting the light beam, configured to illuminate the sample surface with a light beam through the objective lens of the microscope system, the outlet being configured to be located at a beam outlet surface conjugate to the sample surface, and the light beam being displaced laterally from the principal optical axis of the objective lens and incident on the objective lens, such that the objective lens irradiates the sample surface with the light beam at an oblique angle with respect to the principal optical axis of the objective lens.

[0018] In a third aspect, the present invention provides: a method for illuminating a sample surface of a microscope system through an objective lens, comprising: positioning the light beam exit of a multimode optical fiber at a beam exit surface conjugate to the sample surface; illuminating the sample surface with a light beam from the multimode optical fiber; and displacing the light beam laterally from the principal optical axis of the objective lens when the light beam is incident on the objective lens. Preferably, the method comprises focusing the beam exit on the sample surface and / or imaging the beam exit. Preferably, the method comprises collimating the light from the beam exit and relaying the collimated light to the back focal plane of the objective lens, preferably, the relay comprising relaying the light from the back focal plane of at least one collimating lens to the back focal plane of the objective lens.

[0019] From a fourth aspect, the present invention provides the following: A microscope system for imaging a sample placed on a sample surface, comprising: an objective lens of a microscope; and an illumination optical system configured to illuminate the sample surface with a light beam through the objective lens, the illumination optical system having a multimode optical fiber having an outlet for emitting the light beam, wherein the illumination optical system is configured to image the outlet onto the sample surface and / or focus the outlet, and the illumination optical system is configured such that the light beam is displaced laterally from the principal optical axis of the objective lens and incident on the objective lens so that the objective lens irradiates the sample surface with the light beam at an oblique angle with respect to the principal optical axis of the objective lens.

[0020] From a fifth aspect, the present invention provides: an illumination optical system for a microscope system for imaging a sample placed on a sample surface, comprising a multimode optical fiber having an outlet for emitting the light beam, configured to illuminate the sample surface with a light beam through the objective lens of the microscope system, and configured to image the outlet onto the sample surface and / or focus the outlet, wherein the light beam is displaced laterally from the principal optical axis of the objective lens and incident on the objective lens so that the objective lens irradiates the sample surface with the light beam at an oblique angle with respect to the principal optical axis of the objective lens.

[0021] A sixth aspect of the present invention provides the following: a method for illuminating a sample surface of a microscope system through an objective lens, comprising: illuminating the sample surface with a light beam from a multimode optical fiber; imaging and / or focusing the beam exit of the multimode optical fiber onto the sample surface; and displacing the light beam laterally from the principal optical axis of the objective lens when the light beam is incident on the objective lens.

[0022] Conventionally, when using a single-mode fiber, the laser analysis results are Gaussian, and this state is maintained (only the size changes) on the sample plane, the back focal plane, and any equivalent plane. This is a unique property of single-mode fibers and Gaussian beams (the Fourier transform of a Gaussian beam is another Gaussian beam). Therefore, in the conventional configuration where the tip of a single-mode fiber is imaged onto the BPF of the objective lens, the analysis results of the Gaussian distribution illumination at the fiber tip are imaged onto the BPF of the objective lens and reproduced on the sample plane. On the other hand, multimode fibers do not have the same properties, so where and how the beam from the multimode fiber is imaged is more important in order to produce the desired illumination (preferably non-Gaussian) on the sample plane. In a preferred embodiment of the present invention, the tip of the multimode fiber is imaged onto the sample plane. This is in contrast to the conventional configuration where the tip of a single-mode fiber is imaged onto the BPF of the objective lens.

[0023] In a preferred embodiment, oblique illumination, and optionally total internal reflection (TIR) ​​illumination, is performed through a microscope utilizing multimode optical fibers. Advantageously, the exit or tip of the fiber is positioned conjugate to the sample plane of the microscope (e.g., to provide critical illumination of the sample). A preferred arrangement is such that the light emitted from the fiber is focused at or above a critical angle towards the periphery of the objective lens (preferably with a numerical aperture of 1.4 or greater) to produce total internal reflection (TIR) ​​at the substrate-sample interface, or to illuminate the sample at an oblique angle with respect to the principal optical axis of the microscope.

