Differential phase-contrast microscope

Abstract

To provide a microscope for differential phase-contrast (DPC) microscopy which allows a user to access a specimen area without restrictions by placing an illumination source at a working distance from an object, and uses a standard objective lens, reducing the cost thereby.SOLUTION: A microscope 100 for performing differential phase contrast (DPC) microscopy comprises an infinity-corrected microscope objective lens 7, a tube lens 10, and at least one lens configured to image a back focal plane of the microscope objective lens on a conjugate back focal plane outside the microscope objective lens. An aperture stop is located in the conjugate back focal plane. An object plane is located between the objective lens and an illumination source. The illumination source can be configured to illuminate an object from any one of a plurality of locations that are angularly displaced about an axis that is perpendicular to the object plane.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an optical microscope, and particularly to an optical microscope adapted to differential phase contrast microscopy.

Background Art

[0002] Differential phase contrast (DPC) is an optical microscopy method for enhancing contrast, particularly in samples or specimens with low contrast. A circular aperture is arranged on the pupil plane. The sample is illuminated according to the order of phase-shifted illumination by a symmetrically split light source. The local phase gradient in the sample causes diffracted incident light proportional to the steepness of the local gradient. The incident light encountering the local phase gradient is diffracted so as to be blocked at the aperture of the pupil plane. The non-diffracted light passes through the aperture. In this way, the conversion from phase to amplitude is performed. The amplitude modulation signal is restored by subtracting two images taken in the order of continuous illumination.

[0003] Examples of differential phase contrast (DPC) microscopes can be found in the following references. Hamilton et al., "Differential Phase Contrast in Scanning Optical Microscopy", Journal of Microscopy, Vol. 133, Pt 1, January 1984, p. 27-39. Wallra et al., "Quantitative Differential Phase Contrast Imaging in LED Array Microscopy", Optics Express, Vol. 23, No. 9, April 2015.

[0004] In conventional phase contrast microscopy, an expensive dedicated objective lens having a phase plate incorporated in the back focal plane (BFP) is required. In addition, an electric stage is required on the phase plate conjugate surface of the condenser turret to switch the illumination according to different objective lenses. The disadvantages of such a system are the cost and the limitation of user access due to the short focal length in conventional condensers.

[0005] To use DPC, a pupil plane filter must be implemented. In the references above, this filtering is performed by the microscope's objective lens. However, this is not a practical implementation because typical users require phase contrast imaging on micro-titre plates, also known as "multi-well" plates. The depth and lateral dimensions of the wells in these plates limit the range of illumination angles available for diffraction by the sample. The illumination light within this reduced set of angles represents a proportional reduction in the pupil plane area of ​​a standard objective lens. Therefore, the efficiency of phase-to-amplitude conversion by a standard objective lens is significantly reduced, making it possible to detect only very large phase gradients (resulting in reduced contrast).

[0006] Furthermore, the user's default requirement is access to a range of magnifications within the user's selected numerical aperture (NA). The pupil size at the rear aperture of the objective lens varies depending on the objective lens's focal length (which is inversely proportional to the magnification) and the NA. For the user to utilize DPC with a range of objective lenses, the pupil plane filter must be further adapted to any combination of selected magnification and NA. [Overview of the project] [Problems that the invention aims to solve]

[0007] It is desirable to provide a microscope that mitigates at least some of the problems outlined above. [Means for solving the problem]

[0008] The microscope provided by the present invention is a microscope for imaging an object placed on an object plane. The microscope comprises an illumination source and an imaging optical system configured to image the object along an optical path to an imaging device. The imaging optical system includes an infinity-corrected microscope objective lens, a tube lens, at least one lens configured to image the back focal plane of the microscope objective lens to a conjugate back focal plane outside the microscope objective lens, and an aperture diaphragm located on the conjugate back focal plane and intersecting the optical path. The object plane is located between the objective lens and the illumination source. The illumination source can be configured to illuminate the object from any one of a plurality of positions that are angularly offset around an axis perpendicular to the object plane.

[0009] In a preferred embodiment, the microscope objective lens and the tube lens are configured to image the object at an intermediate image plane. The at least one lens has an optical relay configured to project an image of the object from the intermediate image plane to the imaging device and to image the back focal plane of the objective lens at a conjugate back focal plane.

[0010] The optical relay may have a first relay lens and a second relay lens spaced apart along the optical path. The first relay lens is configured to image the back focal plane of the objective lens onto the conjugate back focal plane. The conjugate back focal plane is located between the first relay lens and the second relay lens.

[0011] The conjugate post-focal plane may be positioned at a focal distance of 1 from each of the first relay lens and the second relay lens.

[0012] The first relay lens may be positioned at least one focal length away from the intermediate image plane.

[0013] In a preferred embodiment, the illuminator is operable to illuminate the object using a series of two or more lighting settings. In each lighting setting, the illuminator illuminates the object from a different lighting angle.

