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WO2024246492A1 - Système de microscopie - Google Patents

Système de microscopie Download PDF

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Publication number
WO2024246492A1
WO2024246492A1 PCT/GB2024/051323 GB2024051323W WO2024246492A1 WO 2024246492 A1 WO2024246492 A1 WO 2024246492A1 GB 2024051323 W GB2024051323 W GB 2024051323W WO 2024246492 A1 WO2024246492 A1 WO 2024246492A1
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WO
WIPO (PCT)
Prior art keywords
light field
sample
sample holder
waveguide
fluorescence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/GB2024/051323
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English (en)
Inventor
Ian EPERON
Andrew Hudson
Carlos BUENO ALEJO
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University of Leicester
Original Assignee
University of Leicester
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Filing date
Publication date
Application filed by University of Leicester filed Critical University of Leicester
Publication of WO2024246492A1 publication Critical patent/WO2024246492A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor

Definitions

  • a MICROSCOPY SYSTEM Field The present disclosure relates to a microscopy system, and in particular to an interferometric scattering (ISCAT) microscopy system, and a method of inspecting a fluorescently labelled component of a sample.
  • ISCAT interferometric scattering
  • Background Interferometric scattering microscopy (which may be referred to as ISCAT microscopy or ISCAMS) is a useful technique for determining the mass of single particles.
  • the method can only be applied to substantially pure macromolecules with distinguishable molecular masses.
  • studies on the assembly of complex multi- component complexes are hindered by the heterogeneity of the system (such as crude nuclear or cytoplasmic extracts), where it is difficult or impossible to determine whether any given particle contains the macromolecule of interest.
  • an interferometric scattering microscopy system for inspecting a sample comprising a fluorescently labelled component
  • the interferometric scattering microscopy system comprising: a sample holder mount configured to mount a waveguide sample holder comprising: a first surface for positioning the sample; and a second surface opposite the first surface; a coupling arrangement configured to couple a fluorescence excitation light field to the waveguide sample holder to guide the fluorescence excitation light field along the waveguide sample holder by total internal reflection from the first surface and the second surface; an interferometric scattering microscope arranged to: illuminate the sample with a source light field; and detect interference between: a reference light field comprising a reflection of the source light field; and a scattered light field comprising scatter of the source light field from the sample; and a fluorescence microscope arranged to collect a fluorescence emission light field emitted from the fluorescently labelled component in response
  • the system can advantageously perform both total internal reflection fluorescence (TIRF) microscopy and ISCAT microscopy on the same sample 116, without movement or reconfiguration of the sample 116.
  • the two techniques can be performed sequentially, alternately or simultaneously.
  • the addition of TIRF microscopy data with ISCAT microscopy data enables further information to be gleaned about the sample under test.
  • the system can advantageously measure the masses of fluorescently identifiable molecules in complex mixtures.
  • the coupling arrangement may be configured to couple the fluorescence excitation light field to a first optical path comprising one or more guided modes of the waveguide sample holder.
  • the interferometric scattering microscope may be arranged to provide the source light field via a second optical path different to the first optical path.
  • the waveguide sample holder may comprise a rectangular waveguide sample holder.
  • the first optical path may be substantially parallel to a plane of the first surface and a plane of the second surface.
  • the second optical path may comprise a path traversing the second surface into the waveguide sample holder and traversing the first surface out of the waveguide sample holder.
  • the second optical path may be orthogonal to the plane of the first surface and the plane of the second surface.
  • the second optical path may comprise an interferometric scattering microscope objective lens.
  • the fluorescence microscope may be arranged to collect the fluorescence emission light field via a third optical path.
  • the interferometric scattering microscope may be arranged to detect the interference via a fourth optical path different to the third optical path.
  • the third optical path may comprise a fluorescent microscope objective lens.
  • the interferometric scattering microscope may be arranged to illuminate the sample with a source light field by directing the source light field through the second surface and the first surface of the waveguide sample holder towards the sample.
  • the reference light field may comprise a reflection of the source light field from the first surface and/or the second surface of the waveguide sample holder.
  • the fluorescence microscope may be arranged to be on the same side of the waveguide sample holder as the sample.
  • the fluorescence microscope may be arranged closer to the first surface of the waveguide sample holder than the second surface of the waveguide sample holder, when the waveguide sample holder is mounted in the sample holder mount.