[0024] In a preferred embodiment, laser light from one or more lasers is generated and scrambled in a laser engine including a homogenizer. The laser light is coupled to a multimode optical fiber, and the exit tip of the fiber is positioned at the focal point of a first lens, preferably a collimating lens. A second lens, preferably with a longer focal length than the first lens, creates an image of the fiber tip, preferably a magnified image, on the equivalent sample plane. The image of the fiber tip is relayed through the objective lens of a microscope, typically via a beam splitter such as a dichroic mirror, and projected or focused onto the sample plane (the surface on which the target object or sample, e.g., the object under inspection on a substrate, is located). Light emitted from the sample, e.g., fluorescence, is captured by the objective lens and focused onto the detection element of a photodetector via the tube lens of a microscope (typically via a beam splitter and emission filter). Advantageously, the surface of the fiber tip, the equivalent sample plane, the sample plane, and the surface of the detection element are all conjugate to each other. Typically, to position the laser beam in the peripheral area of ​​the back focal plane (BFP) of the objective lens, one or more optical elements are used that can translate the beam laterally relative to the optical axis of the microscope, preferably so that the laser beam undergoes total internal reflection at the substrate-sample interface. In some embodiments, this lateral translation of the beam is achieved by a rotating flat optical system (TO) positioned between a first lens and a second lens. In other embodiments, the lateral translation of the beam is achieved by translating a third lens positioned between the second lens and the objective lens laterally relative to the optical axis of the microscope.

[0025] Using multimode optical fibers for laser irradiation in TIRF microscopes and oblique illumination microscopes solves the aforementioned problems associated with single-mode optical fibers. The scrambled or homogenized output of multimode optical fibers provides flat, uniform, and interference-free illumination analysis results when used as an illumination source. This makes it easier to prevent and eliminate image artifacts caused by interference fringes superimposed on fluorescence images of biological samples, for example, when using a coherent free-space emitted laser beam in a conventional TIRF illumination system or a coherent laser beam emitted from a single-mode optical fiber. Furthermore, multimode fibers are easy to adjust, have a broad spectrum of acceptable laser wavelengths (extending from UV to NIR), and allow for higher laser power input to the sample.

[0026] Advantageously, several embodiments of the present invention facilitate the implementation of imaging applications requiring high-laser-power TIR illumination (e.g., super-resolution imaging of biological membrane surfaces). The present invention is not limited to use in fluorescence applications. For example, embodiments of the present invention may be used in measurements via reflected light imaging for physical science. Embodiments of the present invention may be adapted to be used as standalone illumination modules fitted into existing microscopes, or they may be integrated into microscopes or other imaging or analytical systems. [Brief explanation of the drawing] [Figure 1] Figure 1 is a schematic diagram of a microscope system embodying one aspect of the present invention, which includes an illumination system embodying another aspect of the present invention. [Figure 2A] Figure 2A is a side view of a first exemplary sample holder for holding a sample to be imaged by a microscope system. [Figure 2B] Figure 2B is a side view of a second exemplary sample holder for holding a sample to be imaged by a microscope system. [Figure 3] Figure 3 is a block diagram of a light source module suitable for use in the microscope system shown in Figure 1. [Figure 4] FIG. 4 is an alternative schematic view of the illumination system of FIG. 1 and includes rays of light indicating the passage of light from the tip of the fiber to the sample surface.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Referring now to FIGS. 1, 2A, and 2B of the drawings, there is shown a microscope system 100 embodying one aspect of the present invention, which is typically designated 100. The microscope system 100 includes an optical microscope, which in a preferred embodiment is a wide-field microscope, although as will be apparent to those skilled in the art, other types of microscopes may be used.

[0029] The microscope system 100 includes a support or holder 2 for receiving an object 3 to be imaged. The object 3 typically includes a sample, such as a biological specimen or a chemical substance, for example. The object or sample 3 may be placed on a substrate 4, such as on a slide 4A in the example of FIG. 2B, or within a container 9 in the example of FIG. 2A. Usually, at least a portion of the sample is present at the bottom of the container 9. The sample may be immersed in a medium, typically a liquid medium such as water or an aqueous biological medium. In the example of FIG. 2A, the sample 3 is supplied to a liquid medium 11 within the container 9. A cover slide 5 may be placed over the sample or non-inspected object as needed, and is typically placed between the sample and the objective lens of the microscope system. In the example of FIG. 2B, the sample 3 is placed between the slide 4A and the cover slide 5 and may be immersed in any suitable medium. The object, or sample 3, is placed on a sample surface 6.