[0014] The illumination source is preferably configured to illuminate the object at an angle to the object's surface. The illumination angles are offset from each other in the angular direction around an axis perpendicular to the object's surface.

[0015] A series of lighting settings preferably has one or more pairs of lighting settings. The lighting settings in each pair are used in sequence, causing the lighting source to illuminate the object from lighting angles that are 180° apart in the angular direction from each other.

[0016] In a preferred embodiment, the illumination source has a spatially partitionable illumination field for illuminating the object from different angles relative to the object's surface.

[0017] The illumination source preferably has an illumination field. The illumination source preferably has an array of light sources that can be controlled individually and / or as two or more groups in order to selectively illuminate one or more of the multiple zones of the illumination field.

[0018] The illumination source preferably has an illumination field. The illumination source is preferably operable to illuminate the object using a series of spatially offset zones within the illumination field.

[0019] Preferably, the spatially offset series of zones have at least one pair of zones that are offset by 180° from each other in the angular direction around the center of the illumination field.

[0020] Advantageously, the illumination source is positioned at a distance from the object plane corresponding to optical infinity or substantially corresponding to the object plane.

[0021] In a preferred embodiment, the microscope has means for adjusting the distance between the illumination source and the object surface.

[0022] The microscope optionally includes an illumination optical system configured to irradiate the object by directing light from the light source toward the object along at least a part of the optical path, preferably through the objective lens.

[0023] The illumination optical system may include a confocal spinning disk. The light source has at least one laser device arranged to direct a laser beam toward the confocal spinning disk. The confocal spinning disk is movable between a use state in which it intersects the optical path and a non-use state in which it does not intersect the optical path.

[0024] The confocal spinning disk is preferably arranged at the intermediate image plane in the use state.

[0025] The microscope preferably further has a transport mechanism for moving the confocal spinning disk between the use state and the non-use state.

[0026] The confocal spinning disk may be included in a spinning disk assembly. The spinning disk assembly is movable between the use state and the non-use state.

[0027] The light source may be arranged to direct the light into the optical path through a beam splitter arranged between the tube lens and the optical relay. The beam splitter is arranged to direct light from the tube lens toward the optical relay.

[0028] Advantageously, the aperture stop is configured to function as a spatial filter, preferably as a pupil plane spatial filter.

[0029] The aperture stop preferably has an aperture of a size not larger than the size of the pupil imaged on the conjugate rear focal plane projected from the rear focal plane.

[0030] The aperture diaphragm defines the opening. Preferably, the aperture diaphragm is movable to adjust the size of the opening.

[0031] In a preferred embodiment, the aperture diaphragm has an iris device that defines the opening, and preferably is movable to adjust the size of the opening.

[0032] The iris device may be positioned on the conjugate plane of the back focal plane of the objective lens such that the aperture intersects the optical path.

[0033] A preferred embodiment of the present invention has a configurable or segmented illumination source positioned to illuminate an object at an oblique angle. The illumination source can be positioned at working distance from the object, allowing the user unrestricted access to the sample area. Advantageously, the microscope may use a standard objective lens, which reduces costs.

[0034] In a preferred embodiment, the pupil space filter is positioned on the conjugate plane of the back focal plane of the objective lens. In embodiments where the object is mounted on a multiwell plate, the size of the pupil filter may be selected to accommodate the constraints of the illumination angle. Advantageously, the size of the pupil filter is adaptable to the combination of NA and magnification in the user's chosen objective lens. [Brief explanation of the drawing]

[0035] Embodiments of the present invention will be described by reference to the accompanying drawings. In the accompanying drawings, similar numbers are used to indicate similar components.

[0036] [Figure 1] Figure 1 is a schematic diagram of a microscope that realizes the present invention. [Figure 2] Figure 2 is a schematic diagram of a light source suitable for use in embodiments of the present invention and capable of configuring multiple zones. [Figure 3A]Figure 3A shows a suitable light source that is suitable for use in embodiments of the present invention and capable of configuring multiple zones, and illustrates an apparatus that realizes bright-field illumination. [Figure 3B] Figure 3B shows the light source from Figure 3A, which has been divided and offset in the angular direction to create eight semicircular lights. [Figure 3C] Figure 3C shows the light source from Figure 3A, which has been divided and offset in the angular direction to create eight semicircular illuminations. [Figure 4] Figure 4 is an end view of the variable iris assembly, which is part of the microscope shown in Figure 1. [Figure 5] Figure 5 is an end view of the confocal spinning disk assembly, which is part of the microscope shown in Figure 1, and shows the confocal spinning disk assembly in a state where the spinning disk intersects with the optical path of the microscope. [Figure 6] Figure 6 is an end view of the confocal spinning disk assembly, which is part of the microscope shown in Figure 1, and shows the confocal spinning disk assembly in a state where the spinning disk does not intersect with the optical path of the microscope. [Figure 7] Figure 7 is an enlarged view of an embodiment having a multiwell plate. [Modes for carrying out the invention]

[0037] Referring to the drawings, a microscope that implements one aspect of the present invention is usually indicated by reference numeral 100. Microscope 100 is an optical microscope, and in a preferred embodiment, a spinning disk confocal microscope. However, as will be apparent to those skilled in the art, the microscope that implements the present invention may be of other types.

