GB2576870A - Apparatus for high stability imaging - Google Patents

Apparatus for high stability imaging Download PDF

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Publication number
GB2576870A
GB2576870A GB1811454.6A GB201811454A GB2576870A GB 2576870 A GB2576870 A GB 2576870A GB 201811454 A GB201811454 A GB 201811454A GB 2576870 A GB2576870 A GB 2576870A
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United Kingdom
Prior art keywords
sample
lens
objective lens
sample holder
holder
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.)
Withdrawn
Application number
GB1811454.6A
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GB201811454D0 (en
Inventor
Schwertner Michael
Van Nugteren Martijn
Vader Hildo
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Linkam Scient Instruments Ltd
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Linkam Scient Instruments Ltd
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Priority to GB1811454.6A priority Critical patent/GB2576870A/en
Publication of GB201811454D0 publication Critical patent/GB201811454D0/en
Publication of GB2576870A publication Critical patent/GB2576870A/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B7/00—Mountings, adjusting means, or light-tight connections, for optical elements
    • G02B7/02—Mountings, adjusting means, or light-tight connections, for optical elements for lenses
    • G02B7/021—Mountings, adjusting means, or light-tight connections, for optical elements for lenses for more than one lens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/02—Objectives
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/26—Stages; Adjusting means therefor
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B7/00—Mountings, adjusting means, or light-tight connections, for optical elements
    • G02B7/02—Mountings, adjusting means, or light-tight connections, for optical elements for lenses
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/28—Base structure with cooling device
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/33—Immersion oils, or microscope systems or objectives for use with immersion fluids

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

A microscope 2, which may be particularly suited for super-resolution or fluorescence microscopy, comprises an objective lens 9, itself having a lens barrel 53 defining a longitudinal axis, and a front lens 61 mounted on the axis within the barrel. The microscope additionally comprises a sample holder 15 and a holder mount 17. The mount is couplable to the objective lens and configured to be removably mounted to either the sample holder or objective lens such that the sample holder is disposed on the longitudinal axis. The mount may comprise a first collar with first screw thread 29a on an internal surface, whilst the objective lens comprises a corresponding first screw thread 29b one an external surface. The apparatus may comprise an actuator 35 comprising elements coupled to objective lens and sample holder, and which moves the first element parallel to the longitudinal axis. A kit of parts containing components for attaching to a microscope so as to produce the previously described apparatus is also described, as is a method of using such apparatus, which may be conducted under cryogenic sample conditions.