[0030] The microscope system 100 includes an imaging optical system 20 for imaging a sample 3 onto an optical detector 14 (typically including a camera). The imaging optical system 20 is configured to focus the image of the sample 3 onto the focal plane 13 of the detector 14, for example, the image sensor of the camera. The imaging optical system 20 may have various configurations and typically includes a row of optical devices that provide an optical path. The optical row usually includes at least one lens positioned to image the sample 3 onto the detector 14, i.e., to form an image of the sample 3 on the detector's focal plane via the optical row. The optical row may optionally include one or more other optical components, e.g., one or more mirrors and / or one or more beam splitters and / or one or more filters. In a preferred embodiment, the optical detector 14 is a digital camera having a digital image sensor 15, such as a CCD sensor. The imaging optical system 20 images the sample 3 onto the image sensor 15. More specifically, it is desirable that the imaging optical system 20 focuses the image of sample 3 onto the sensor 15 (where the image sensing surface of sensor 15 is positioned on the focal plane 13 of the imaging optical system 20).

[0031] The imaging optical system 20 includes a microscope objective lens 7 having a back focal plane (BFP) 8, typically an infinity-corrected microscope objective lens. The objective lens 7 typically has an optical axis perpendicular to the object plane 6.

[0032] The imaging optical system 20 typically comprises at least one tube lens 10 configured, together with the objective lens 7, to form a magnified image of the sample 3 at the detector 14. It will be understood that the optical array of the imaging optical system 20 may comprise one or more additional optical components depending on the embodiment.

[0033] The microscope system 100 includes an illumination optical system 22 for illuminating the sample 3. The illumination optical system 22 includes or is connectable to the light source 24 and is a laser light source comprising one or more laser devices (not shown) in a preferred embodiment, but may alternatively comprise any other suitable conventional light source, such as one or more LEDs. The light source 24 may be configured to produce light, preferably laser light, in one or more frequency bands, as is appropriate for the application and will be obvious to those skilled in the art. For example, if the sample 3 contains a non-test material that can fluoresce (because it can fluoresce intrinsically, i.e., due to autofluorescence, or because one or more fluorescent markers (e.g., proteins or dyes) have been added to the sample), the light source 24 may be configured to provide light in one or more frequency bands to excite the non-test material / markers and cause fluorescence.

[0034] In a preferred embodiment, the illumination optics system 22 is configured to illuminate the sample 3 by directing a light beam 25 (a laser beam in this example), which may consist of one or more wavelengths of light corresponding to the fluorescence properties of the sample / marker, through the objective lens 7 to the sample 3. Preferably, a single light beam 25 is present. The light beam 25 is preferably homogenized. Preferably, the beam 25 contains incoherent light. The illumination optics system 22 comprises rows of optical components, one or more of which are shared with the optical rows of the imaging optics system 20 in a preferred embodiment. Thus, the illumination light 25 may be directed along or parallel to at least a portion of the optical path of the imaging optics system 20. To facilitate this, a beam splitter 12 may be provided in the imaging optics system 20 or otherwise provided to the optical path. The beam splitter 12 is configured such that light from the light source 24 reaches the sample 3 and light 27 emitted from the sample 3 reaches the optical detector 14. In the illustrated embodiment, the beam splitter 12 is configured to reflect or partially reflect (i.e., split) light in one or more frequency bands corresponding to the light generated by the light source 24. The light source 24 is positioned to direct a beam 25 toward the beam splitter 12, and at least a portion of the beam 25 is directed toward the sample 3 via the objective lens 7. In this example, the beam splitter 12 is configured to be transparent to light in one or more frequency bands corresponding to the light reflected or emitted from the sample 3, so that the reflected / emitted light 27 can reach the optical detector 14. The beam splitter 12 may also be said to have one or more transmission bands corresponding to the light emitted from the sample 3 and one or more reflection bands corresponding to the light generated by the light source 24. In the illustrated embodiment, the beam splitter 12 is positioned between the tube lens 10 and the optical detector 14. Typically, the beam splitter 12 includes a dichroic mirror. In alternative embodiments, the tube lens 10 may be positioned elsewhere in the optical path, for example, between the beam splitter 12 and the optical detector 14.

[0035] In some embodiments, sample 3 includes a sample that fluoresces when excited by light from the illumination optical system 22 (either by autofluorescence or by a fluorescent marker (or label) contained in the sample). Therefore, when the microscope 100 operates in imaging mode, it is the fluorescent light emitted from the sample that is imaged by the imaging optical system 20 to the camera 14.

[0036] Typically, an emission filter 17 is provided in the optical path to filter the light 27 emitted from the sample 3, so that only light in one or more target wavelength bands can reach the optical detector 14. For example, the emission filter 17 may be configured to transmit light in one or more wavelength bands corresponding to the fluorescence emitted from the sample during use. If multiple target wavelength bands are to be targeted, multiple different interchangeable emission filters may be provided, for example, in a filter selector device, so that the appropriate emission filter is placed in the optical path depending on the target wavelength bands that may be targeted at any given time. In the illustrated embodiment, the filter 17 is located adjacent to the optical detector 14, but alternatively, it may be located elsewhere in the optical path as convenient.