[0038] The microscope 100 includes a stage 20 for supporting the object 55 to be imaged. Typically, the object 55 has a slide 9 on which a sample, such as a biological sample, is placed. The sample (sometimes called a specimen) may be immersed in a medium, such as water. A cover slide 8 may be placed over the sample, if necessary. The object 55 is positioned on the object surface 56. As will be described in detail below, the microscope 100 includes an illumination source 60 for illuminating the object 55. Typically, the illumination source 60 is positioned to illuminate the object 55 from behind the stage 20, that is, to illuminate the object through the opening of the stage 20 and the slide 9 in this example. For this reason, the slide 9 is made of an optically transparent material, such as glass.

[0039] The microscope 100 includes an imaging optical system 30 for imaging an object 55 onto an imaging device. Typically, the imaging optical system 30 has a camera 14 along the optical path. In a preferred embodiment, it is desirable that the imaging optical system 30 focus the image of the object 55 onto the focal plane of the camera 14. The imaging optical system 30 has a series of optical devices. Typically, the series of optical devices has at least one lens and at least one mirror. The at least one mirror is optionally positioned to image the object 55 onto the camera 14, i.e., to form an image of the object 55 on the camera 14 via the optical array. The imaging optical system 30 has a microscope objective lens 7, preferably an infinity-corrected microscope objective lens. Advantageously, the objective lens 7 is a standard microscope objective lens and does not have an embedded phase plate. The objective lens 7 has a back focal plane (BFP), which is the pupil plane of the objective lens 7. Typically, the objective lens 7 has an optical axis perpendicular to the object plane 56.

[0040] Furthermore, a preferred imaging optical system 30 includes a tube lens 10 configured to form an intermediate image of an object 55 on the intermediate image plane (IIP) together with the objective lens 7 (in particular, the objective lens included in the objective lens 7 or the objective lens assembly 7'). In a preferred embodiment, a confocal spinning pinhole disk 11 is positioned on the intermediate image plane IIP and intersects the optical path. A mirror 19 is optionally provided between the objective lens 7 and the tube lens 10. The mirror 19 is configured so that the excitation light 65 is precisely aligned with the optical axis of the objective lens 7.

[0041] A preferred imaging optical system 30 comprises an optical relay having at least one relay lens. The optical relay is positioned between the tube lens 10 and the camera 14 and is configured to project an intermediate image of the object 55 from the intermediate image plane IIP to the camera 14. In the illustrated embodiment, the optical relay has a first relay lens 13 and a second relay lens 13' between the tube lens 10 and the camera 14. A mirror 21 is optionally provided between the relay lens 13 and the relay lens 13'. The mirror 21 is configured so that the light rays are optimally aligned with the optical axis of the second relay lens 13'. In alternative embodiments (not shown), the imaging optical system may have other suitable arrangements of lenses and, if necessary, mirrors.

[0042] In a preferred embodiment, the camera 14 is a digital camera having a digital image sensor 22, such as a CCD sensor. The imaging optical system 30 forms an image of the object 55 on the image sensor 22. More specifically, it is desirable that the imaging optical system 30 focuses the image of the object 55 on the sensor 22, whose imaging plane is positioned at the focal plane of the imaging optical system 30.

[0043] The microscope 100 includes a focusing system 35 for adjusting the imaging optical system 30 and / or the stage 20 to focus an image of an object 55 onto the camera 14. The focusing system 35 has means for relative movement between the stage 20 and the objective lens 7 in an axial direction corresponding to the optical axis of the objective lens 7. In a typical embodiment, the objective lens 7 is axially movable relative to the stage 20 and the object 55. For this purpose, the objective lens 7 is supported by a movable support structure 15, typically an objective lens turret. In the illustrated embodiment, the turret 15 and the objective lens 7 are movable in the direction indicated by the arrows A-A'. The turret 15 may have a drive system (not shown) for moving the turret 15 in the A-A' direction, such as an electric drive system or a piezoelectric drive system. Alternatively, the turret 15 may be coupled to a drive system. Any suitable conventional electric drive system may be used.

[0044] Movement of the objective lens 7 toward the object 55 in the axial direction and movement away from the object 55 in the axial direction adjust the focus of the image to the camera 14. In this way, the movable objective lens assemblies 7,15 provide part of the focusing system 35. Typically, the stage 20 is stationary during focusing, and the objective lens 7 moves relative to the stage 20. Alternatively, the stage 20 may move axially relative to the objective lens 7. In that case, the objective lens 7 may remain stationary during focusing. More generally, either or both of the objective lens 7 and the stage 20 may be axially movable toward or away from each other in order to adjust the focus.