Description

(54) Title of the Invention: Apparatus for high stability imaging Abstract Title: Apparatus for high stability microscopy imaging (57) A microscope 2, which may be particularly suited for super-resolution or fluorescence microscopy, comprises an objective lens 9, itself having a lens barrel 53 defining a longitudinal axis, and a front lens 61 mounted on the axis within the barrel. The microscope additionally comprises a sample holder 15 and a holder mount 17. The mount is couplable to the objective lens and configured to be removably mounted to either the sample holder or objective lens such that the sample holder is disposed on the longitudinal axis. The mount may comprise a first collar with first screw thread 29a on an internal surface, whilst the objective lens comprises a corresponding first screw thread 29b one an external surface. The apparatus may comprise an actuator 35 comprising elements coupled to objective lens and sample holder, and which moves the first element parallel to the longitudinal axis. A kit of parts containing components for attaching to a microscope so as to produce the previously described apparatus is also described, as is a method of using such apparatus, which may be conducted under cryogenic sample conditions.
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Intellectual
Property
Office
Application No. GB1811454.6
RTM
Date :24 January 2019
The following terms are registered trade marks and should be read as such wherever they occur in this document:
Zerodur
Invar
PEEK
Delrin
Nikon
Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo
- 1 Apparatus for high stability imaging
Field
The present invention relates to microscopes and methods of imaging samples. In particular, the present invention relates to microscopes and methods for stable imaging at high resolution and high magnification.
Background
Current optical microscopy techniques, such as fluorescence imaging and in particular super-resolution imaging typically require high stability of the imaging setup to avoid vibration or drift of the sample when imaging. The effect of vibration and drift can limit the capability of the instrument and affect data quality and integrity, especially at high magnification.
With the widespread use of optical super-resolution techniques, the requirements for stability and reduced drift have increased beyond those for conventional imaging systems. Mechanical stability and low-drift is often a prerequisite for the use of superresolution techniques.
In order to counteract vibration and thermal drift problems in microscopy applications, common solutions include tight temperature control of the room, vibration - insulated tables and desks, active vibration insulation based on piezo-electric actuators and temperature controlled enclosures around the microscope setup or a part of it. Other measures can include the use of well-known low-expansion materials, such as Zerodur (low expansion ceramics) or invar, a metal alloy with very low thermal expansion coefficient. All these measures are effective and can be combined. However, they often lead to a large setup and / or increased cost.
Therefore, there is a need to provide an improved apparatus and method for stable imagining at high resolution and magnification, while ensuring a simplified design and enabling sample manipulation.
Summary
Accordingly, in a first aspect, there is provided a microscope comprising:
an objective lens comprising:
- 2 a lens barrel comprising a proximal end and a distal end and defining a longitudinal axis therebetween; and a front lens mounted on the longitudinal axis within the lens barrel;
a sample holder configured to support a sample thereon; and a holder mount coupled to or configured to be coupled to the objective lens and configured to removably mount the sample holder thereto such that the sample holder is disposed on the longitudinal axis and a sample mounted thereon may be viewed or imaged through the front lens.
Advantageously, the apparatus of the first aspect has a small mechanical loop allowing stable imaging at high resolution and magnification. The mechanical loop can be defined as the shortest mechanical path from the sample location observed to the tip of the objective lens.
The longitudinal axis may be understood to be the optical axis of the microscope.
Accordingly, it can be viewed as an axis along which light travels through the objective lens and/or perpendicular to the focal plane.
Preferably, the holder mount is configured to be coupled directly to the objective lens. 20 Preferably, the holder mount is configured to directly mount the sample holder.
Preferably, the holder mount is coupled to or configured to be coupled at or towards a proximal end of the objective lens. In one embodiment, the objective lens does not comprise an outer cosmetic jacket. Accordingly, the holder mount is preferably coupled 25 to or configured to be coupled directly to the lens barrel. Accordingly, the holder mount is preferably coupled to or configured to be coupled at or towards a proximal end of the lens barrel.
In one embodiment, the holder mount may be fixedly coupled to the objective lens.
Accordingly, the holder mount may be fixedly coupled directly to the lens barrel, preferably at or towards a proximal end thereof. The holder mount may be fixedly coupled using glue, preferably epoxy glue.
In one embodiment, the holder mount is configured to be removably coupled to the 35 objective lens. Accordingly, the holder mount may be configured to be removably coupled directly to the lens barrel, preferably at or towards a proximal end thereof.
-3The holder mount may comprise a first collar sized to fit over the lens barrel, thereby mounting the holder mount to the lens barrel.
The holder mount may be configured to be removably coupled to the objective lens using magnetic means. Accordingly, the holder mount may comprise a magnet and the objective lens may comprise a magnetic material. Alternatively, the objective lens may comprise a magnet and the holder mount may comprise a magnetic material. The magnetic material may comprise a ferrous alloy or magnetic stainless steel. Preferably, 10 the steel is ferritic stainless steel. Alternatively, both the objective lens and the holder mount may comprise a magnet. Preferably, in embodiments where it is present, the first collar of the holder mount comprises the magnet or magnetic material. Preferably, the lens barrel of the objective lens comprises the magnet or the magnetic material. The or each magnet may be an electromagnet. Advantageously, the electromagnet can 15 be deactivated or switched to allow the holder mount to be removed from the objective lens. Preferably, the or each magnet is a permanent magnet. Advantageously, a permanent magnet will not introduce heating and temperature drift.
Alternatively, the first collar may define a first screw thread on an internal surface thereof and the objective lens may define a corresponding first screw thread on an external surface thereof. Accordingly, the first collar maybe screwed to the objective lens. It may be appreciated that to allow the first collar to be screwed to the objective lens, the first screw thread and the corresponding first screw thread should have substantially the same pitch. The first screw thread on the internal surface on the first 25 collar may be disposed at or towards a distal end of the first collar. The corresponding first screw thread on the external surface of the objective lens may be disposed at or towards a proximal end of the objective lens. Preferably, the corresponding first screw thread is defined on an external surface of the lens barrel, more preferably at or towards a proximal end thereof.
The holder mount maybe configured to be removably coupled to the objective lens by clamping means. Accordingly, the holder mount may comprise a ring clamp configured to fit around the objective lens, and more preferably the lens barrel. Preferably, the ring clamp is configured to move between an open configuration, in which it can be 35 inserted over the objective lens or removed therefrom, and a closed configuration, in which it can be clamped to the objective lens. Preferably, the ring clamp comprises first
-4and second clamping portions, wherein, in the open configuration, the first and second clamping portions are spaced apart, and in the closed configuration the first and second clamping portions are disposed substantially adjacent to each other. In one embodiment, the ring clamp comprises a first section, comprising a first end and a second end, and a second section, comprising a first end and a second end, wherein the first and second sections are rotatably coupled at their first ends and the second ends of the first and second sections define the first and second clamping portions. Preferably, the ring clamp further comprises fastening means configured to reversibly fasten the first and second clamping portions. Preferably, when the first and second clamping portions are disposed substantially adjacent to each other an internal surface of the ring clamp substantially defines a cross section of the objective lens, and more preferably the lens barrel. Preferably, the holder mount can be clamped in several positions along the optical axis, effectively allowing to focus the sample.
The fastening means may comprise magnetic fastening means. Accordingly, the first clamping portion may comprise a magnet and the second clamping portion may comprise magnetic material. The magnetic material may comprise a ferrous alloy or magnetic stainless steel. Preferably, the steel is ferritic stainless steel. The or each magnet may be an electromagnet. Advantageously, the electromagnet can be deactivated or switched to allow the holder mount to be removed from the objective lens or positioned along the optical axis for the purpose of focussing. Preferably, the or each magnet is a permanent magnet.
Alternatively or additionally, the fastening means may comprise mechanical fastening 25 means. The mechanical means may comprise a catch or pin configured to reversibly couple the first and second clamping portions.
In one embodiment, the holder mount is configured to prevent the sample holder from moving along the longitudinal axis when it is mounted thereto. In this embodiment, the holder mount may consist of a single element.
In an alternative embodiment, the holder mount may be configured to allow adjustment of the position of the sample holder along the longitudinal axis. Advantageously, varying the position of the sample holder on the longitudinal axis can 35 allow a user to focus the microscope.
-5Accordingly, the first collar may define a second screw thread disposed at or towards a proximal end of the first collar, and the holder mount may further comprise a second collar defining a corresponding second screw thread. Accordingly, the second collar may be screwed to the first collar. It may be appreciated that to allow the second collar 5 to be screwed to the first collar, the second screw thread and corresponding second screw thread should have substantially the same pitch. Preferably, the second collar is configured to removably mount the sample holder thereto.