  • the fluorescence microscope may be arranged above the sample holder mount.
  • the fluorescence microscope may be arranged to face the first surface of the waveguide sample holder.
  • the interferometric scattering microscope may be arranged to be on the opposite side of the waveguide sample holder to the sample.
  • the interferometric scattering microscope may be arranged closer to the second surface of the waveguide sample holder than the first surface of the waveguide sample holder, when the waveguide sample holder is mounted in the sample holder mount.
  • the interferometric scattering microscope may be arranged below the sample holder mount.
  • the fluorescence microscope may be arranged to face the second surface of the waveguide sample holder.
  • the fluorescence microscope may comprise a camera for capturing fluorescence image data comprising the fluorescence emission light field.
  • the interferometric scattering microscope may comprise a camera for capturing interferometric scattering image data representing the interference.
  • the fluorescence microscope may be arranged to collect the fluorescence emission light field emitted from the fluorescently labelled component in response to absorption of an evanescent field of the fluorescence excitation light field guided along the waveguide.
  • the system may comprise a fluorescence excitation source for providing the fluorescence excitation light field.
  • the fluorescence excitation source may comprise a fluorescence excitation laser or a fluorescence excitation LED.
  • the system may comprise an interferometric light source for providing the source light field.
  • the interferometric light source may comprise a laser source or a LED.
  • the system may comprise the waveguide sample holder.
  • the waveguide sample holder may include an edge extending between first surface and the second surface, wherein the edge comprises an optical surface for coupling the fluorescence excitation light field to the waveguide sample holder.
  • the edge may comprise a polished, cleaved and/or etched edge.
  • the waveguide sample holder may comprise one or more reference markers. The one or more reference markers may be for spatial correlation of data output from the interferometric scattering microscope and data output from the fluorescence microscope.
  • the waveguide sample holder may comprise a slide for a microscope or a coverslip.
  • the waveguide sample holder may comprise a glass waveguide sample holder (e.g. a glass slide or a glass coverslip).
  • the interferometric scattering microscope may be configured to output interference data of the sample.
  • the fluorescence microscope may be configured to output fluorescence data of the sample.
  • the interference data may comprise interferometric image data comprising one or more images of a detected interference pattern.
  • the interference data may comprise output mass data derived from the detected interference.
  • the interference data may comprise output mass data derived from a time dependent variation of the detected interference and/or the interferometric image data.
  • the fluorescence data may comprise fluorescence image data of the collected fluorescent light.
  • the fluorescence image data may comprise one or more images.
  • a method of inspecting a fluorescently labelled component of a sample comprising: positioning the sample on a first surface of a waveguide sample holder; coupling a fluorescence excitation light field to the waveguide sample holder to guide the fluorescence excitation light field along the waveguide sample holder by total internal reflection from the first surface and a second surface of the waveguide sample holder opposite the first surface; collecting a fluorescence emission light field emitted from the fluorescently labelled component in response to absorption of the fluorescence excitation light field; and performing interferometric scattering microscopy on the sample by: illuminating the sample with a source light field; and detecting interference between: a reference light field comprising a reflection of the source light field; and a scattered light field comprising scatter of the source light field from the sample.
  • the method may comprise collecting the fluorescence emission light field to identify a position of the fluorescently labelled component on the sample holder.
  • the method may comprise performing the interferometric scattering microscopy on the sample at the identified position of the fluorescently labelled component.
  • the steps of collecting the fluorescence emission light field and performing the interferometric scattering microscopy may be performed simultaneously.
  • the method may comprise aligning a timing of fluorescent image data of the fluorescence emission light field and interferometric scattering image data from the interferometric scattering microscopy.
  • the method may comprise tethering the sample to the first surface of the waveguide.
  • the method may comprise selectively tethering the fluorescently labelled component to the first surface.
  • the sample may comprise a solution-based sample.
  • the sample may comprise a mixture of macromolecules.
  • the sample may comprise a mixture of macromolecules and small molecules.
  • the fluorescently labelled component may comprise one or more of: a ribonucleic acid molecule; a Deoxyribonucleic acid molecule; a protein; a polymer; a long-chain molecule; and a small molecule.