[0045] Furthermore, the focusing system 35 includes a control device 50 for controlling the movement of the objective lens 7 (and / or the stage 20, if applicable) to focus an image on the camera 14. The control device 50 can typically be any conventional form having a appropriately programmed processor, such as a microprocessor or microcontroller. Preferably, the focusing system 35 is configured to autofocus the image on the camera 14. To this end, the camera 14 and / or the microscope 100 may have any conventional autofocus means. For example, the control device 50 may be programmed to perform contrast detection autofocus using any conventional contrast detection autofocusing algorithm.

[0046] In some embodiments, the microscope 100 includes an illumination optical system 45 for irradiating an object 55, particularly a sample contained within the object 55. The illumination optical system 45 includes a light source 25. In a preferred embodiment, the light source 25 has one or more laser devices. However, the light source 25 may alternatively have other suitable conventional light sources, such as one or more light-emitting diodes (LEDs) or one or more incandescent bulbs. The light source 25 may be configured to produce light in one or more frequency bands suitable for the application, as will be apparent to those skilled in the art. For example, if the object 55 has a sample that can fluoresce, the light source 25 may be configured to produce light in one or more frequency bands that excite the sample or marker and cause fluorescence. The sample can fluoresce either by being innately fluorescent (i.e., autofluorescence) or by having one or more fluorescent markers, such as proteins or dyes, attached to the sample.

[0047] In a preferred embodiment, the illumination optical system 45 is configured to illuminate the object 55 by directing light (a laser beam 65 in this example) towards the object along at least a portion of the optical path defined by the imaging optical system 30. The light may have one or more wavelengths from a plurality of wavelengths corresponding to the fluorescence characteristics of the sample or marker. In particular, the illumination optical system 45 is configured to illuminate the object 55 through the objective lens 7. To facilitate this, the imaging optical system 30 may have a beam splitter 12.

[0048] The beam splitter 12 is configured to transmit light in one or more frequency bands corresponding to the light generated by the laser device 25. The laser device 25 is positioned so that the laser beam 65 is directed into the optical path through the beam splitter 12 and then directed towards the object 55 through the objective lens 7. The beam splitter 12 is configured to at least partially reflect light in one or more frequency bands corresponding to the light reflected or emitted from the object 55. The beam splitter 12 may have one or more reflection bands corresponding to the light emitted by the object 55 and a transmission band corresponding to the light generated by the laser device 25.

[0049] In the illustrated embodiment, the beam splitter 12 is positioned between the tube lens 10 and the first relay lens 13 to reflect the light that has passed through the tube lens 10 to the first relay lens 13. The beam splitter 12 is positioned between the intermediate image plane and the optical relay. Typically, the beam splitter 12 has a dichroic mirror. In alternative embodiments, such as those in which the laser device 25 is not required, the beam splitter 12 may be replaced with, for example, a simple mirror.

[0050] In the illustrated embodiment, the microscope 100 can perform spinning disk confocal laser microscopy. In the illustrated embodiment, the illumination optical system 45 has a confocal spinning disk 11 to which a laser beam 65 is directed. The spinning disk 11 has an array of pinholes (not shown). The spinning disk 11 may be part of a spinning disk assembly having a spinning illumination beam collector disk (not shown) with microlenses.

[0051] In a preferred embodiment, the diameter of the pinhole does not exceed 2 Airy units. The spinning disk 11 or spinning disk assembly functions as a scanner, causing an array of laser beams generated from the laser beam 65 to irradiate the object 55. The spinning disk 11 is preferably positioned in the intermediate image plane IIP. In the illustrated embodiment, the spinning disk 11 is positioned between the tube lens 10 and the beam splitter 12.

[0052] In a preferred embodiment, the spinning disk 11 (or whichever of the spinning disk assembly applies) is movable between an in-use state and an unused state. In the in-use state, the spinning disk 11 (or whichever of the spinning disk assembly applies) having the pinhole array is in the intermediate image plane and intersects with the imaging and illumination field area. That is, it is positioned in the optical paths of the imaging optical system 30 and the illumination optical system 45. In the unused state, the spinning disk 11 (or whichever of the spinning disk assembly applies) having the pinhole array does not intersect with the imaging and illumination field area. That is, it is not positioned in the optical paths of the imaging optical system 30 or the illumination optical system 45, and is preferably removed from the intermediate image plane.

[0053] When the spinning disk 11 (or spinning disk assembly) is in use, the microscope 100 is in a confocal mode capable of performing spinning disk confocal laser microscopy. When the spinning disk 11 (or spinning disk assembly) is not in use, the microscope 100 can perform other types of microscopy, including differential phase-contrast microscopy, bright-field microscopy, or epifluorescence microscopy.