It maybe appreciated that the second collar could be sized to fit over the first collar, and in this embodiment the second screw thread would be defined on an external surface of the first collar and the corresponding second screw thread would be defined on an internal surface of the second collar. Alternatively, the second collar could be sized to fit inside the first collar, and in this embodiment the second screw thread would be defined on an internal surface of the first collar and the corresponding second 15 screw thread would be defined on an external surface of the second collar.
Preferably, the first screw thread has a different pitch to the second screw thread. The difference in pitch is preferably at least 0.001 mm, more preferably at least 0.01 mm, at least 0.02 mm, at least 0.03 mm, at least 0.04 mm or at least 0.05 mm, and most preferably at least 0.06 mm, at least 0.07 mm, at least 0.08 mm, at least 0.09 mm or at least 0.1 mm. The difference in pitch is preferably less than 100 mm, more preferably less than 10 mm, less than 7.5 mm, less than 5 mm, or less than 2.5 mm, and most preferably less than 1 mm, less than 0.8 mm, less than 0.6 mm, less than 0.4 mm or less than 0.2 mm. The difference in pitch maybe between 0.01mm and 0.1mm, preferably between 0.02mm and 0.8mm, more preferably between 0.03 mm and 0.6mm, even more preferably between 0.04 and 0.4mm, and most preferably between 0.05 and 0.2mm.
Preferably, the microscope further comprises fixing means configured to prevent the 30 second collar from rotating independently of the object lens, while allowing relative movement in the longitudinal direction. The fixing means may comprise an elongate member comprising a proximal end and a distal end extending between the objective lens and the second collar. The distal end of the elongate member may be coupled to the objective lens and the proximal end of the elongate member may be disposed in a 35 recess in the second collar. Alternatively, the distal end of the elongate member may be disposed in a recess on the objective lens and the proximal end of the elongate member
-6may be coupled to the second collar. In a further alternative, the distal end of the elongate member may be disposed in a recess on the objective lens and the proximal end of the elongate member may be disposed in a recess in the second collar.
Advantageously, when the first collar is rotated, the second collar and the sample holder mounted thereto move along the longitudinal axis.
Alternatively, the holder mount may comprise an actuator comprising:
a first element coupled or configured to be coupled to the objective lens; and 10 a second element coupled to the first element and configured to removably mount the sample holder thereto, wherein the actuator is configured to move the second element, relative to the first element.
The first element maybe configured to be removably coupled to the objective lens.
Preferably, the actuator is configured to move the second element, relative to the first element, in a direction substantially parallel to the longitudinal axis. Preferably, the actuator is a piezoelectric actuator. The piezoelectric actuator may be a stacked piezo actuator or a “walking type” drive. Preferably, the piezoelectric actuator is a “walking 20 type” drive.
The first element may comprise a first collar sized to fit over the lens barrel, thereby mounting the holder mount to the lens barrel. The second element may comprise a second collar configured to fit over or within the first collar.
Preferably, the sample holder and/or the holder mount comprise mounting means configured to removably mount the sample holder directly to the holder mount. Preferably, the sample holder is mounted directly to a proximal end of the holder mount.
Preferably, the mounting means allows the sample holder to move in a plane which is substantially perpendicular to the longitudinal axis. Advantageously, this allows the position of a sample supported on the sample holder to be adjusted relative to the longitudinal axis.
-ΊThe mounting means may comprise magnetic mounting means. Accordingly, the sample holder may comprise a magnet and the holder mount may comprise a magnetic material. Alternatively, the holder mount may comprise a magnet and the sample holder may comprise a magnetic material. The magnetic material may comprise a ferrous alloy or magnetic stainless steel. Preferably, the steel is ferritic stainless steel.
Alternatively, both the sample holder and the holder mount may comprise a magnet. The or each magnet may be an electromagnet. Advantageously, the electromagnet can be deactivated or switched to allow the sample holder to be removed from the objective lens. It may be understood that the proximal end of the holder mount may comprise 10 the magnet or the magnetic material. Alternatively, or additionally, in embodiments where they are present, the second collar or second element may comprise the magnet or the magnetic material.
According to a second aspect of the invention, there is provided a microscope comprising:
an objective lens comprising:
a lens barrel comprising a proximal end and a distal end and defining a longitudinal axis therebetween; and a front lens mounted on the longitudinal axis within the lens barrel; and 20 a sample holder configured to support a sample thereon;
characterised in that the sample holder and/or the objective lens comprises mounting means configured to removably mount the sample holder directly to the objective lens on the longitudinal axis such that a sample supported on the sample holder may be viewed or imaged through the front lens.
Advantageously, the apparatus of the second aspect also has a small mechanical loop allowing stable imaging at high resolution and magnification.
The longitudinal axis may be understood to be the optical axis of the microscope.
Accordingly, it can be viewed as an axis along which light travels through the objective lens and/or perpendicular to the focal plane.
It maybe understood that the proximal end of the lens barrel is configured to be disposed substantially adjacent to a sample. Similarly, it may be understood that the 35 distal end of the lens barrel is configured to be disposed spaced apart from a sample.
-8The microscope may be an optical microscope.
Preferably, the objective lens does not comprise an outer cosmetic jacket.
Preferably, the sample holder and/or the lens barrel comprise mounting means configured to removably mount the sample holder directly to the lens barrel.
Preferably, the sample holder is mounted directly to the proximal end of the lens barrel.
Preferably, the sample holder, lens barrel and front lens define a cavity when the 10 sample holder is mounted to the objective lens.
The mounting means may comprise mechanical mounting means. Accordingly, the sample holder may comprise a catch and the objective lens may comprise a corresponding staple configured to receive the latch. Alternatively, the objective lens 15 may comprise a catch and the sample holder may comprise a corresponding staple configured to receive the latch.
However, in a preferred embodiment, the mounting means comprise magnetic mounting means. Accordingly, the sample holder may comprise a magnet and the 20 objective lens may comprise a magnetic material. Alternatively, the objective lens may comprise a magnet and the sample holder may comprise a magnetic material. The magnetic material may comprise a ferrous alloy or magnetic stainless steel. Preferably, the steel is ferritic stainless steel. Alternatively, both the sample holder and the objective lens may comprise a magnet. The or each magnet maybe an electromagnet. 25 Advantageously, the electromagnet can be deactivated to allow the sample holder to be removed from the objective lens.
Preferably, the mounting means prevent the sample holder from moving along the longitudinal axis when it is mounted to the objective lens. Preferably, the mounting 30 means allows the sample holder to move in a plane which is substantially perpendicular to the longitudinal axis when it is mounted to the objective lens. Preferably, the mounting means only allows movement of the sample holder to move in a plane which is substantially perpendicular to the longitudinal axis when a predetermined force is applied. Advantageously, this allows the position of a sample mounted on the sample 35 holder to be adjusted and moved into the longitudinal axis while preventing drift.
-9Advantageously, the mounting means reduces drift in a plane substantially perpendicular to the longitudinal axis when the sample holder is mounted to the objective lens.
The objective lens may be configured for dry use. Alternatively, the objective lens may be configured for use with an immersion liquid. The liquid may comprise water or oil.
Alternatively, or additionally, the objective lens maybe configured for use with with a solid immersion lens (SIL). Accordingly, the microscope may comprise an SIL. The 10 sample holder may be configured to support the SIL thereon. Accordingly, the SIL can be mounted directly on the sample in order to increase the effective numerical aperture (NA).
The microscope may further comprise a sample manipulator configured to move the 15 sample holder in a plane which is substantially perpendicular to the longitudinal axis.
Preferably, the sample manipulator is configured to reversibly contact the sample holder and apply a force thereto to thereby cause movement of the sample holder in a plane which is substantially perpendicular to the longitudinal axis. Preferably, the 20 sample manipulator is configured to not contact the sample holder when the sample holder it is in a desired location. Advantageously, this prevents vibrations from the sample manipulator from being transferred to the sample holder while the sample is being viewed or imaged.
Alternatively, the sample manipulator may be configured to removably couple to the sample holder. Advantageously, this allows the sample manipulator to be coupled to the sample holder when it is necessary to move the sample but allows the sample manipulator to be decoupled when the sample is in a desired location to prevent vibrations from the sample manipulator from being transferred to the sample holder 30 while the sample is being imaged
Accordingly, the sample manipulator and/or the sample holder may comprise coupling means configured to allow the sample manipulator to removably couple to the sample holder. The coupling means may comprise magnetic coupling means. Accordingly, the 35 sample manipulator may comprise a magnet and the sample holders may comprise a magnetic material. Alternatively, the sample holder may comprise a magnet and the
- 10 sample manipulator may comprise a magnetic material. The magnetic material may comprise a ferrous alloy or magnetic stainless steel. Preferably, the steel is ferritic stainless steel. Alternatively, both the objective lens and the holder mount may comprise a magnet. The or each magnet may be an electromagnet. Advantageously, the electromagnet can be deactivated or switched to allow the holder mount to be removed from the objective lens. In one embodiment, the sample holder comprises a magnetic material and the sample manipulator comprises an electromagnet.