  • the computer program may be a software implementation, and the computer may be considered as any appropriate hardware, including a digital signal processor, a microcontroller, and an implementation in read only memory (ROM), erasable programmable read only memory (EPROM) or electronically erasable programmable read only memory (EEPROM), as non-limiting examples.
  • the software may be an assembly program.
  • the computer program may be provided on a computer readable medium, which may be a physical computer readable medium such as a disc or a memory device, or may be embodied as a transient signal. Such a transient signal may be a network download, including an internet download.
  • Figure 1 illustrates an interferometric scattering microscopy system according to an embodiment of the present disclosure
  • Figure 2 illustrates a method according to an embodiment of the present disclosure
  • Figure 3 illustrates example total internal reflection fluorescence microscopy images captured by a system according to an embodiment of the present disclosure
  • Figure 4 illustrates time-traces of total internal fluorescence emission derived from images captured by a system according to an embodiment of the present disclosure
  • Figure 5 illustrates a validation of performing TIRF microscopy and ISCAT mass photometry at the same position of a sample using a microscopy system according to an embodiment of the present disclosure.
  • FIG. 1 illustrates an interferometric scattering (ISCAT) microscopy system 100 for inspecting a sample 116 comprising a fluorescently labelled component according to an embodiment of the present disclosure.
  • the system comprises an ISCAT microscope 102, a fluorescence microscope 104, a sample holder mount 106 and a coupling arrangement 108.
  • the sample holder mount 106 is configured to mount a waveguide sample holder 110 (which may be referred to herein simply as the waveguide 110 or the sample holder 110) comprising a first surface 112 for positioning the sample 116 and a second surface 114 opposite the first surface 112.
  • a waveguide sample holder 110 which may be referred to herein simply as the waveguide 110 or the sample holder 110
  • the coupling arrangement 108 is suitable for coupling a fluorescence excitation light field 118 to the waveguide sample holder 110 to guide the fluorescence excitation light field 118 along the waveguide sample holder 110 by total internal reflection (TIR) from the first surface 112 and the second surface 114.
  • the fluorescence microscope 104 is arranged to collect a fluorescence emission light field 120 emitted from the fluorescently labelled component of the sample 116 (positioned on the first surface 112) in response to absorption of (an evanescent field of) the fluorescence excitation light field 118.
  • the ISCAT microscope 102 is arranged to illuminate the sample with a source light field 126.
  • the ISCAT microscope may direct the source light field 126 through the second surface 114 and the first surface 112 of the waveguide sample holder 110 towards the sample 116.
  • the ISCAT microscope 102 detects interference between: (i) a reference light field 128; and (ii) a scattered light field 130 comprising scatter of the source light field 126 from the sample 116.
  • the reference light field 128 may comprise a reflection of the source light field 126 from the first surface 112 and/or the second surface 114 of the waveguide sample holder 110.
  • the system 100 is configured to advantageously perform both total internal reflection fluorescence (TIRF) microscopy and ISCAT microscopy on the same sample 116, without movement or reconfiguration of the sample 116. The two techniques can be performed sequentially, alternately or simultaneously.
  • TIRF microscopy data with ISCAT microscopy data enables further information to be gleaned about the sample under test, as explained further below.
  • the system can advantageously measure the masses of fluorescently identifiable molecules in complex mixtures.
  • the addition of TIRF microscopy provides an orthogonal label that allows specific molecules to be identified and thus their mass assigned even in highly heterogeneous mixtures. This is achieved by labelling the molecules of interest with a fluorophore, and then detecting the masses of the fluorescently identified molecules using ISCAT.
  • the apparatus enables new insights into the mass of complexes in functional environments and greatly expands the capability of ISCAT methods, for example, the study of complex processes of protein assembly in real time.
  • the apparatus is particularly suited to solution-based samples and can avoid the requirement of a purification step prone to the unwanted formation of aggregates or condensates prior to the microscopy, as described above.
  • the system 100 is configured to perform TIRF microscopy by exciting the fluorescently labelled component of the sample 116 by guiding the fluorescence excitation light field 118 along the waveguide sample holder 110 and monitoring the resultant fluorescence emission light field 120.
  • the TIRF microscopy can advantageously be performed independently of (i.e. without influencing) operation of the ISCAT microscope 102.