[0037] Figure 3 shows an example of a light source module that can be used as a light source 24. The light source module comprises one or more primary light sources 324 for generating and emitting light. In a typical embodiment, the light is in the ultraviolet (UV), visible, or near-infrared (NIR) frequency band. In a preferred embodiment, the light is laser light, and each light source 324 comprises a laser device. However, each light source may include, but is not limited to, light-emitting diodes (LEDs), laser diodes, solid-state devices, superluminescent diodes (SLDs), or arc lamps, and any other light source capable of generating and emitting light. Each light source 324 is configured to generate and emit light, or emit light, at one or more wavelengths. In the example in Figure 3, two laser devices 324 are shown, but it will be understood that in alternative embodiments, there may be a single light source or more than two light sources. Each laser device 324 may be a single-mode laser device or a multi-mode laser device. In a preferred embodiment, the light source module emits light, preferably laser light, at multiple wavelengths. As illustrated, this can be achieved by providing module 24 with multiple light sources 324, each generating light at a different wavelength or frequency band. Alternatively, the light source module 24 may emit light at a single wavelength.

[0038] In embodiments where the light source module has two or more light sources 324, it may include an optical coupling unit 326 having one or more optical elements arranged in any conventional manner to combine the respective beams from each light source 324 to produce an output light beam 25. The optical coupling unit 326 may include, for example, one or more mirrors and / or beam splitters and / or dichroic beam couplers.

[0039] Optionally, the light source module includes an optical control and adjustment unit 328 for manipulating the cross-sectional shape and / or dimensions of the beam 25 emitted from the module 24. In embodiments where two or more light sources 324 are present, the control and adjustment unit 328 may include a separate control and adjustment subunit (not shown) for each light source 324. The control and adjustment unit 328 (or each of its subunits, if applicable) may include any optical elements or combinations of optical elements (not shown) for controlling and adjusting the emitted beam in any desired conventional manner. For example, the control and adjustment unit 328 (or each of its subunits, if applicable) may include a Galilean telescope beam expander, or an anamorphic prism or cylindrical optical system, and / or any other conventional beam shaping means, and / or an acousto-optic tunable filter (AOTF), or a mechanical shutter.

[0040] In the illustrated embodiment, the optical coupling unit 326 is positioned before the optical adjustment unit 328 so that it is adjusted after the light from the laser light source 324 is coupled. In an alternative embodiment (not shown), the optical coupling unit 326 is positioned after the optical adjustment unit 328 so that it is adjusted before the light from the laser light source 324 is coupled.

[0041] The light source module 24 may include an optical coupler 329 from which the output light beam 25 is emitted. The optical coupler 329 includes one or more optical elements (not shown) for directing or optically coupling the beam to an optical fiber 30 (see Figure 1). Typically, the optical coupler 329 includes one or more lenses (e.g., a collimator lens, a focusing lens, and / or a micro-objective lens) for focusing the output beam to the end of the optical fiber 30. The optical fiber 30 may be mechanically coupled to the module 24 using any convenient coupling device or means (not shown). Alternatively, the optical coupler 26 may emit the beam 25 into free space, thereby directing the beam 25 to the optical fiber 30.

[0042] The optical fiber 30 has an entry end 32 for receiving a beam 25 from a light source 24. The entry end 32 may receive the beam 25 from free space. In a preferred embodiment, an optical coupler 329 is configured to collect the beam 25 at the entry end 32 of the fiber 30. The respective cross-sectional shapes and sizes of the beam 25 and the optical core of the fiber 30 are interchangeable so that the beam 25 is transmitted along the fiber 30. This interchangeability may be achieved, at least in part, by a control and adjustment unit 328.

[0043] The optical fiber 30 may be conventional, typically comprising an optically transparent core (not shown) for transmitting the beam 25, the core being surrounded by a transparent cladding material (not shown) having a lower refractive index than the core. The core and cladding may be formed from any suitable material, such as glass (silica) or plastic. The fiber 30 may also be described as an optical waveguide, and may be of any known form, such as a type known as an optical pipe, which comprises an optically transparent core (e.g., formed from glass or plastic) but without cladding.