The sample manipulator may be configured to be moved manually. Alternatively, the microscope may comprise a motorised system configured to move the sample manipulator. The motorised system may comprise a stepper motor or a piezo motor. The microscope may further comprise a sensor configured to sense the position of the sample holder. The sensor may comprise a readhead configured to sense the position of a target disposed on a proximal side of the sample holder. The readhead may be an optical readhead and the target may be an optical target. The sensor may comprise a capacitive sensor or a magnetic encoder.
Preferably, the front lens is disposed concentrically within the lens barrel.
Preferably, the front lens is disposed towards the proximal end of the lens barrel. Accordingly, it maybe understood that the front lens is disposed closer to the proximal end than the distal end of the lens barrel.
The microscope may comprise an optical sub-system comprising optical microscope components aside from the objective lens.
The optical microscope components may comprise an excitation filter, a dichroic filter, an emission filter, a light detector, a beam scanner, a pinhole, a grating, a spatial light modulator (SLM), an adaptive optic (AO) component and/or a tuneable lens.
Advantageously, a tuneable lens can be used to adjust focus. Furthermore, a spatial light modulator (SLM) and/or an adaptive optics system (AO) can be used to change focus and potentially to correct for optical aberrations.
The microscope may be configured for use in cryogenic sample conditions. The skilled 35 person may understand cryogenic sample conditions to relate to the imagining of vitrified and/or frozen samples at temperatures of -14O°C or colder. Advantageously,
- 11 many of the fluorescence super-resolution methods can benefit from the very low photo-bleaching and high signal to noise naturally found under cryogenic sample conditions.
Accordingly, the microscope may comprise a first cryogenic cup configured to hold a cryogenic coolant. The cryogenic coolant may be liquid nitrogen or liquid helium. The first cryogenic cup may comprise a thermally insulating material, such as foam, or comprise an evacuated vessel, preferably comprising glass or stainless steel. Alternatively, the first ciyogenic cup may comprise a thermally conductive material, such as a metal. More particularly the thermally conductive material may comprise aluminium, silver or copper. Furthermore, the microscope may comprise a second cryogenic cup configured to hold a cryogenic coolant, and the first cryogenic cup may be configured to be disposed in the second cryogenic cup. The cryogenic coolant in the second cryogenic cup is preferably the same as the cryogenic coolant in the first ciyogenic cup. Preferably, the second cryogenic cup comprises a thermally insulating material, such as foam, or comprises an evacuated vessel, preferably comprising glass or stainless steel. Advantageously, this arrangement prevents or reduces bubbles from forming in the first cryogenic bath.
In one embodiment, the front lens may comprise a cryo-immersion lens. Accordingly, the sample holder and the front lens may be disposed in the first cryogenic bath. Advantageously, in use, the sample holder and the front lens will be disposed in the ciyogenic liquid.
Alternatively, the sample holder may comprise a thermally conductive material such as a metal. More particularly the thermally conductive material may comprise aluminium, silver or copper, and the microscope may comprise a thermal bridge extending between the first cryogenic bath and the sample holder. The thermal bridge may comprise an elongate member or a braid. The thermal bridge may comprise a thermally conductive material, such as copper or aluminium.
The microscope may comprise an objective lens mount on which the objective lens is configured to be removably coupled. In some embodiments, the objective lens is configured to be removably coupled directly to the objective lens mount. Preferably, the objective lens comprises mounting means disposed at a distal end of the lens barrel configured to removably mount the objective lens on the objective lens mount. The
- 12 mounting means may comprise a projection with a circular cross-section defining a screw thread on an outer surface thereof, and the objective lens mount may comprise a corresponding recess with a circular cross-section defining a screw thread and configured to receive the projection.
In some embodiments, the microscope may comprise:
a thermally insulating lens adapter on which the objective lens is configured to be removably coupled; and an objective lens mount on which the insulating adapter is configured to be removably coupled, wherein the insulating lens adapter is configured to thermally insulate the objective lens from the objective lens mount.
The thermally insulating objective lens adapter maybe configured to be removably coupled to the objective lens mount. The objective lens may be configured to be removably coupled to the insulating lens adapter. Preferably, the objective lens comprises a first mounting means disposed at a distal end of the lens barrel configured to removably couple to the objective lens on the insulating lens adapter. The first mounting means may comprise a first projection with a circular cross-section defining a 20 screw thread on an outer surface thereof, and the insulating lens adapter may comprise a corresponding recess on a proximal side thereof with a circular cross-section defining a screw thread and configured to receive the first projection. Preferably, the insulating lens adapter comprises a second mounting means disposed at a distal side thereof configured to removably mount the insulating lens adapter on the objective lens mount.
The second mounting means may comprise a second projection with a circular crosssection defining a screw thread on an outer surface thereof, and the objective lens mount may comprise a corresponding recess with a circular cross-section defining a screw thread and configured to receive the second projection.
The insulating lens adapter preferably comprises a plastic or ceramic material of low thermal conductivity.
The objective lens may further comprise a lens heater assembly configured to control the temperature of the objective lens. Preferably, the lens heater assembly is configured to be coupled to the lens barrel. More preferably, the lens heater assembly is configured to be coupled directly on the lens barrel. The lens heater assembly may
-13comprise a sleeve configured to be coupled over an outer surface of the lens barrel. Preferably, the lens heater assembly comprises a temperature sensor configured to sense the temperature of the lens barrel. Preferably, the lens heater assembly comprises a heating element configured to heat the lens barrel when the temperature 5 sensor senses a temperature which falls below a predetermined minimum temperature.
Preferably, the heating element is configured to stop heating the lens barrel when the temperature sensor senses a temperature which falls above a predetermined maximum temperature. Preferably, the temperature is between 10 and 4O°C, more preferably between 14 and 35°C and most preferably between 18 and 3O°C. Preferably, the lens heater assembly comprises a material for the thermal coupling to the lens barrel, preferably the material is kapton film.
The microscope of the second aspect may comprise an enclosure disposed around the sample holder and the objective lens. Similarly, the microscope of the first aspect may 15 comprise an enclosure disposed around the sample holder, the holder mount and the objective lens. The enclosure maybe configured to hermetically seal the sample holder and the objective lens therein. In embodiments where it is present, the holder mount may also be hermitically sealed in the enclosure. Alternatively, the enclosure may comprise a vent configured to allow air to flow out of the enclosure. Preferably, the 20 vent comprises a filter. The enclosure may also comprise an inlet to allow air to flow into the enclosure. Preferably, the inlet comprises a filter. Preferably, the or each filter is configured to remove moisture, particulate matter and/or gaseous matter from the air. Advantageously, the enclosure provides a stable imaging environment.
It may be appreciated that the microscope of the first aspect could be obtained by components being retrofitted onto an existing microscope. Similarly the microscope of the second aspect could be obtained by components being retrofitted onto an existing microscope.
Accordingly, in a third aspect, there is provided a kit of parts for retrofitting a microscope, the kit of parts comprising:
a sample holder configured to mount a sample; and a holder mount configured to be coupled to an objective lens of the microscope and configured to removably mount the sample holder thereto such that the sample 35 holder is disposed on a longitudinal axis of the objective lens and a sample supported thereon maybe viewed or imaged through a front lens of the objective lens.
-14The sample holder and holder mount may be as defined in relation to the first and second aspects.
The kit of parts may further comprise an objective lens, a sample manipulator, a sensor configured to sense the position of the sample holder, a first cryogenic bath, a second cryogenic bath, a thermally conductive bridge, an insulating lens adapter and/or a lens heater assembly. These components may also be as defined in relation to the first and second aspects.
In accordance with a fourth aspect, there is provided a kit of parts for retrofitting a microscope, the kit of parts comprising:
an objective lens comprising:
a lens barrel comprising a proximal end and a distal end and defining a longitudinal axis therebetween; and a front lens mounted on the longitudinal axis within the lens barrel; and a sample holder configured to support a sample thereon;
characterised in that the sample holder and/or the objective lens comprises mounting means configured to removably mount the sample holder directly to the objective lens 20 on the longitudinal axis such that a sample supported on the sample holder may be viewed or imaged through the front lens.
The objective lens and sample holder maybe as defined in relation to the second aspect.
The kit of parts may further comprise a sample manipulator, a sensor configured to sense the position of the sample holder, a first cryogenic bath, a second cryogenic bath, a thermally conductive bridge, an insulating lens adapter and/or a lens heater assembly. These components may also be as defined in relation to the first and second aspects.
In accordance with a fifth aspect, there is provided a method of viewing or imaging a sample, the method comprising:
supporting a sample on a sample holder;
removably mounting the sample holder on a holder mount, wherein the holder mount is coupled, or configured to be coupled, to an objective lens of a microscope, wherein the objective lens comprises a lens barrel comprising a