[0054] The spinning disc 11 (or whichever of the spinning disc assembly applies) may be moved between an in-use state and an unused state by any convenient transport mechanism, preferably under the control of a control device 50. Figures 5 and 6 show an example of a suitable transport mechanism having a transport table 82 movably coupled to a base 84. In the illustrated example, the transport table 82 is coupled to the base 84 by a linear sliding mechanism 83. The linear sliding mechanism 83 allows the spinning disc 11 to move relative to the base 84 in the direction indicated by arrow A.

[0055] In Figure 5, the spinning disk 11 is in use, intersecting the imaging and illumination field area 88. In Figure 6, the spinning disk 11 is in unused mode, not intersecting the imaging and illumination field area 88. Preferably, a drive mechanism is provided for moving the transport table 82 relative to the base 84. The illustrated drive mechanism includes a motor 85 and a lead screw 86 provided on the base 84. The lead screw 86 is coupled to the transport table 82 by a lead screw coupler 87. The transport mechanism and / or drive mechanism may be of other convenient conventional forms.

[0056] In alternative embodiments where the microscope 100 is not suitable for spinning disk confocal microscopy, the spinning disk 11 may be omitted. In embodiments where the microscope illuminates the object 55 by laser scanning, any conventional laser scanning system may be provided.

[0057] In some embodiments, the object 55 emits fluorescence either by autofluorescence or by a fluorescent marker (or label) present on the sample when excited by light from the illumination optical system 45. Therefore, when the microscope 100 is operating in imaging mode, the fluorescence emitted from the sample is imaged by the imaging optical system 30 and captured by the camera 14.

[0058] The microscope 100 has an aperture diaphragm configured to function as a spatial filter in the imaging optical system 30, preferably in the form of an iris device 70. The iris device 70 defines an aperture 71 and is preferably operable to adjust the size or diameter of the aperture 71. Conveniently, the iris device is controlled by a control device 50. The iris device 70 may be any conventional type.

[0059] The iris device 70 or spatial filter is positioned on the conjugate plane BFP' of the back focal plane BFP of the objective lens 7. In a preferred embodiment, the relay lens 13, together with the tube lens 10, re-images the back focal plane BFP onto its conjugate plane BFP'. Since the back focal plane BFP is the pupil plane of the objective lens 7, the re-imaged conjugate plane BFP' can be said to be the pupil plane of the re-imaged objective lens. Generally, the back focal plane BFP can be re-imaged onto its conjugate plane BFP' by arbitrarily and appropriately arranging the lenses. In a preferred embodiment, the conjugate plane BFP' is positioned at a focal distance of 1 from each relay lens 13, 13'. Furthermore, it is preferable that the relay lens 13 is at least 1 focal distance away from the intermediate image plane IIP.

[0060] The precise location of the conjugate plane BFP' can be changed in any convenient way, for example, by providing a suitable field lens (e.g., a field lens with a desired focal length) (not shown) between the tube lens 10 and the relay lens 13. Advantageously, the conjugate back focal plane BFP' is accessible outside the objective lens 7 in the optical row, such that the iris device 70 can be positioned to intersect the conjugate back focal plane BFP'. The iris device 70 is positioned such that the aperture 71 aligns with the axis of the optical path, in particular with the optical axis of the relay lens 13 as shown, i.e., the axis passes through the aperture.

[0061] The iris device 70, particularly the aperture 71, functions as a spatial filter and is called a pupil plane spatial filter. The iris device 70 may be configured to function as a spatial filter by setting the size of the aperture 71. The size of the aperture 71 may be set to be less than or equal to the size of the pupil projected from the back focal plane BFP and re-imaged to the conjugate back focal plane BFP'. In particular, when the object 55 is illuminated by the illumination source 60 using the entire illumination field (i.e., bright-field illumination), it is preferable that the size (e.g., width or diameter) of the aperture 71 be set to be less than or equal to the size (e.g., width or diameter) of the pupil projected from the objective lens BFP and re-imaged by the relay lens 13. In alternative embodiments (not shown), other spatial filter devices, preferably those with an adjustable aperture size, may be used instead of the iris device 70.

[0062] In embodiments where the object is placed in a multiwell plate, the size of the aperture 71 may be selected or adjusted to accommodate the constraints of the illumination angle. Preferably, the size of the aperture 71 is adaptable to combinations of NA and magnification of the microscope 100 selected in a convenient and conventional manner by any user. The size of the aperture 71 may be automatically adjusted by the control device 50 in response to changes in the configuration of the objective lens 7 and / or the tube lens 10.