  • the fluorescence excitation light field 118 is provided via a first optical path 119 (the waveguided path or mode of the waveguide 110) that differs from a second optical path 132 of the source light field 126, which in this example is provided via an ISCAT microscope objective lens 122.
  • the fluorescence emission light field 120 is collected via a third optical path 134 (in this example provided by a fluorescence microscope objective lens 124) which is different to a fourth optical path 136 via which the interference of the reference light field 128 and the scattered light field 130 is detected.
  • the fourth optical path 136 comprises the ISCAT microscope objective lens 122 and is coincident with the second optical path 132.
  • an ICAT microscopy path comprising the second and fourth paths 132, 136 is independent of a fluorescence microscopy path comprising the first and third paths 119, 134.
  • the fluorescence excitation / emission can be provided/collected without affecting the ISCAT microscopy path.
  • special optics would be required that balance the different requirements of ISCAT and fluorescence microscopy and would be optimised for neither, e.g. dichroic mirrors or an objective lens 122 with a numerical aperture that balances ISCAT and fluorescence requirements.
  • the fluorescence microscope 104, sample holder mount 106 and coupling arrangement can be combined with an existing ISCAT microscope 102 and require little or minimal adaptation of the design of the ISCAT microscope 102.
  • the ISCAT microscope objective 122 and other coupling optics (not shown) internal to the ISCAT microscope 102 can remain optimised for ISCAT microscopy and utilise the most appropriate source light field (optimum light source in terms of power, wavelength, linewidth etc.).
  • a green or blue wavelength for ISCAT can provide optimum sensitivity due to an optimum combination of wavelength and available source quality (intensity, linewidth, beam quality etc).
  • the third optical path 134 can be optimised for TIRF microscopy, for example utilising a high numerical aperture objective lens 124 that is typical of TIRF microscopy. Such high NA objective lenses are less suitable for ISCAT microscopy.
  • the fluorescence microscopy set-up can also use the optimum excitation and emission wavelengths without any requirements for compatibility with the ISCAT microscopy wavelength, for example the same colour/wavelength could be used for both ISCAT and fluorescence detection. It can be challenging to use the same or closely spaced wavelengths on a common or shared path because some form of discrimination is required to couple the light to/from the different detectors/sources of the ISCAT and fluorescence set-ups.
  • the sample holder mount 106 is configured to mount the waveguide sample holder 110.
  • the waveguide sample holder 110 comprises a rectangular waveguide sample holder 110.
  • the rectangular waveguide 110 (which may also be referred to as a slab waveguide) refers to a waveguide with a rectangular (or square) cross-section.
  • the rectangular waveguide 110 includes a first surface 112 (or face) for positioning the sample 116 on and a second surface 114 (or face) opposite the first surface 112.
  • the first surface 112 and second surface 114 may be polished or otherwise sufficiently flat/smooth to provide specular reflection.
  • the rectangular waveguide 110 may comprise a cuboid or slab of material that is transparent to a fluorescence excitation light field 118.
  • the material may have a refractive index greater than 1.0 such that the material-air interface can provide total internal reflection (TIR) to, and guide, the fluorescence excitation light field 118 along the rectangular waveguide 110.
  • TIR total internal reflection
  • the material may have a refractive index greater than 1.33 to avoid light being coupled out of the waveguide by an aqueous sample.
  • the rectangular waveguide 110 may comprise a layered structure parallel to the first and second surfaces 112, 114, such that the fluorescence excitation field is guided by TIR from an interface of one or more of the layers.
  • the rectangular waveguide 110 comprises a cover glass (which may also be referred to as a coverslip or glass slip), and the waveguiding / TIR properties are provided by the glass-air interfaces (or glass-liquid interface in the presence of a sample) at the first and second surfaces 112, 114.
  • the cover glass may comprise a standard cover glass used in ISCAT microscopy and may have a thickness in the range of 0.1-0.2mm or 0.13-0.16 mm.
  • the waveguide 110 may be comprise a greater thickness, e.g. from 0.2 to 1.5 mm.
  • the waveguide 110 may comprise a slide for a microscope or a similar cuboid glass structure.
  • a thicker waveguide may have greater tolerance for coupling the fluorescence excitation light field 118 and may capture a higher amount of the fluorescence excitation light field 118.
  • a maximum thickness may be limited by the power of the ISCAT microscope objective lens 122.