-ι5proximal end and a distal end and defining a longitudinal axis therebetween and a front lens mounted on the longitudinal axis within the lens barrel and the sample holder is mounted on the longitudinal axis; and viewing or imaging the sample through the front lens.
Preferably, the fifth aspect is carried out using the apparatus of the first aspect.
The method of the fifth aspect may comprise removably coupling the holder mount onto the objective lens. The holder mount maybe removably coupled on the objective 10 lens prior to the sample holder being mounted on the holder mount. Alternatively, the holder mount may be removably coupled on the objective lens after the sample holder has been mounted on the holder mount.
In accordance with a sixth aspect, there is provided a method of viewing or imaging a sample, the method comprising:
supporting a sample on a sample holder;
removably mounting the sample holder directly on an objective lens of a microscope, wherein the objective lens comprises a lens barrel comprising a proximal end and a distal end and defining a longitudinal axis therebetween and 20 a front lens disposed on the longitudinal axis within the lens barrel and the sample holder is mounted on the longitudinal axis; and viewing or imaging the sample through the front lens.
Preferably, the sixth aspect is carried out using the apparatus of the second aspect.
After the sample holder has been mounted, the methods of the fifth and sixth aspects may comprise moving the sample holder in a plane substantially perpendicular to the longitudinal axis to thereby position the sample in the longitudinal axis. The method may comprise sensing the position of the sample holder.
The methods of the fifth and sixth aspects may comprise focusing the microscope on the sample. The microscope can be focused using optical means. Alternatively, the method of the fifth aspect may comprise moving the sample holder along the longitudinal axis to thereby focus the microscope.
-16The methods of the fifth and sixth aspects may be carried out under cryogenic sample conditions. Accordingly, the methods may comprise cooling the sample holder to a temperature at least below -14O°C, and to maintain the temperature at these low levels. In the context of cryo-microscopy of vitrified frozen samples, which can be considered a 5 typical application, it is required to maintain a sample temperature below the devitrification threshold of (water) ice as disclosed in JOURNAL DE PHYSIQUE, Colloque C7, supptement an noq, Tome 45, septembre 1984, page C7-85, CRYOELECTRON MICROSCOPY OF VITRIFIED WATER, J. Dubochet and J. Lepault.
The methods may comprise positioning the sample holder and the front lens in a cryogenic coolant.
Alternatively, the method may comprise providing a cryogenic coolant in a first cryogenic cup and providing a thermal bridge between the cryogenic coolant and the 15 sample holder. Preferably, the thermal bridge is configured to thermally conduct heat from the sample holder to the cryogenic coolant. Optionally, the method further comprises disposing the first cryogenic cup in a second cryogenic cup comprising a cryogenic coolant.
All features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
Brief Description of the Figures
For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:Figure 1 is a cross-sectional side view of a microscope apparatus;
Figure 2 is a top plan view of a sample manipulator;
Figure 3 is a top plan view of an alternative sample manipulator;
Figure 4 is a cross-sectional side view of an objective lens of a microscope and a sample holder;
Figure 5 is a cross-sectional side view of an objective lens of a microscope and a sample holder;
Figure 6 is a cross-sectional side view of a further microscope apparatus;
-17Figure 7 is a cross-sectional side view of a cryogenic imaging microscope;
Figure 8 is a cross-sectional side view of a further cryogenic imaging microscope;
Figure 9 is a cross-sectional side view of an inverted system for cryogenic imaging microscope; and
Figure 10 is a cross-sectional side view of a lens.
Figure 11 is a top plane view of a clamping mechanism.
Figure 12 is a cross-sectional side view of a further cryogenic microscope where a solid immersion lens is used;
Figure 13 shows an image of the microscope objective lens, where an external thread 10 has been cut in the proximal portion of the objective lens.
Figure 14 shows an image of a holder mount of the invention.
Figure 15 shows an image of a sample holder of the invention.
Figure 16 shows an image of the holder mount with the sample mount magnetically coupled in place from the top plane.
Figure 17 shows an image of the sample holder and holder mount attached to the tip of the objective lens.
Figure 18 depicts a single image showing the fluorescent bead recorded in 16 bit monochromatic mode.
Figure 19 shows the XY plot of the movement of a fluorescent bead that was traced 20 over 10 minutes, using the microscope of the present invention.
Figure 20 shows the X component of the trace of figure 18.
Figure 21 shows the Y component of the trace of figure 18
Detailed Description
A microscope (2) generally comprises a sample holder (15) for holding a sample (13) to be studied.
The microscope (2) further comprises an objective lens (9) configured to magnify a sample (13) placed on the sample holder (15). The present invention is concerned with 30 reducing the size of the mechanical loop between the sample holder (15) and the objective lens (9) to minimise susceptibility to vibration and thermal drift problems.
The mechanical loop (7) can be defined as the shortest mechanical path from the sample location observed to the tip of the objective lens.
-18Example 1
Figure i shows an example of a microscope (2) according to the invention, where ultrastable imaging is achieved.
The microscope (2) comprises an objective lens (9) comprising a lens barrel (53) defining a proximal end (64) and a distal end (66). An optical axis (5) runs longitudinally between the proximal end (64) and distal end (66) of the lens barrel. A front lens (61) is mounted within the lens barrel (53), on the optical axis (5), substantially adjacent the proximal end. The objective lens (9) of the microscope (2) shown in figure 1 does not comprise an outer cosmetic jacket (55) disposed over the lens barrel (53). The lens (9) comprises a projection (6) disposed on the distal end (66) of the lens barrel (53). The projection (6) has a circular cross-section defining a screw thread (not shown) on an outer surface thereof. The projection (6) is sized to screw into a corresponding recess (60) in an objective lens mount (59). Thereby mounting the objective lens (9) to the microscope (2).
A sample (13) is mounted on a sample holder (15), and a holder mount (17) mechanically connects the sample holder (15) to the lens barrel (53) of the objective lens, such that the sample (13) is mounted in the immediate proximity of the front-lens 20 (61), such that the distance between the from lens and sample is between 0.1mm to
5mm.. The mechanical loop (7) created by the sample (13), sample holder (15), holder mount (17) and objective lens (9) is indicated in figure 1 for illustration.
The microscope (2) comprises a light source (not shown) configured to illuminate the 25 sample (13). A cone of light (10) between the objective lens (9) and the sample (13) is shown indicating the light path for illumination and/or imaging. In embodiments where the sample (13) is illuminated with fluorescent light, the light source may be an LED or mercury lamp coupled to the microscope (2). The sample (13) will then emit fluorescence into all directions, e.g. the hemisphere above the sample. The objective 30 lens can then capture the emitted fluorescence light only up to a certain maximum angle defined by the numerical aperture (NA) and illustrated by the cone of light (10).
The sample holder (15) attaches to the holder mount (17) by means of magnetic force.
The sample holder (15) can be detached from the holder mount (17) to allow the sample 35 (13) to be disposed thereon. This can be achieved by using a magnetic material for the
-19sample holder (15) and/or the holder mount (17), or by embedding magnets into one or both of elements (15) and (17).
The sample holder (15), together with the holder mount (17), forms a magnetic stick5 slip plate assembly, where the sample holder (15) can be positioned in an XY plane perpendicular to the optical axis (5) for the navigation of the sample.
The sample holder (15) is moved by means of the sample manipulator (33) and interacts with it, such that the sample manipulator is in contact with the sample holder 10 (15). To further increase stability, the sample manipulator (33) is not coupled to the sample holder (15), and can be retracted therefrom by moving the sample manipulator (33) away from the sample holder (15). While retracted, the sample manipulator (33) does not touch the sample holder (15), which prevents the potential transmission of vibrations from the sample positioning system into the sample holder (15) and further 15 isolates the sample from vibrations.
To further describe and illustrate the magnetic stick-slip mechanism for the sample positioning within the XY plane perpendicular to the optical axis (5) we turn to Figure
2. Depicted is the sample holder (15) with the sample (13) and the sample manipulator 20 (33). The sample manipulator (33) comprises a contacting element (82) configured to touch the sample holder (15) and can move it along in the X direction. A user, or motorised system, can manipulate the sample manipulator (33) using the manipulator arm (83). The equivalent principle is used in the Y direction, although not shown in the figure. When the XY motion is completed and before imaging with high stability is 25 started, the sample manipulator (33) moves in order to produce a gap between sample holder (15) and sample manipulator (33) - so as to retract the sample holder from the sample manipulator (33). The sample manipulator (33) is driven in the XY plane either by a manual system, for example based on micrometres, or is motorised using a stepper motor or a piezo motor.
An alternative embodiment of the sample manipulator (33) is shown in Figure 3. It will be appreciated that this sample manipulator (33) could also be used with microscope shown in Figure 1. As shown in the Figure, the sample holder (15) comprises a connecting portion (16) which extends from the base of the sample holder and is configured to connect to the sample manipulator (33). The sample holder’s connecting portion (16) attaches magnetically to the sample manipulator (33) via a magnetic
- 20 coupling (34), such that either the sample holder’s connecting portion (16) comprises a magnet and the sample manipulator (33) comprises magnetic material, or the sample holder’s connecting portion (16) comprises magnetic material and the sample manipulator (33) comprises a magnet. When connected, the sample manipulator (33) 5 controls the position of the sample mount (15) in the XY plane, as explained above. To prevent the transmission of vibrations and improve stability the sample manipulator is equipped with an electromagnetic coil (36) to perform decoupling. When a current is fed into the electromagnetic coil (36), the electromagneitic coil overrides the magnetic force such that the magnetic coupling (34) can be opened and the sample holder (15) 10 detaches from the sample manipulator (33). Alternatively, the electromagnetic coil (36) provides a magnetic force such that the sample holder (15) and sample manipulator are coupled to one another when a current is fed to the electromagnetic coil (36), and decoupled when no current is fed to the electromagnetic coil (36).