[0063] Figure 4 shows an end view of a variable iris assembly having an exemplary variable iris device 70. The exemplary variable iris device 70 has an iris shutter 72 that is operable to control the size of the opening 71. In this example, the shutter 72 is operable by a ring gear drive 73. The ring gear drive 73 is driven by a motor 74 via a worm gear 75. The iris device 70, motor 74, and worm gear 75 are conveniently supported by a common support structure 76. The motor 74 may be controlled by a control device 50.

[0064] The illumination source 60 can be configured to illuminate the object 55 from one of several positions that are angularly offset around an axis perpendicular to the object plane 56 (typically coinciding with the axis of the objective lens). Therefore, the illumination source 60 can operate to illuminate the object 55 obliquely from different positions, allowing for differential phase-contrast microscopy. These different positions are angularly offset around an axis perpendicular to the object plane. A preferred illumination source 60 is a light source capable of configuring multiple zones, and is called a split-field illumination source. In a split-field illumination source, the entire illumination field of the illumination source 60 is divided or spatially partitioned. Therefore, the illumination source 60 can illuminate from different zones or regions of the illumination field. As a result, the illumination source 60 can be configured to illuminate the object 55 from one of several different azimuth angles with respect to the object plane and its perpendicular axis.

[0065] Advantageously, the distance between the illumination source 60 and the object 55 can be arbitrarily set to balance, for example, the need for light efficiency and ease of access to the object. In a preferred embodiment, the distance between the illumination source 60 and the object 55 is relatively long to facilitate user access to the sample areas 20, 55. The illumination source 60 is arbitrarily positioned at a long optical distance from the object surface 56. In other words, the illumination source 60 is positioned at effective or substantially optical infinity such that the axial variation of the pupil position in the iris is minimized (e.g., + / - 10 mm or less), i.e., to simulate an illumination source positioned at infinity. This is facilitated in a preferred embodiment by an illumination source 60 without a capacitor.

[0066] The apparatus 100 may have any convenient means (not shown) for moving the illumination source 60 relative to the stage 20 in order to adjust the distance between the illumination source 60 and the object surface 56, for example, a movable transport platform for the illumination source 60 and / or the stage 20. The transport platform or other means of movement may be manually movable and / or power-operated by any convenient drive mechanism, preferably under the control of the control device 50.

[0067] Another advantage of the configurable distance between the illumination source 60 and the object 55 is that the distance can be set to accommodate cases where the object 55 has a sample or other material contained in a multiwell plate (also known as a microplate). Figure 7 shows a sample holder 20 that holds a multiwell plate 90. The multiwell plate 90 has a number of wells 92 for containing a sample. Illumination of the contents of each well 92 is affected by the dimensions of the well, in particular the size of the well opening, the depth of the well, and the angle at which the sides of the well extend from the opening. Wells are typically cylindrical or cubic, but can also be conical or narrow in the direction away from the opening. Typically, the opening of the well 92 is parallel to the object plane 56 and in a plane perpendicular to the shortest line of sight (LOS) between the illumination source 60 and the object 55.

[0068] The efficiency of illumination of the contents of each well by light is determined by the angle θ (Figure 7) at which light from the illumination source enters the opening of each well 92. The angle of incidence may depend not only on the distance from the multiwell plate to the illumination source 60, but also on the width of the illumination source 60 in a direction perpendicular to the distance from the multiwell plate to the illumination source 60 or along a plane parallel to the object 55.

[0069] In a preferred embodiment, when a multiwell plate is used, the width of the illumination source (and in particular the width of its illumination field) is configured in relation to the vertical distance (shortest distance) between the illumination source 60 and the object 55 in the multiwell plate. In this configuration, the angle of incidence of the light from the illumination source on the object surface (preferably all the light from the illumination source, i.e., light from the side edges of the illumination source) is within an elevation angle (less than or equal to θ in the example of Figure 7) in which the contents of the wells can be efficiently illuminated. Typically, the angle of incidence is such that the light reaches the bottom of the well, preferably such that the light reaches the bottom of the well without reflection from the sides.

[0070] Figure 3A shows an end view of a preferred embodiment of the illumination source 60. The illumination source 60 has a plurality of light sources 61 (e.g., LED units or lamps) arranged in an array to selectively partition the illumination field. The plurality of light sources 61 can be selectively turned on or off individually or in groups. This allows the illumination source 60 to bring light from different areas or zones of the illumination field.

[0071] In the illustrated example, the illumination field of the illumination source 60 has an inner central portion 67A surrounded by an annular outer portion 67B. The central portion 67A has multiple segments 68A spaced angularly or radially. In this example, the central portion 67A has eight segments 68A. The outer portion 67B has multiple segments 68B spaced angularly or radially. In this example, the outer portion 67B has eight segments 68B. Figure 3A shows the illumination source 60 in a state where all segments are lit, corresponding to bright-field illumination. Conveniently, each segment 68A, 68B corresponds to its respective light source 61 or, conveniently, multiple light sources.