  • the rectangular waveguide 110 may include four edges extending between the first surface 112 and the second surface 114. One or more of the edges may comprise an optical surface which is sufficiently smooth/flat for coupling the fluorescence excitation light source 118 into the rectangular waveguide 110.
  • the optical surface may be provided by polishing, cleaving and/or etching and it may or may not be passivated to prevent adsorption of macromolecules and contain ligands for the binding of specific macromolecules.
  • the rectangular waveguide 110 may include one or more reference markers on the first surface 112 and/or the second surface 114.
  • the one or more reference markers can enable alignment of the respective images received from the ISCAT microscope 102 and the fluorescent microscope 104.
  • the markers may be used to define and calibrate a common coordinate system of the two microscopes 102, 104 for each sample 116.
  • the markers may be arranged as a grid to enable registration of ISCAT and TIRF images from the respective ISCAT and fluorescence microscopes 102, 104.
  • the system 100 may include the rectangular waveguide sample holder 110.
  • the sample holder mount 106 may include one or more locating features for locating the rectangular waveguide sample holder 110 onto the sample holder mount 106.
  • the one or more locating features may include protrusions or indentations on the surface of the sample holder mount 106 and/or couplings or fastenings for positioning and/or securing the rectangular waveguide 110 in a predetermined location in the system 100.
  • Providing the rectangular waveguide 110 in a predetermined location can assist repeatable coupling of the fluorescence excitation light field 118 to a wave-guided mode of the rectangular waveguide 110.
  • the coupling arrangement 108 enables coupling of the fluorescence excitation light field 118 to the rectangular waveguide 110 to guide the fluorescence excitation light field 118 along the rectangular waveguide 110 by TIR from the first and second surfaces 112, 114.
  • the guiding of the fluorescence excitation light field 118 along the rectangular waveguide 110 by TIR may be considered as coupling the fluorescence excitation light field 118 to one or more guided modes of the rectangular waveguide 110.
  • the one or more guided modes may comprise the first optical path 119.
  • the coupling arrangement 108 is simply a laser holder for mounting a fluorescence excitation source comprising a fluorescence excitation laser.
  • the fluorescence excitation source may comprise any known light source, such as a visible laser or LED, suitable for providing the fluorescence excitation light field 118 to excite the fluorescently labelled component of the sample 116.
  • the coupling arrangement 108 may comprise a plurality of fluorescence excitation sources (e.g.
  • the coupling arrangement 108 may comprise a mount for a single excitation source which allows for interchanging of different excitation sources.
  • the coupling arrangement 108 may include coupling optics for coupling or launching the fluorescence excitation light field 118 from the fluorescence excitation light source into one or more guided modes of the rectangular waveguide 110.
  • the one or more guided modes of the rectangular waveguide 110 may be considered as the first optical path 119 for the fluorescence excitation light field 118.
  • the coupling arrangement 108 may comprise a pair of cylindrical lenses in a Keplerian configuration for expanding the beam along the single axis and a further cylindrical lens for focusing the beam on the same axis.
  • Such an arrangement can be advantageous for coupling to waveguides 110 with an elongated end-face such as a cover glass or slide.
  • the ISCAT microscope 102 is arranged in relation to the sample holder mount 106 such that the ISCAT microscope 102 will be closer to the second surface 114 of the rectangular waveguide 110 than the first surface 112, when the rectangular waveguide 110 is located in the sample holder mount 106.
  • the ISCAT microscope 102 is positioned below the sample holder mount 106, i.e. on the opposite side of the rectangular waveguide sample holder 110 to the sample 116.
  • the sample holder mount 106 includes a planar surface with an aperture to provide the second and fourth optical paths 132, 136 from the ISCAT microscope 102 to the sample 116.
  • the ISCAT microscope 102 includes the ISCAT microscope objective lens 122 that provides the second optical path 132 and the fourth optical path 136.
  • the rectangular waveguide sample holder 110 may be mounted directly onto the ISCAT microscope objective lens 122 such that the second surface 114 is in contact with an outer surface of the objective lens 122. Oil immersion may be used between the objective lens 122 and the second surface 114 to increase the effective NA of the objective lens 122.
  • ISCAT microscopy is a known technique and is summarised briefly here.
  • the ISCAT microscope provides the source light field 126 along the second optical path 132.