For accurate positioning and tiling and/or stitching of larger sample areas, an optical readhead (41) is provided which determines the position of an optical target (39) disposed on a proximal side (14) of the sample holder (15). Together, the pair of components (41) and (39) form an optical encoder system, which can be an incremental or an absolute encoder. Such encoder system could be an integrated two-axis XY non20 contact encoder, or independent encoder systems for both X and Y directions. Further, the optical readhead (41), together with the optical target (39) is also configurable to measure the angle of rotation of the sample holder (15) in the XY plane, if required. The information from the optical readhead (41) and optical target (39) can be used to move the sample (13) into a desired position.
Apart from navigating the sample in a plane substantially perpendicular to the optical axis, the microscope (2) typically also requires means for focussing the sample. The attachment of the holder mount (17) to the objective lens (9) is preferably implemented by means of clamping, where changing the clamping position of the holder mount (17) 30 on the objective lens (9) in a direction along the optical axis (5) is equivalent to changing or pre-adjusting the focus position. The holder mount (17) can be designed to act as a clamp around the objective lens barrel (53), as shown in Figure 11. The holder mount (17) can grip or release the lens barrel (53) due to electromagnetic means (78) or a piezo device (not shown) contracting the holder mount (17) and causing it to grip the 35 lens barrel (53). Releasing the holder mount (17), repositioning it and then reactiving the electromagnetic means (78) allows the position of the holder mount (17) to be
- 21 adjusted along the optional axis of the objective lens barrel (53) such as to enable the focusing of a sample. Effectively, in this embodiment, the holder mount (17) comprises a remotely actuated clamping mechanism.
Alternatively, in the embodiment shown in figure 1, the holder mount (17) is a single element, mechanically linking the sample holder (15) to the tip of the objective lens (9). The holder mount (17) may be attached to the lens barrel (53) by glue, by the use of grub-screws or by a fixed clamping mechanism. Accordingly, the objective lens (9) may be focused by optical means, which does not require physical movement along the optical axis (5).
Focussing can be accomplished by moving an optical detector disposed in the optical sub-system (19), such as a CCD or CMOS camera, along the optical axis. However, this will typically introduce spherical aberrations and can only be used for very small changes in focus, as described in C. J. R. Sheppard and C. J. Cogswell (“Effects of aberrating layers and tube length on confocal imaging properties”, Optik 87, No. 1 (1991) P- 34-38).
The microscope (2) comprises an optical sub-system (19) which comprises optical microscope components aside from the objective lens (9). Typical optical components that can be present inside the optical sub-system (19) include excitation filters, dichroic filters, emission filters, light detectors, a tube lens, beam scanners, pinholes, gratings, spatial light modulators (SLM) and adaptive optics (AO) components as well as tuneable lenses.
It will be appreciated that in embodiments where the optical sub-system (19) comprises a tuneable lens this can be used to adjust focus. The tuneable lens may be a fluid filled elastic lens, whereby the lens is manipulated by an electromagnet to alter focus by compressing the lens.
In a more complex setup for optical focussing, the optical sub-system (19) comprises a spatial light modulator (SLM) or an adaptive optics system (AO) in order to change focus and potentially to correct for optical aberrations as well. Suitable spatial light modulators and adaptive optics systems would be known to those skilled in the art, as described in https://wvvw.thoriabs.com/nevvgrouppage9.cfm7objectgiOup id-sosb and rmps://hGk)eje.com/sp;0;aMLhLmodm;dGr</.
- 22 Another known solution for aberration-free focussing is described in E.J. Botcherby et al. (“An optical technique for remote focusing in microscopy”, Optics Communications 281, (2008) 880-887, doi:io.ioi6/j.optcom.2oo7.io.oo7) and would be known to the skilled person. Here focussing is achieved by means of moving a separate reference mirror disposed in the optical sub-system (19). Interestingly, the optical focussing effect is achieved without introducing additional optical aberration. However, this design requires a second copy of the objective lens inside the optical sub-system (19).
It will be appreciated that the microscope (2) shown in Figure 1 is a dry lens configuration. However, immersion fluid can be provided in the gap between sample (13) and objective lens (9) to obtain an immersion lens configuration. Still another option is to use a Solid Immersion Lens (SIL). Here a lens is mounted together with the sample (13) on the sample holder (15). The SIL and the objective lens (9) are then both part of the optical system to image the sample.
Example 2
An objective lens (9) of a further microscope (2) is depicted in Figure 4. In this embodiment, the objective lens (9) comprises a lens barrel (53) defining a proximal end (64). A front lens (61) is mounted in the lens barrel (53), spaced apart from the proximal end (64) to define a cavity (62), such that the distance between the front lens (61) and sample (13) is between 0.1mm to 5mm.
Accordingly, by positioning the sample (13) together with the sample holder (15) it is possible to mount the sample holder (15) directly to the proximal end (64) of the lens barrel (53) to achieve a very small mechanical loop (7). The sample holder may be mounted to the lens by magnetic means. The imaging stability of this setup is very high because of the miniaturised and direct mechanical link between the lens barrel (53) of the objective lens (9) and the sample holder (15).
The sample holder (15) can be moved to a desired position using a sample manipulator (33) as discussed in example 1.
Sealing the cavity (62) can further improve the stability of the imaging because it reduces the flow of fluids around the sample (13), or the exchange of air with the
-23environment, which can have a detrimental effect on the imaging. The cavity maybe sealed by mounting of the sample holder to the lens.
The microscope (2) would comprise an optical sub-system (19), and can be focused using optical focusing techniques, as described in example 1.
Example T
An objective lens (9) of a further microscope (2) is depicted in Figure 5. In this embodiment, the objective lens (9) comprises a lens barrel (53) containing a front lens 10 (61). The lens barrel (53) defines a first screw thread (29a) on an outer surface thereof, substantially adjacent to a proximal end (64) of the lens barrel (53).
A sample (13) is mounted on a sample holder (15), and a holder mount (17) mechanically connects the sample holder (15) to the lens barrel (53) of the objective 15 lens (9), such that the sample (13) is mounted in the immediate proximity of the front lens (61).
As shown in the figure, the holder mount (17) comprises a focus ring (25), a threaded mount (23) and a fixation pin (27).
The focus ring (25) comprises a ring shaped cross-section which extends between a distal end (28) and a proximal end (30). The focus ring (25) defines first screw threads (29b) on an internal surface thereof substantially adjacent to the distal end (28), and second screw thread (31b) on an internal surface thereof substantially adjacent to the 25 proximal end (30),
Meanwhile, the threaded mount (23) also defines a ring shaped cross-section, and defining a second screw thread (31a) on an outer surface thereof.
The focus ring (25) is shaped so that it can be mounted to the lens barrel (53) of the objective lens (9) and held in place due to the mating of the corresponding screw threads (29a, 29b). Similarly, the threaded mount (23) is shaped so that it can be mounted within the focus ring (25) and held in place due to the mating of the corresponding screw threads (31a, 31b). The sample holder (15) is mounted to the threaded mount (23) by means of magnetic force. Accordingly, this mechanically connects the sample holder (15) to the lens barrel (53), as shown in Figure 5. The
-24sample holder (15) can be positioned in a plane perpendicular to the optical axis using a sample manipulator (33) as discussed in example 1.
The fixation pin (27) extends between the lens barrel (53) and threaded mount (23), such that a distal end (24) of the fixation pin (27) is disposed in a recess (8) in the lens barrel (53) and a proximal end (26) of the fixation pin (27) is disposed in a recess (22) in the focus ring (23). Accordingly, the fixation pin (27) allows the threaded mount (23) to move relative to lens barrel (53) along the optical axis (5), but prevents the threaded mount (23) and hence the sample (13) from rotating in the XY plane.
The pitch parameters of first pair of threads (29a, 29b) and the second pair of screw threads (31a, 31b) are different. For example, the pitch of the first screw threads (29a, 29b) can be 0.4 mm per turn while the pitch of second screw threads (31a, 31b) is 0.5 mm per turn. Accordingly, turning the focus ring (25) allows a precise motion of the sample along the Z-axis for focussing, while the fixation pin (27) prevents rotation of the holder mount (23). In the example described with 0.4 mm per turn and 0.5 mm per turn, respectively, the differential focus action of the resulting assembly is 0.1 mm/turn. The threaded mount (25) is adjusted either manually or by means of a motor or piezo-drive.
The microscope (2) would comprise an optical sub-system (19), and can also be focused by changing the distance between sample (13) and objective lens (9) and/or by using optical focusing techniques implemented in the optical sub-system (19), as described in example 1.
Example 4
A further microscope (2) is depicted in Figure 6. Similar to example 1, the microscope (2) comprises an objective lens (9) comprising a lens barrel (53) mounting a front lens (61). A sample (13) is mounted on a sample holder (15), and a holder mount (17) mechanically connects the sample holder (15) to the lens barrel (53) of the objective lens, such that the sample (13) is mounted in the immediate proximity of the front-lens (61).
However, in the embodiment illustrated in Figure 6, the holder mount (17) is a piezo actuator, comprising a static component of the piezo actuator (35), which is mounted to the lens barrel (53) of the objective lens (9). The piezo actuator also comprises a