[0044] Advantageously, the optical fiber 30 is a multimode optical fiber. A multimode optical fiber is an optical fiber that supports multiple optical propagation paths or modes. Multiple optical paths or modes may be supported simultaneously over a wide wavelength spectrum. In contrast, a single-mode optical fiber supports only one mode. Multimode fibers typically have a wider core diameter than single-mode fibers (typically 50 to 400 micrometers). The core diameter of a multimode optical fiber is greater than the wavelength of light being transmitted. The core of the multimode fiber 30 preferably has a square or rectangular cross-section, but may alternatively have a circular cross-section or any other suitable cross-section, and the beam 25 may be shaped accordingly. As an example, the optical fiber 30 may have a core diameter of 50 μm and a numerical aperture (NA) of 0.12. The multimode fiber 30 can be said to have an extended light source, unlike a point source. This makes illumination of the sample surface easier because the extended light source provides a wider field of illumination than a point source.

[0045] The optical fiber 30 has a beam outlet 33 at its end or tip, from which an optical beam 25 is emitted. The beam 25 may be emitted into free space from the outlet 33.

[0046] In a preferred embodiment, the beam 25 emitted from the exit 33 of the multimode optical fiber 30 is homogenized or scrambled to provide a flat, uniform, interference-free illumination analysis result. For this purpose, any conventional beam homogenizer or optical diffuser (not shown) may be provided. For example, the optical tuning unit 328 may include a beam homogenizer or optical diffuser, but any such device(s) may be provided at any other suitable location in the illumination system. Advantageously, the homogenized beam 25 contains incoherent light. Furthermore, compared to single-mode optical fiber, the multimode fiber 30 is easier to tune, has a broader spectrum of acceptable laser wavelengths (e.g., extending to UV and NIR), and allows for irradiation of the sample 3 with higher laser power.

[0047] Referring to Figures 1 and 4, the illumination optics system 22 is advantageously configured such that the beam 25 at the exit or tip 33 of the optical fiber 30, more specifically at the exit 33, is imaged onto the sample surface 6. Preferably, the arrangement is such that an image of the tip 33 of the fiber, or optionally an enlarged image, is focused onto the sample surface 6. In a preferred embodiment, the illumination optics system 22 is configured to image the beam exit 33 onto an intermediate optical surface which may be called an equivalent sample surface (ESP) 43. Thus, an image of the exit 33, or optionally an enlarged image, is provided at the ESP 43. The illumination optics system 22 is further configured to image the intermediate image at the ESP 43 onto the sample surface 6. The ESP 43 is conjugate to the sample surface 6 and conjugate to the surface 39 where the tip 33 of the fiber, or the beam exit, is located.

[0048] In a preferred embodiment, the illumination optics system 22 includes a first lens 40, preferably a collimating lens. Its arrangement is such that the beam exit 33 is located at the front focal plane 39 of the lens 40. The first lens 40 includes a back focal plane (BFP) 41 that collimates the beam 25 from the exit 33 toward the BFP 41. It should be noted that the lens 40 does not form an image of the exit 33 at the BFP 41. This is shown in Figure 4, from which it can be seen that the rays LR from the exit 33 are not collected in such a way as to form an image of the exit 33 at the BFP 41. Instead, the light from the exit 33 is preferably collimated, or substantially collimated, at the BFP 41.

[0049] The illumination optical system 22 is configured to relay light from the BFP 41 to the BFP 8 of the objective lens 7. In a preferred embodiment, this is achieved using a second lens 42 and a third lens 10 arranged to function as a set of image relay lenses. In the embodiment of Figure 1, the third lens 10 is a tube lens of the microscope system 100. However, in other embodiments (not shown), the third lens 10 may be any suitable lens provided between the ESP 43 and the BPF 8 (which may be provided in addition to the tube lens), rather than a tube lens.

[0050] In a preferred illumination optical system 22, a second lens 42, which may include any suitable imaging lens, is positioned so that it forms an intermediate image at the ESP 43, either at the exit 33 or at the tip of the fiber. This is indicated by the collection of rays LR from the exit 33 at the ESP 43. Preferably, the second lens 42 is positioned so that its front focal plane coincides with the BFP 41. A preferred arrangement is such that the lens 42 provides a magnified image of the beam 25 at the exit 33 in the ESP 43, with a magnification ratio such that the magnified beam 25 fills or substantially fills the field of view of the detector. Preferably, the focal length of the second lens 42 is greater than the focal length of the first lens 40. A third lens 10 collimates the beam 25 from the ESP 43 to the BPF 8 of the objective lens. The BPF 8 of the objective lens preferably corresponds to the BPF of the third lens 10. It should be noted that the third lens 10 does not form an image at the exit 33 in the BFP8. This is shown in Figure 4, from which it can be seen that the light rays LR are not collected to form an image in the BFP8. Instead, the light is preferably collimated or substantially collimated in the BFP8.