  • the source light field 126 may be provided by a laser or LED.
  • the laser may comprise a green solid-state laser, such as a frequency-doubled solid-state laser.
  • the source light field 126 passes from the ISCAT objective lens 122 and traverses the second surface 114 and passes into the rectangular waveguide 110.
  • Reflections of the source light field 126 from the second surface 114 may provide the reference light field 128.
  • the source light field 126 continues traversing the rectangular waveguide 110 and traverses the first surface 112 to irradiate the sample 116. Reflections of the source light field 126 from the first surface 112 may provide the reference light field 128.
  • the sample 116 scatters the source light field 126 via Rayleigh scattering to produce the scattered light field 130.
  • the intensity of the scattered light field 130 depends on the number, orientations and density of polarizable bonds in the molecule / sample 116.
  • the scattered light field radiates in all directions.
  • the portion of the scattered light field 130 that irradiates along the fourth optical path 136 optically interferes with the reference light field 128.
  • the ISCAT microscope 102 detects this interference.
  • the intensity of the reference light field 128 may be orders of magnitude greater than the intensity of the scattered light field 130 scattered into the fourth optical path 136 (even with attenuation of the reference light field 128, for example through use of AR coatings on the sample holder 110 or a polarising beam splitter).
  • the ISCAT microscope 102 may capture a sequence of images or a video (using a camera (not shown)). From the sequence of images, an image difference between a first image and a second image can be determined to detect changes in the interference pattern with time. Changes in the interference pattern may relate to changes in the sample, for example the binding of a protein.
  • the change in interference representative of the change in scattering
  • the present disclosure combines ISCAT microscopy with TIRF microscopy of fluorescently labelled components within the ISCAT sample to provide further insight, for example to observe protein agglomerate formation, as described further below.
  • the fluorescence microscope 104 is arranged in relation to the sample holder mount 106 such that the fluorescence microscope 104 will be closer to the first surface 112 of the rectangular waveguide 110 than the second surface 114, when the rectangular waveguide 110 is located in the sample holder mount 106.
  • the fluorescence microscope 104 is positioned above the sample holder mount 106, i.e. on the same side of the rectangular waveguide sample holder 110 as the sample 116.
  • the fluorescence microscope 104 comprises a fluorescence microscope objective lens 124 configured to collect the fluorescence emission light field 120 and provide the third optical path 134.
  • the fluorescence microscope objective lens 124 may include a longer working distance than the ISCAT objective lens 122.
  • the fluorescence microscope objective lens 124 can focus on the bottom interior surface of the chamber while maximising the NA.
  • the sample holder comprises a second coverslip 138 for covering/enclosing the sample 116 and providing and interface between the fluorescence microscope objective lens 124 and the sample 116. Water immersion may be used between the fluorescence microscope objective lens 124 and the second coverslip 138 to increase the effective NA of the objective lens 124.
  • the fluorescence microscope 104 comprises a camera 140 for capturing images and/or video of the fluorescence excitation light field emitted from the sample 116.
  • the camera 140 and the fluorescence microscope objective lens 124 may each be mounted on xyz translation stages to aid alignment of the light path and focussing of the camera.
  • the coupling arrangement 108 couples the fluorescence excitation light field 118 into a guided mode of the rectangular waveguide 110.
  • the fluorescence excitation light field 118 is guided along the rectangular waveguide by TIR from the first surface 112 and the second surface 114.
  • An evanescent field of the fluorescent excitation light field 118 penetrates through the first surface 112 and into the sample 116.
  • the evanescent field excites the fluorescently labelled component in the sample 116 which emits the fluorescence emission light field in response.
  • the fluorescence emission light field 120 radiates in all directions.
  • the high NA fluorescence objective lens captures the portion of the fluorescence emission light field radiating into the NA. Excitation via the evanescent field can advantageously only excite labelled components that are close to (e.g.
  • Figure 1 illustrates the ISCAT microscope 102 and the fluorescence microscope 104 on opposite sides of the waveguide sample holder 110, in other examples both microscopes may be positioned on the same side.
  • both microscopes 102, 104 may share a common objective such as the ISCAT objective lens 122.
  • Such an example may comprise a dichroic mirror positioned under the ISCAT objective lens 122 to send the fluorescence emission light field 120 to a fluorescence camera while the ISCAT signal continues to an ISCAT camera.