[0072] In a preferred embodiment, the illumination field of the illumination source 60 is 2N when the integer N is 1 or greater. It is divided into opposing semicircular regions in the diametrical direction. Each semicircular region can be arbitrarily divided into 2M concentric ring regions when the integer M is 1 or greater.

[0073] The ability of the illumination source 60 to selectively partition its illumination field is advantageous because it allows for adjustment of the spatial frequency response of the imaging system. This adjustment of the spatial frequency response of the imaging system enables optimization for feature sharpness.

[0074] Figure 3B shows eight exemplary settings of the illumination field of the illumination source 60. In each setting, segments 68A and 68B are configured to transmit light only through half of the available illumination field. In this example, since the illumination field is circular, each half is semicircular. The illustrated settings are arranged in pairs a-a', b-b', c-c', and d-d'. In each pair, the light-transmitting half of one setting is shifted 180° angularly around the center point of the illumination field relative to the other setting. That is, each pair is configured to be a mirror image of the other. Furthermore, the settings of the b-b', c-c', and d-d' pairs are shifted 90°, 45°, and 135° angularly, respectively, relative to the a-a' pair. Thus, Figure 3B shows the illumination source 60 realizing four pairs of divided and angularly shifted semicircular illumination settings. Each illumination setting illuminates the object 55 from a different illumination angle.

[0075] Figure 3C further illustrates eight exemplary configurations of the illumination source 60. In each configuration, segments 68A and 68B are configured to transmit light only through the annular portion of the available illumination field. The illustrated configurations are arranged in pairs e-e', f-f', g-g', and h-h'. In each pair, the light-transmitting annular portion is shifted 180° angularly around the center point of the illumination field relative to the other configurations. That is, each pair is configured to be a mirror image of the others. Furthermore, the f-f', g-g', and h-h' configurations are shifted 90°, 45°, and 135° angularly, respectively, relative to the e-e' configuration. Thus, Figure 3C shows the illumination source 60 implementing four pairs of divided and angularly shifted annular illumination configurations. Each illumination configuration illuminates the object 55 from a different illumination angle.

[0076] Therefore, the illumination source 60 can be configured to bring light from different zones or regions of the illumination field depending on the settings of the light source 61. The center point of the illumination field is on an axis perpendicular to the object plane 56. Therefore, the different zones or regions of the illumination field are shifted angularly around the axis perpendicular to the object plane 56. Thus, it can be said that the illumination source 60 can be configured to illuminate the object 55 from any one of several different azimuth angles with respect to the object plane and its perpendicular axis.

[0077] In a preferred embodiment, the illumination source 60 is controlled by the control device 50 to determine how the illumination field is partitioned, i.e., which segments or areas are to be turned on or off, and how the object 55 is illuminated. In embodiments where each segment or area of ​​the illumination field corresponds to a controllable light source 61, the control device 50 may optionally be configured to adjust the intensity of the light sources. Adjusting the intensity of the light sources may optionally include turning each light source on or off as needed. Also, when the microscope 100 is not performing differential phase-contrast microscopy, i.e., in confocal microscopy mode, the illumination source 60 may be turned off or disabled.

[0078] In a preferred embodiment, for DPC microscopy, the object 55 is illuminated sequentially using one or more pairs of illumination settings that are shifted by 180° in the angular direction. For example, one or more pairs of settings such as a-a', b-b', c-c', d-d', e-e', f-f', g-g', h-h' may be used. The individual settings in each pair are used sequentially, for example, b followed by b'. Thus, the object 55 is sequentially illuminated by zones of light source 60 that are shifted in the angular direction. Consequently, the object 55 is illuminated from different illumination angles (called azimuth illumination angles) depending on the settings of the light source 60. As a result, the object 55 is sequentially illuminated from two or more different azimuth angles, preferably one or more pairs of azimuth angles that are shifted by 180°.

[0079] Figure 2 shows an alternative illumination source 60' having a light source 61' and a spatial light modulator 67. The spatial light modulator 67 may be any conventional type and is positioned between the light source 61' and the object 55. The spatial light modulator 67 can be configured to selectively block or mask a portion of the light emitted from the light source 61' in order to control how the object 55 is illuminated by the illumination source 60', including controlling the angle at which the object 55 is illuminated. Thus, the spatial light modulator 67 selectively partitions the illumination field of the illumination source 60' by blocking or masking one or more areas or segments of the illumination field. In a preferred embodiment, the illumination source 60' can also operate in bright-field mode, which provides bright-field illumination to the object 55. The light source 61' may have any conventional light source, for example, one or more incandescent bulbs or one or more LEDs. A collector lens 62 is optionally positioned between the light source 61' and the spatial light modulator 67. A condenser lens 63 is optionally positioned between the spatial light modulator 67 and the object 55.

[0080] The present invention is not limited to the embodiments described herein and may be modified or altered without departing from the scope of the invention.