[0051] The objective lens 7 images the image at the exit 33, more specifically the image of the beam 25 at the exit 33, onto the sample surface 6. In a preferred embodiment, the objective lens 7 focuses the light collimated by the BFP 8 onto the sample surface 6.

[0052] A preferred illumination optical system 22 is configured to project or image an intermediate image of the beam 25 in the ESP 43 onto the sample surface 6. In a preferred embodiment, this is achieved by a third lens 10 and an objective lens 7. In the embodiment of Figure 1, the beam 25 is directed from the ESP 43 to the objective lens 7 via a beam splitter 12.

[0053] In a preferred embodiment, the first lens 40 provides a collimated spot of light to the BPF 41. This spot of light is relayed to the BPF 8 of the objective lens 7, where it forms a corresponding spot of light. The objective lens 7 focuses the light spot to the BPF 8 to form an image of the fiber tip 33 on the sample surface 6. Preferably, the illumination optics system 22 is configured to relay the smallest available collimated spot to the BPF 8. This can be achieved by arranging lenses 40 and 42 such that the BPF 41 of lens 40 coincides with the front focal plane of lens 42. It is also preferable to focus the smallest available collimated spot from the BPF 8 onto the sample surface 6. This can be achieved by arranging lenses 10 and 7 such that the BPF 41 of lens 10 coincides with the BPF 8 of objective lens 7. In an alternative embodiment, the respective focal planes do not need to coincide, but this may negatively affect the sharpness of the image formed on the sample surface 6.

[0054] A preferred arrangement of the lenses 40 and 42 in the illumination optical system 22, particularly in the illustrated embodiment, is such that the surface 39 on which the exit 33 is located (corresponding to the front focal plane of lens 40 in the illustrated example) and the equivalent sample plane (ESP) 43 (corresponding to the BFP of the second lens 42 in the illustrated example) are conjugate to each other. A preferred arrangement of the lenses 10 and 7 in the illumination optical system 22, particularly in the illustrated embodiment, is such that the sample plane 6 and the ESP 43 are conjugate to each other. In a preferred embodiment, the surface 39 of the exit 33, the ESP 43, and the sample plane 6 are conjugate to each other. Furthermore, it is preferable that the focal plane 13 of the detector is conjugate to the sample plane 6, the ESP 43, and the exit surface 39.

[0055] The illumination optical system 22 is configured such that when the illumination beam 25 is incident on the objective lens 7, the illumination beam 25 is displaced laterally from the principal optical axis 7A of the objective lens 7. Depending on the arrangement of the illumination optical system 22, the beam 25 may be parallel or substantially parallel to the optical axis 7A (for example, as shown in Figure 1), or it may be oblique to the optical axis 7A when it strikes the objective lens 7. Preferably, the beam 25 is incident on the peripheral part of the objective lens 7. This arrangement facilitates the objective lens 7 to project the beam 25 onto the sample surface 6 at an oblique angle with respect to the optical axis. The configuration may be such that oblique illumination of the sample surface 6, and therefore oblique illumination of the sample, is achieved. Optionally, the configuration is such that the beam 25 is projected from the objective lens 7 onto the sample surface 6 at an angle that causes total internal reflection (TIR) ​​of the beam 25 above a relevant critical angle. To facilitate TIR, the objective lens 7 preferably has a relatively large numerical aperture, preferably 1.4 or more. TIR occurs at the sample interface, which may be located at or substantially located at the sample surface 6. For example, referring to Figure 2A, the sample interface may include the inner bottom surface of the container 9. Alternatively, in the example of Figure 2B, the sample interface may be the surface of the coverslide 5 facing the sample 3. For example, in the case of an inverted microscope with a high numerical aperture objective lens, the sample substrate is typically a glass surface with a so-called "coverslip thickness." The objective lens is designed to image through glass of this thickness. TIR can occur at the substrate (coverslip)-sample interface or the glass-water interface. The entire sample may be a biological sample sandwiched between a thick glass slide 4 and a coverslip 5 (with water for the sample in between) and placed upside down on an inverted microscope. Alternatively, it may be a "dish" style sample (Figure 2A) consisting of a chamber with walls and a bottom with a coverslip thickness. The sample is typically cultured on a glass coverslip in the chamber and filled with water or another aqueous liquid in a dish that opens to the top.