  • FIG. 2 illustrates a method 242 of inspecting a fluorescently labelled component of a sample 116 according to an embodiment of the present disclosure. The method may be performed by the apparatus of Figure 1, which will continue to be referred to.
  • a first step 244 comprises positioning the sample 116 on a first surface 112 of a rectangular waveguide sample holder 110.
  • a second step 246 comprises coupling a fluorescence excitation light field 118 to the rectangular waveguide sample holder 110 to guide the fluorescence excitation light field 118 along the rectangular waveguide sample holder 110 by total internal reflection from: (i) the first surface 112; and (ii) a second surface 114 of the rectangular waveguide sample holder 110 opposite the first surface 112.
  • a third step 248 comprises collecting a fluorescence emission light field 120 emitted from the fluorescently labelled component in response to absorption of the fluorescence excitation light field 118.
  • a fourth step 250 comprises performing interferometric scattering microscopy on the sample by: (i) illuminating the sample with a source light field; and (ii) detecting interference between: a reference light field comprising a reflection of the source light field; and a scattered light field comprising scatter of the source light field from the sample.
  • the steps may be performed in the illustrated order or a different order.
  • the fourth step 250 may be performed before the second step 246.
  • the method may comprise performing the second step 246 and the third step 248 to identify the position of the fluorescently labelled component on the rectangular waveguide 110.
  • the fourth step 250 may then be performed at the identified position of the fluorescently labelled component.
  • the sample may comprise a macromolecule mixture including proteins and a labelled RNA component as a site for protein agglomeration.
  • the method may comprise performing TIRF microscopy (second and third steps 246, 248) to identify where a labelled RNA component of the sample 116 is positioned on the sample holder 110.
  • the method may comprise performing ISCAT microscopy (fourth step 250) at the identified position of the RNA component to observe protein agglomeration on the RNA component and the resulting mass accumulation provided from the data of the ISCAT microscopy.
  • the method may be performed in the reverse order to that described above.
  • the fourth step 250 may be performed prior to the second and third steps 246.
  • the method may comprise performing ISCAT microscopy (fourth step 250) to observe mass changes during protein agglomeration provided from the data of the ISCAT microscopy.
  • the method may then comprise performing TIRF microscopy (second and third steps 246, 248) to identify whether the labelled RNA component of the sample 116 was present in the observed protein agglomeration.
  • the third and fourth steps 248, 250 may be performed simultaneously.
  • the method may comprise aligning the timing of fluorescent image data from the camera 140 of the fluorescent microscope 104 and ISCAT image data from the camera of the ISCAT microscope 102.
  • the method may comprise tethering the sample 116 to the first surface of the rectangular waveguide 110. Tethering the sample 116 may comprise selectively tethering the fluorescently labelled component. Tethering the sample 116 may comprise known tethering methods such as providing a layer of polyethylene glycol and a biotinylated derivative thereof on the first surface of the coverslip 110 for selective tethering of the labelled component.
  • the method may comprise aligning the fluorescent microscope and the ISCAT microscope to a common image coordinate system using one or more reference markers on the rectangular waveguide 110.
  • Figure 3 illustrates TIRF images, labelled A-H, collected using a microscopy system according to an embodiment of the present disclosure, such as the system of Figure 1.
  • Images A and B correspond to respective samples each comprising 40nm red emission polystyrene beads.
  • Images C and D correspond to respective samples each comprising single stranded DNA oligo labelled with Alexa-647.
  • Images E and F correspond to respective samples each comprising Streptavidin labelled with Alexa-647.
  • Images G and H correspond to respective samples each comprising RNA labelled with Cyanine 5. Single molecules were tethered to the first surface using biotin-avidin chemistry.
  • Each image illustrates fluorescent emission from the respective labelled sample particles / proteins upon TIRF excitation by the fluorescent excitation light field.
  • Figure 4 illustrates fluorescence emission time traces, labelled A to I, corresponding to the images of Figure 3 and derived from images collected by a microscopy system according to an embodiment of the present disclosure, such as the system of Figure 1.
  • Each time trace is from a particular emission region of an image, for example a pixel or group of pixels, corresponding to a labelled protein.