Claims

1. This is a microscope for imaging an object placed on an object surface. Light source and The system comprises an imaging optical system configured to form an image of the object along an optical path leading to an imaging device, The aforementioned imaging optical system is An infinity-corrected microscope objective lens, Tube lens and, At least one lens configured to focus the back focal plane of the infinity-corrected microscope objective lens onto a conjugate back focal plane located outside the infinity-corrected microscope objective lens, It has an aperture diaphragm positioned on the conjugate postfocal plane and intersecting the optical path, The object surface is located between the infinity-corrected microscope objective lens and the illumination source. A microscope in which the illumination source is configured to illuminate the object from one of a plurality of positions that are angularly offset around an axis perpendicular to the object's surface.

2. The infinity-corrected microscope objective lens and the tube lens are configured to form an image of the object on the intermediate image plane. The at least one lens has an optical relay configured to project an image of the object from the intermediate image plane to the imaging device, and to focus the back focal plane of the infinity-corrected microscope objective lens onto the conjugate back focal plane. The microscope according to claim 1.

3. The optical relay has a first relay lens and a second relay lens spaced apart along the optical path. The first relay lens is configured to image the back focal plane of the infinity-corrected microscope objective lens onto the conjugate back focal plane. The conjugate post-focal plane is located between the first relay lens and the second relay lens. The microscope according to claim 2.

4. The microscope according to claim 3, wherein the conjugate postfocal plane is positioned at a focal distance of 1 from each of the first relay lens and the second relay lens.

5. The microscope according to claim 3 or 4, wherein the first relay lens is positioned at least one focal length away from the intermediate image plane.

6. The lighting source is operable to illuminate the object using a series of two or more lighting settings, The microscope according to any one of claims 1 to 5, wherein in each lighting setting, the lighting source illuminates the object from each different lighting angle.

7. A series of lighting settings consists of one or more pairs of lighting settings. The lighting settings in each pair are used sequentially, causing the lighting sources to illuminate the object from lighting angles that are 180° apart in the angular direction from each other. The microscope according to claim 6.

8. The microscope according to any one of claims 1 to 7, wherein the illumination source has a spatially partitionable illumination field for illuminating the object from different angles with respect to the object's surface.

9. The aforementioned lighting source has a lighting field, The illumination source has an array of first light sources that can be controlled individually and / or as groups of two or more in order to selectively illuminate one or more of the multiple zones of the illumination field. The microscope according to any one of claims 1 to 8.

10. The aforementioned lighting source has a lighting field, The illumination source is operable to illuminate the object using a series of spatially offset zones in the illumination field. A microscope according to any one of claims 1 to 9.

11. The microscope according to claim 10, wherein the spatially offset series of zones have at least one pair of zones that are offset by 180° from each other in the angular direction around the center of the illumination field.

12. The microscope according to any one of claims 1 to 11, wherein the illumination source is positioned away from the object plane corresponding to optical infinity.

13. The microscope according to any one of claims 1 to 12, further comprising means for adjusting the distance between the illumination source and the object surface.

14. The microscope according to any one of claims 1 to 13, comprising an illumination optical system having a second light source and configured to irradiate an object by directing light from the second light source to the object along at least a portion of the optical path.

15. The illumination optical system has a confocal spinning disk, The second light source has at least one laser device positioned to direct a laser beam toward the confocal spinning disk, The confocal spinning disk is movable between an operating state in which it intersects the optical path and an unused state in which it does not intersect the optical path. The microscope according to claim 14.

16. The microscope according to claim 15, which is dependent on claim 2, wherein the confocal spinning disk is positioned in the intermediate image plane in the operating state.

17. The microscope according to claim 15 or claim 16, further comprising a transport mechanism for moving the confocal spinning disk between the used state and the unused state.

18. The confocal spinning disk is included in the spinning disk assembly, The spinning disk assembly is movable between the used state and the unused state. The microscope according to any one of claims 15 to 17.

19. The second light source is arranged to direct the light into the optical path via a beam splitter placed between the tube lens and the optical relay. The beam splitter is positioned to direct the light from the tube lens towards the optical relay. A microscope according to any one of claims 14 to 18, which is dependent on claim 2.

20. The microscope according to any one of claims 1 to 19, wherein the aperture diaphragm is configured to function as a spatial filter.

21. The microscope according to claim 20, wherein the aperture diaphragm has an opening that is less than or equal to the size of the pupil projected from the back focal plane and imaged on the conjugate back focal plane.

22. The microscope according to any one of claims 1 to 21, wherein the aperture diaphragm is movable to define and adjust the size of the opening.

23. The aforementioned aperture diaphragm has an iris device that defines the opening, The microscope according to any one of claims 1 to 22, wherein the iris device is movable to adjust the size of the opening.

24. The microscope according to claim 23, wherein the iris device is arranged on the conjugate plane of the back focal plane of the infinity-corrected microscope objective lens such that the aperture intersects the optical path.

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