-25movable component (37), which is able to travel back and forth along an axis substantially aligned with the optical axis (5). The sample holder (15) is removably mounted to the movable component (37) of the piezo actuator by magnetic means. Accordingly, the holder mount (17) can move the sample holder (15) along the optical axis (5) to focus it.
It maybe appreciated that several different designs of piezo actuators can be selected, for example stacked piezo actuators with solid-state joints or the squiggle-type “walking” piezo where travel over longer distance can be achieved by exploiting a stick10 slip mechanism in the piezo drive. The required sensors for closed-loop position control, such as capacitive sensors or optical encoder systems are also embedded where necessary but are not further discussed here.
Example 5
A further microscope (2) is depicted in Figure 7. Similar to the embodiment described in example 3, the holder mount (17) makes use of a differential thread to focus the sample.
Unlike the apparatus described in example 3, the apparatus shown in figure 7 is configured for the use in cryo-conditions. Accordingly, the sample holder (15) is made from a material of high thermal conductivity, such as a metal, preferably copper, aluminium, silver or gold, and comprises a cryo - fin (43) which extends from a proximal side (14) of the sample holder (15) to a cryogen bath (45). A cryogenic coolant, such as liquid nitrogen or helium, is disposed in the cryogen bath (45).
It will be appreciated that in some embodiments, a thermally conductive braid, preferably made from copper or another material with very good thermal conductivity, could complement or replace the cryo-fin (43) and would extend between the sample holder (15) and the cryogen bath (45).
Furthermore, for the purpose of cryo-imaging, measures are taken to reduce the flow of heat from the lens barrel (53) to the sample (13). The measures can include the use of materials with low thermal conductivity along the path, such as PEEK ceramics, Delrin or PTFE and a reduced material cross section.
In some embodiments, the setup will just contain a single cryogen bath (45). However, in the embodiment illustrated in Figure 7, there is a dual cryogenic bath design
- 26 comprising the cryogen bath (45) and a secondary cryogenic bath (47) is disposed around the cryogen bath (45) and cryogenic coolant is also disposed therein. The cryogenic coolant in the cryogen bath (45) and in the secondary cryogenic bath (47) is the same. The cup (48) containing the cryogen bath (45), is made out of a metal with 5 high thermal conductivity, such as copper, aluminium, silver or gold. This bath is in direct thermal contact with both the cryogenic coolants disposed on its inside and its outside. This arrangement prevents or reduces bubbles from forming in the cryogenic coolant in the cryogen bath (45), and instead they will form on the external surface of the cup (48) thereby reducing vibrations transmitted to cryo fin (43) disposed in the 10 cryogen coolant in the cryogen bath (45).
The fact that this microscope (2) can be used under cryogenic sample conditions is an advantage because many of the fluorescence super-resolution methods can benefit from the very low photo-bleaching and high signal to noise naturally found under cryo15 conditions.
While not discussed, it will be appreciated that the microscopes (2) discussed in examples 1 to 4 could also be adapted for use with a single cryogen bath (45) or a dual cryogen bath comprising (45) and (47).
Example 6
A further microscope (2) is depicted in Figure 12. It is similar to the design discussed above, however, a solid immersion lens (12) is installed. The solid immersion lens (12) is mounted on or above the sample (13). In a preferred embodiment the sample (13) 25 and the solid immersion lens (12) are jointly mounted on the sample holder (15). The solid immersion lens can be a hemispherical design, a spherical or aspherical design or a diffractive type solid immersion lens.
In Figure 12 a microscope layout capable to operate under cryo-conditions is shown.
However, the solid immersion lens (12) can as also be used under conditions close to ambient temperature. The purpose of the solid immersion design is to allow a larger effective numerical aperture and improved resolution.
Example 7
In an alternative embodiment to that discussed in examples 5 and 6, the front lens (61) comprises a cryo-immersion lens. As shown in Figure 8, in this embodiment, there is
-27no need for a cryo fin (43) or thermally conductive braid. Instead the whole assembly including the proximal end (64) of the objective lens (9), the sample (13) and the sample holder (15) are all immersed in a cryogenic coolant in a cryogen bath (45). As explained in the previous example, the cryogen bath (45) can be disposed in a secondary cryogen bath (47) which also comprises the cryogenic coolant.
Example 8
Sometimes it is preferable to use an inverted microscope setup, where the objective lens (9) observes the microscope sample from underneath. For imaging at room temperature or above, this may be achieved by turning the whole arrangement discussed in examples 1 to 5 upside down.
However, for a microscope adapted for use under cryo conditions the cryo fin (43) and/or the thermally conductive braid (51) need to be adapted to work with the inverted microscope setup, as shown in Figure 9.
Example Q
As it is well known, temperature changes of a mechanical system can lead to thermal drift due to thermal expansion of the components. Apart from selecting suitable materials with a low coefficient of thermal expansion (CTE), critical components of an assembly can be temperature controlled in order to keep them at an accurately defined temperature and/or to avoid condensation.
Figure 10 shows a further embodiment of an objective lens (9) and objective lens mount 25 (59). In this embodiment, a thermally insulating lens adapter (63) comprises a projection (68) disposed on the distal side (69) thereof. The projection (68) has a circular cross-section defining a screw thread (not shown) on an outer surface and is sized to screw into the recess (60) in the objective lens mount (59). The thermally insulating lens adapter (63) also defines a recess (70) in a proximal end side (71) 30 thereof. The recess (70) has a circular cross-section defining a screw thread (not shown) on an internal surface and is sized to receive the projection (6) of the objective lens (9), thereby mounting the objective lens (9) to the microscope (2). The thermally insulating lens adapter (63) is preferably made from plastic or ceramics and reduces the heat transfer between the objective lens mount (59) and the objective lens (9).
-28The objective lens (9) also comprises a lens heater assembly (65) comprising a temperature sensor and a heating element, operating together in closed loop control mode to stabilise the temperature of the objective lens (9). For good thermal contact it is essential that the lens heater assembly (65) is attached directly to the inner lens barrel (53). The lens may be held at room temperature, or at any temperature at which the objective lens is designed for. A fixed set-point avoids temperature drift.
Example 10
To demonstrate the effectiveness of the arrangement and method according to the 10 invention, experimental position stability data was recorded with a setup of the invention.
Figure 13 shows a microscope objective lens (9) where an external thread (74) has been cut in the proximal end (64) of the tip. Figure 14 shows the holder mount (17), which 15 can be attached onto the proximal end (64) of the lens (9) by using the internal thread (72) of the holder mount (17). The holder mount (17) is equipped with embedded magnets (84). In this example the holder mount (17) is mounted to the lens barrel by means of a single thread. It is similar to the differential thread shown in figure 5, but the sample will rotate around the optical axis when it is focussed mechanically. Figure 20 15 shows the sample holder (15) with the sample (13) attached. In this example, case the sample holder (15) is an assembly comprising a central copper element (75) with the sample (13) at the centre as well as an attached magnetic stainless steel plate (76) to enable the XY adjustment of the sample. Figure 16 shows the holder mount (17) with the sample holder (15) magnetically clipped in place from the top. Figure 17 shows the 25 sample holder (15) and holder mount (17) attached to the proximal end (64) of the objective lens, where the objective lens (9) is mounted onto a microscope (Nikon LV 100ND).
In this example, the sample consisted of an electron microscopy grid where green fluorescent beads of 1 um diameter were mounted. The fluorescent beads were excited with a blue LED mounted to the illumination axis of the Nikon LV100 ND microscope, also containing the fluorescence filter cube and having a CMount camera attached (Point Grey Grasshopper GS3-U3-23S6C-C) with a pixel size of 5.86 um.
-29A fluorescent single fluorescent bead was brought to focus by turning the holder mount and a region of interest of 64 x 64 pixels was selected containing a single fluorescent bead.
Figure 18 depicts a single image showing the fluorescent bead recorded in 16 bit monochromatic mode. The pixel size was calibrated to be 95.9 nm and the field of view depicted in figure 15 is 6.14 um. Exposure time was set to 99 ms. In order to measure stability in the XY plane, a total of 600 frames were recorded, one frame per second.
The image sequence was then imported into ImageJ (https://imagej.nih.gov/ij/ NIH image processing package) and thresholding with an intensity level of 6000 was applied to separate foreground and background. The brightest pixels had a grey value of about 41000. Subsequently the centre of gravity of the intensity distribution was calculated using an ImageJ script. The experiment was performed at room temperature and during the 10 minutes of the experiment the fluorescent bead observed bleached to about 78% of the initial intensity. The centre of gravity results were expressed in nanometers using the previously obtained calibration factor of 95.9 nm per pixel. Figure 19 shows the XY plot where the movement of the bead is traced over 10 minutes, while Figure 20 shows the X component and Figure 21 shows the Y component, respectively. The standard deviation of the 600 position values in was 4.8 nm and 4.3 nm in the X and Y directions, respectively.
The standard deviation in the order of only 5 nanometers is a very good result, which would usually require a temperature controlled environment and/or vibration insulation measures. This application example has demonstrated that very good stability can be achieved with a device according to the invention. Furthermore, it has been shown how such design can be retro-fitted to an existing microscope platform.
Summary
The above examples provide microscopes with particularly small mechanical loops and optional features for imaging under cryo sample-conditions. Accordingly, the microscopes described above all allow samples to be imaged with exceptional stability at high resolution and magnification. Furthermore, the sample holder can be moved in the XY plane for all of the designs ensuring that the desired portion of the sample can be imaged easily.