  • the time traces show the fluorescence emission from the same emission region of the image across a sequence of consecutive images (e.g. a video) captured by the microscopy system.
  • Traces A-C correspond to a sample of DNA oligo labelled with Alexa-647.
  • Traces D-F correspond to a sample of Streptavidin labelled with Alexa-647.
  • Traces G-I correspond to RNA labelled with Cy5.
  • the traces demonstrate that a consistent (detectable) fluorescence emission signal is obtained over a sequence of several hundred images before the fluorescent molecule is bleached by the excitation source.
  • the single bleaching step indicates that molecules containing a single fluorophore were detected.
  • regions of an image that contain a labelled molecule can be identified using TIRF and ISCAT microscopy can subsequently be performed on the regions with a confidence that the target molecule will remain at the respective region.
  • Figure 5 illustrates a validation of performing TIRF microscopy and ISCAT mass photometry at the same position of a sample using a microscopy system according to an embodiment of the present disclosure, such as the system of Figure 1.
  • the Figure illustrates a first TIRF image 552 captured prior to ISCAT based mass photometry measurement and a second TIRF image 554 captured subsequent to the ISCAT measurement.
  • the first TIRF image 552 and the second TIRF image 554 are substantially identical illustrating that the microscope 104 remains focussed on the same site while mass photometry images are acquired.
  • an accurate mass photometry spectrum was obtained for bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • any reference to “close to”, “before”, “shortly before”, “after” “shortly after”, “higher than”, or “lower than”, etc, can refer to the parameter in question being less than or greater than a threshold value, or between two threshold values, depending upon the context.

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Abstract

L'invention concerne un système de microscopie à diffusion interférométrique pour inspecter un échantillon comprenant un composant marqué par fluorescence, le système comprenant : un support de porte-échantillon configuré pour monter un porte-échantillon de guide d'ondes comprenant : une première surface pour positionner l'échantillon ; et une seconde surface opposée à la première surface ; un agencement de couplage configuré pour coupler un champ de lumière d'excitation de fluorescence au porte-échantillon de guide d'ondes pour guider le champ de lumière d'excitation de fluorescence le long du porte-échantillon de guide d'ondes par réflexion interne totale à partir de la première surface et de la seconde surface ; un microscope à diffusion interférométrique conçu pour : éclairer l'échantillon avec un champ de lumière source ; et détecter une interférence entre : un champ de lumière de référence comprenant une réflexion du champ de lumière source ; et un champ de lumière diffusée comprenant la diffusion du champ de lumière source à partir de l'échantillon ; et un microscope à fluorescence conçu pour collecter un champ de lumière d'émission de fluorescence émis par le composant marqué par fluorescence en réponse à l'absorption du champ de lumière d'excitation de fluorescence.
PCT/GB2024/051323 2023-05-26 2024-05-22 Système de microscopie Pending WO2024246492A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040091862A1 (en) * 2000-01-21 2004-05-13 Albrecht Brandenburg Method and device for detecting temperature-dependent parameters, such as the association/dissociation parameters and/or the equilibrium constant of complexes comprising at least two components
WO2011151307A2 (fr) * 2010-06-02 2011-12-08 Laser-Laboratorium Göttingen E.V. (Llg) Dispositif d'injection servant à injecter la lumière dans un guide d'ondes planaire
WO2018011591A1 (fr) * 2016-07-13 2018-01-18 Oxford University Innovation Limited Microscopie à diffusion interférométrique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040091862A1 (en) * 2000-01-21 2004-05-13 Albrecht Brandenburg Method and device for detecting temperature-dependent parameters, such as the association/dissociation parameters and/or the equilibrium constant of complexes comprising at least two components
WO2011151307A2 (fr) * 2010-06-02 2011-12-08 Laser-Laboratorium Göttingen E.V. (Llg) Dispositif d'injection servant à injecter la lumière dans un guide d'ondes planaire
WO2018011591A1 (fr) * 2016-07-13 2018-01-18 Oxford University Innovation Limited Microscopie à diffusion interférométrique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JAIME ORTEGA ARROYO ET AL: "Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging", NATURE PROTOCOLS, vol. 11, no. 4, 3 March 2016 (2016-03-03), GB, pages 617 - 633, XP055360160, ISSN: 1754-2189, DOI: 10.1038/nprot.2016.022 *

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