[0056] The desired positioning of the beam 25 can be achieved using any convenient positioning means. For example, in Figure 1, the illumination optics system 22 includes a translation optics device 50 between the first and second lenses 40, 42. This is configured or can be configured to adjust the position of the beam 25 relative to the principal axis of the lenses 40, 42, particularly the second lens 42 (e.g., by moving it laterally and / or adjusting its tilt angle) in order to control the position and / or angle of the beam 25 when it reaches the beam splitter 12, so that the beam 25 is positioned as desired when it reaches the objective lens 7. The translation optics device 50 can take any conventional form, e.g., a rotatable optical or optical translation stage or mount. Alternatively or additionally, the desired positioning of the beam 25 may be achieved by positioning either or both of the exit 33 and the first lens 40 such that the beam 25 emerges from the first lens 40 along an axis displaced from the principal axis of the second lens 42. This is shown in Figure 4, where the exit 33 and the first lens 40 are displaced laterally with respect to the second lens 42, and a preferred arrangement is such that the principal axis of the first lens 40 is parallel to and away from the principal axis of the second lens 42. More generally, the beam positioning means may be implemented by one or more optical devices and / or optical configurations that allow the lateral position and / or inclination angle of the beam 25 with respect to the principal optical axis or other reference axis to be set in a desired manner and adjusted or tuned at the discretion of the user. For example, in an alternative embodiment (not shown), the illumination optical system 22 may include one or more lenses that are movable laterally (i.e., perpendicular to its principal axis) to position the beam 25 as needed.

[0057] In the illustrated embodiment, light generated by a light source is coupled to a multimode optical fiber 30, and the exit 33 of the fiber 30 is set to the focal point of a first lens 40. A second lens 42 creates an image (optionally magnified image) of the exit 33 at the equivalent sample plane (ESP) 42. The image of the beam 25 at the exit 33, or the magnified image, is then relayed through a microscope 100 and projected onto the sample plane 6 of the target sample (typically a micro-sample on a substrate 4) via the objective lens 7, and in this example via the beam splitter 12. Light 27 (typically fluorescence) emitted from the sample 3 is captured by the objective lens 7 and collected onto the optical detector 14 by the microscope's tube lens 10 (via the beam splitter 12 and emission filter 17 in the illustrated embodiment). Advantageously, the exit plane 39, ESP 43, sample plane 6, and preferably the focal plane 13 of the detector are all conjugate to each other.

[0058] A preferred illumination optics system 22 is configured such that the beam 25 is positioned at the periphery of the back focal plane (BFP) 8 of the objective lens, either because the beam 25 undergoes total internal reflection at the sample interface or because it obliquely illuminates the sample surface 6. The positioning of the beam 25 may be achieved by one or more optical elements that can translate the beam laterally with respect to the optical axis of the microscope. Advantageously, the illumination optics system 22 is configured to direct a homogenized or incoherent beam 25 from the fiber 30 to the sample surface 6. Advantageously, the illumination optics system 22 is configured to image the tip or exit of the fiber 30 onto the sample surface 6.

[0059] In alternative embodiments (not shown), either or both of the imaging optical system 20 and the illumination optical system 22 may have different optical components and / or different arrangements of optical components than those shown in Figures 1 and 4. For example, the beam splitter 12 may be positioned between the tube lens 10 and the objective lens 7. One or more additional lenses (not shown) may be provided between the ESP 43 and the objective lens 7 (for example, between the ESP 43 and the beam splitter 12, or between the beam splitter 12 and the objective lens 7) to function as a third lens (shown as lens 10 in Figure 4, as described above) instead of the tube lens 10 in the example of Figure 1. Optionally, the additional lenses, or each additional lens, may be movable laterally with respect to the optical axis, as described above, in order to position the beam 25 as desired.

[0060] In alternative embodiments (not shown), one or more of the lenses described herein may be implemented as a single lens or as an array of lenses, as will be obvious to those skilled in the art.

[0061] Advantageously, in a preferred embodiment, the illumination optics system 22 is configured to project a structure-free pattern of the homogenized beam 25 from the multimode fiber 30 onto the sample surface, particularly to form an image. Thus, the sample surface, and therefore the sample itself, is illuminated uniformly across the entire field of view of the microscope, without interference fringes that degrade image quality.

[0062] The present invention is not limited to the embodiments described herein and can be modified or altered without departing from the scope of the invention.