Claims (25)

  1. Claims
    1. A microscope comprising:
    an objective lens comprising:
    a lens barrel comprising a proximal end and a distal end and defining a
    5 longitudinal axis therebetween; and a front lens mounted on the longitudinal axis within the lens barrel;
    a sample holder configured to support a sample thereon; and a holder mount coupled to or configured to be coupled to the objective lens and configured to removably mount the sample holder thereto such that the sample holder io is disposed on the longitudinal axis and a sample mounted thereon may be viewed or imaged through the front lens.
  2. 2. The microscope according to claim 1, wherein the holder mount is fixedly coupled to or configured to be fixedly coupled to the objective lens.
  3. 3. The microscope according to claim 1, wherein the holder mount is removably coupled to or configured to be removably coupled to the objective lens.
  4. 4. The microscope according to claim 3, wherein the holder mount is configured to 20 be removably coupled to the objective lens by clamping means.
  5. 5. The microscope according to either claim 1 or claim 3, where in the holder mount comprises a first collar which defines a first screw thread on an internal surface thereof, and the objective lens defines a corresponding first screw thread on an external
    25 surface thereof, such that the first collar is configured to be screwed to the objective lens.
  6. 6. The microscope according to claim 5, wherein the first collar defines a second screw thread, and the holder mount comprises a second collar defining a corresponding
    30 second screw thread, such that the second collar is configured to be screwed to the first collar, wherein the second collar is configured to removably mount the sample holder thereto.
  7. 7. The microscope according to claim 6, wherein the first screw thread has a
    35 different pitch to the second screw thread, optionally wherein the difference in pitch is between 0.01mm and 0.1mm.
  8. 8. The microscope according to either claim 6 or claim 7, wherein the microscope further comprises fixing means configured to prevent the second collar from rotating independently of the object lens, while allowing relative movement in the longitudinal
    5 direction.
  9. 9. The microscope according to any one of claims 1 to 3, wherein the holder mount comprises an actuator comprising:
    a first element coupled or configured to be coupled to the objective lens; and
  10. 10 a second element coupled to the first element and configured to removably mount the sample holder thereto, wherein the actuator is configured to move the second element, relative to the first element in a direction substantially parallel to the longitudinal axis.
    15 10. The microscope according to claim 9, wherein the actuator is a piezoelectric actuator.
  11. 11. A microscope comprising an objective lens comprising:
    20 a lens barrel comprising a proximal end and a distal end and defining a longitudinal axis therebetween; and a front lens mounted on the longitudinal axis within the lens barrel; and a sample holder configured to support a sample thereon;
    characterised in that the sample holder and/or the objective lens comprises mounting 25 means configured to removably mount the sample holder directly to the objective lens on the longitudinal axis such that a sample supported on the sample holder may be viewed or imaged through the front lens.
  12. 12. The microscope according to claim 11, wherein the sample holder and/or the
    30 lens barrel comprise mounting means configured to removably mount the sample holder directly to the lens barrel, optionally wherein the sample holder is mounted directly to the proximal end of the lens barrel.
  13. 13. The microscope according to claim 12, wherein the mounting means is a
    35 mechanical mounting means or a magnetic mounting means.
    -3214· The microscope according to any one of claims 11 to 13, wherein the lens barrel and front lens define a cavity when the sample holder is mounted to the objective lens.
  14. 15. The microscope according to any preceding claim, further comprising a sample 5 manipulator configured to move the sample holder in a plane which is substantially perpendicular to the longitudinal axis.
  15. 16. The microscope according to claim 15, wherein the sample manipulator is configured to reversibly contact the sample holder and apply a force thereto to thereby
    10 cause movement of the sample holder in a plane which is substantially perpendicular to the longitudinal axis, and is further configured to not contact the sample holder when the sample holder it is in a desired location.
  16. 17. The microscope according to either claim 15 or claim 16, wherein the sample 15 manipulator is configured to removably couple to the sample holder.
  17. 18. The microscope according to claim 17, wherein the sample holder comprises coupling means, optionally wherein the coupling means comprises magnetic coupling means.
  18. 19. The microscope according to any preceding claim, wherein the microscope is configured for use in cryogenic sample conditions, wherein the microscope comprises a first cryogenic bath configured to hold a cryogenic coolant, optionally wherein the microscope comprises a second cryogenic bath configured to hold a cryogenic coolant,
    25 and the first cryogenic bath is be configured to be disposed in the second ciyogenic bath.
  19. 20. The microscope according to any one of claims 1-19, further comprising a thermally insulating lens adapter on which the objective lens is configured to be
    30 removably coupled; and an objective lens mount on which the insulating adapter is configured to be removably coupled, wherein the insulating lens adapter is configured to thermally insulate the objective lens from the objective lens mount.
    A kit of parts for retrofitting a microscope, the kit of parts comprising: a sample holder configured to mount a sample; and
  20. 21.
    -33a holder mount configured to be coupled to an objective lens of the microscope and configured to removably mount the sample holder thereto such that the sample holder is disposed on a longitudinal axis of the objective lens and a sample supported thereon may be viewed through a front lens of the objective lens.
  21. 22. A kit of parts for retrofitting a microscope, the kit of parts comprising:
    an objective lens comprising:
    a lens barrel comprising a proximal end and a distal end and defining a longitudinal axis therebetween; and io a front lens mounted on the longitudinal axis within the lens barrel; and a sample holder configured to support a sample thereon;
    characterised in that the sample holder and/or the objective lens comprises mounting means configured to removably mount the sample holder directly to the objective lens on the longitudinal axis such that a sample supported on the sample holder may be 15 viewed through the front lens.
  22. 23. The kit according to claim 21 or 22, further comprising a sample manipulator, a sensor configured to sense the position of the sample holder, a first cryogenic bath, a second cryogenic bath, a thermally conductive bridge, an insulating lens adapter and/or
    20 a lens heater assembly.
  23. 24. A method of viewing or imaging a sample, the method comprising:
    supporting a sample on a sample holder;
    removably mounting the sample holder directly on an objective lens of a
  24. 25 microscope, wherein the objective lens comprises a lens barrel comprising a proximal end and a distal end and defining a longitudinal axis therebetween and a front lens disposed on the longitudinal axis within the lens barrel and the sample holder is mounted on the longitudinal axis; and viewing or imaging the sample through the front lens.
    25. A method of viewing or imaging a sample, the method comprising:
    supporting a sample on a sample holder;
    removably mounting the sample holder on a holder mount, wherein the holder mount is coupled, or configured to be coupled, to an objective lens of a
    35 microscope, wherein the objective lens comprises a lens barrel comprising a proximal end and a distal end and defining a longitudinal axis therebetween and
    -34a front lens mounted on the longitudinal axis within the lens barrel and the sample holder is mounted on the longitudinal axis; and viewing or imaging the sample through the front lens.
  25. 27. The method according to either claim 24 or claim 25, wherein the methods are conducted under cryogenic sample conditions. .
    Intellectual
    Property
    Office
    Application No: GB1811454.6
    Examiner: Sophie Cartmell
GB1811454.6A 2018-07-12 2018-07-12 Apparatus for high stability imaging Withdrawn GB2576870A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB1811454.6A GB2576870A (en) 2018-07-12 2018-07-12 Apparatus for high stability imaging

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
GB1811454.6A GB2576870A (en) 2018-07-12 2018-07-12 Apparatus for high stability imaging

Publications (2)

Publication Number Publication Date
GB201811454D0 GB201811454D0 (en) 2018-08-29
GB2576870A true GB2576870A (en) 2020-03-11

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Application Number Title Priority Date Filing Date
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Country Status (1)

Country Link
GB (1) GB2576870A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020015225A1 (en) * 2000-04-19 2002-02-07 Olympus Optical Co., Ltd. Focus stabilizing apparatus
JP2005003992A (en) * 2003-06-12 2005-01-06 Olympus Corp Specimen support device

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020015225A1 (en) * 2000-04-19 2002-02-07 Olympus Optical Co., Ltd. Focus stabilizing apparatus
JP2005003992A (en) * 2003-06-12 2005-01-06 Olympus Corp Specimen support device

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