Claims

1. 1. A microscope system for imaging a sample disposed in a sample plane, comprising: The objective lens of the microscope, an illumination optical system configured to illuminate the sample surface with a light beam through the objective lens, the illumination optical system comprising a multimode optical fiber having an outlet for emitting the light beam; the illumination optical system is configured such that the outlet is located at a beam exit plane conjugate with the sample surface; A microscope system, wherein the illumination optical system is configured such that the light beam is incident on the objective lens at a lateral displacement from the main optical axis of the objective lens so that the objective lens irradiates the light beam on the sample surface at an oblique angle relative to the main optical axis of the objective lens.

2. The microscope system of claim 1 , wherein the illumination optical system is configured so that the light beam is incident on a peripheral portion of the objective lens.

3. 3. The microscope system of claim 1, wherein the illumination optical system is configured to image the beam exit surface onto an equivalent sample surface that is intermediate between the beam exit surface and the sample surface and conjugate to the sample surface.

4. The microscope system of claim 3 , wherein the illumination optical system comprises at least one lens configured to image the beam exit surface to the equivalent sample surface.

5. the illumination optical system comprises at least one collimating lens having front and back focal planes coincident with the beam exit surface; The microscope system of claim 1 , wherein the illumination optical system is configured to relay a back focal plane of the at least one collimating lens to a back focal plane of the objective lens.

6. the at least one lens comprises the at least one collimating lens and at least one imaging lens; A microscope system according to claim 5 when dependent on claim 4, wherein the at least one imaging lens is configured to form an image of the beam exit at the equivalent sample plane.

7. 4. The microscope system of claim 3, wherein the illumination optical system comprises at least one lens disposed between the equivalent sample plane and the objective lens and configured to collimate light from the sample plane to a back focal plane of the objective lens.

8. The microscope system of claim 7 , wherein the at least one lens comprises a microscope tube lens.

9. 2. The microscope system of claim 1, wherein the illumination optical system comprises a positioning means for positioning the light beam so that the light beam is incident on the objective lens laterally displaced from a main optical axis of the objective lens.

10. 10. The microscope system of claim 9, wherein the positioning means comprises one or more optical components configured to move the light beam relative to a main optical axis of the microscope system, optionally tilt the light beam and / or move the light beam laterally relative to the main axis.

11. The microscope system of claim 5 , wherein the at least one collimating lens is positioned such that its optical axis is laterally displaced from a main optical axis of the microscope system.

12. The microscope system of claim 1 , wherein the multimode optical fiber is coupled to a light source for generating the light beam.

13. 13. The microscope system of claim 12, wherein the light source is a laser light source and the light beam is a laser beam, preferably a homogenized laser beam.

14. The microscope system of claim 1 , wherein the illumination optical system is configured such that the angle causes total internal reflection at a sample interface.

15. The microscope system of claim 1 , wherein the illumination optical system is configured to focus the beam outlet onto the sample plane.

16. 2. The microscope system of claim 1, wherein the illumination optical system comprises at least one collimating lens arranged to collimate light from the beam outlet, and at least one relay lens arranged to relay light collimated by the at least one collimating lens to a back focal plane of the objective lens.

17. 17. The microscope system of claim 16, wherein the at least one relay lens is configured to relay light from a back focal plane of the at least one collimating lens to a back focal plane of the objective lens.

18. The microscope system of claim 1 , wherein the illumination optical system is configured to image the beam outlet onto the sample surface.

19. The microscope system according to claim 1 , wherein the multimode optical fiber has a square or rectangular cross-sectional shape.

20. The microscope system of claim 1 , wherein the illumination optical system comprises means for homogenizing the light beam.

21. 1. An illumination optical system for a microscope system for imaging a sample disposed in a sample plane, comprising: configured to illuminate the sample surface with a light beam through an objective lens of the microscope system; a multimode optical fiber having an outlet for emitting said light beam; the outlet is configured to be located at a beam exit plane conjugate to the sample surface; An illumination optical system configured such that the light beam is incident on the objective lens laterally displaced from a main optical axis of the objective lens such that the objective lens irradiates the light beam on the sample surface at an oblique angle relative to a main optical axis of the objective lens.

22. 1. A method for illuminating a sample plane of a microscope system through an objective lens, comprising: positioning a light beam exit of a multimode optical fiber at a beam exit plane conjugate with said sample face; illuminating the sample surface with a light beam from the multimode optical fiber; and laterally displacing the light beam from a main optical axis of the objective lens when the light beam is incident on the objective lens.

23. 23. The method of claim 22, further comprising focusing and / or imaging the beam outlet onto the sample surface.

24. Collimating light from the beam outlet; relaying the collimated light to a back focal plane of the objective lens; 24. The method of claim 22 or 23, wherein said relaying preferably further comprises relaying light from a back focal plane of at least one collimating lens to a back focal plane of said objective lens.

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