EP-3980832-B1 - AN OPTICAL MICROSCOPE

Inventors

• BROZIK, James Alan
• THOMPSON, ANDREW JAMES

Dates

Publication Date

20260506

Application Date

20200603

Claims (12)

1. An optical microscope (10) having a resolution of less than 100nm, the optical microscope comprising: an image sensor configured to capture a plurality of images of a sample on a sample stage over time; a computer configured to store and process the plurality of images; a first optical microscope; and a second optical microscope with a different mode of operation to the first optical microscope; wherein the first optical microscope is a confocal microscope and the second optical microscope is a total internal reflection fluorescence microscope, wherein the optical microscope is configured such that the first optical microscope and the second optical microscope simultaneously view a sample, and wherein the optical microscope is configured to use the second optical microscope to correct drift from the first optical microscope and/or the sample in the X,Y plane based on the stored plurality of images using at least one reference element located relative to the sample and without movement of the sample stage.
2. An optical microscope (10) according to claim 1, wherein the first optical microscope uses a first light source (R) and the second optical microscope uses a second light source (Q); and the first light source is different to the second light source.
3. An optical microscope (10) according to claim 1 or claim 2, further comprising an objective lens (H), wherein the first light source (Q) and the second light source (R) pass through the objective lens.
4. An optical microscope (10) according to claim 3, wherein the objective lens (H) has a numerical aperture of at least 1.37.
5. An optical microscope (10) according to claim 3 or claim 4, wherein the optical microscope is configured such that a sample for imaging is located below the objective lens.
6. An optical microscope (10) according to any preceding claim, wherein the optical microscope is housed in a single housing.
7. An optical microscope according to claim 6, wherein the single housing is in one and only one piece.
8. An optical microscope (10) according to any preceding claim, wherein the computer processes the plurality of images to provide an output image.
9. An optical microscope (10) according to any preceding claim, wherein the at least one reference element has a diffraction limited intensity distribution of emitted light.
10. An optical microscope (10) according to any preceding claim, wherein the first optical microscope is used to correct drift from the first optical microscope and/or the sample in the Z direction or vertical direction.
11. An optical microscope (10) according to any preceding claim, further comprising a beam splitter (Z) and at least two detectors (C, A) configured to detect light from the sample split by the beam splitter from the first optical microscope.
12. An optical microscope (10) according to claim 11, wherein the detectors (C, A) are at a calibrated focal plane within an axial confocal volume of the first optical microscope.

Description

FIELD OF THE DISCLOSURE The present disclosure relates to an optical microscope, particularly for high resolution or super resolution microscopy, a super resolution optical microscope, a super resolution optical microscopy sample enclosure, and a sample surface. BACKGROUND OF THE DISCLOSURE Microscopes are instruments that allow things to be seen that are too small to be seen by a human eye. Optical microscopy is a microscopy technique that uses light to produce an image. Optical microscopes are available that operate in different modes. That is to say, while they all use light to capture magnified images of small objects, the underlying methodology is different. For example, the basic types of optical microscopes are simple microscopes that use a single lens for magnification, and compound microscopes that use a plurality of lenses. In a compound microscope, a so-called objective lens close to the object being viewed is used to collect light. This provides the viewer with an enlarged inverted virtual image of the object. This focuses a real image of the object being imaged inside the microscope. This image is then magnified by a second lens or group of lenses in an eye piece of the microscope. Other modes of optical microscopy include confocal microscopy and total internal fluorescence (TIRF) microscopy. In confocal microscopy, a small beam of light is focussed at one narrow depth of a sample at a time. This is in contrast to the basic types of microscope mentioned above in which light travels through the sample as far as it can penetrate. In confocal microscopy, a spatial pinhole is used to block out-of-focus light in image formation. Only light produced by fluorescence very close to the focal plane is detected. However, as much of the light from sample fluorescence is blocked at the pinhole, there is low signal intensity and so a sensitive detector such as a photomultiplier tube or avalanche photodiode are used. Together, this results in optical resolution much better than that of the basic types of microscope mentioned above. In TIRF, a sample is located on a glass slide in a fluid or liquid, such as water. An evanescent wave is used to selectively illuminate and excite fluorophores in the sample. An evanescent wave travels along the glass-fluid or glass-liquid interface with an amplitude that falls off exponentially with distance from the interface. Fluorophores are fluorescent chemical compounds that re-emit light once they are excited by light a restricted region of the sample immediately adjacent to a glass-fluid interface. This re-emitted light is collected by an objective lens of the microscope. The evanescent wave is generated only when the incident light is totally internally reflected at the glass-fluid interface. As the evanescent wave decays exponentially from the interface, it penetrates to only approximately 100 nm into the sample. As discussed, in TIRF a glass-fluid interface is required. The fluid is located in a light transparent glass bath with the sample located on the base of the open bath. The objective lens is located below the bath. TIRF can be used to observe the fluorescence of a single molecule. The use of optical microscopy to observe biological processes in particular has undergone huge advances in the last decade. There have been significant advances in resolution including high resolution, often called, super resolution microscopy with resolutions as low as 100nm or less. Many of the developments have focused on achieving higher resolution. However, the instruments are often complex, are often built in-house, and require considerable user knowledge to operate. In addition, movements within the resultant images can compromise precision, and become an increasing challenge as the magnification is raised. To emphasise super resolution optical microscopes with resolutions as low as 100nm or less are very sensitive to even the most minute movements in the environment in which they operate. High resolution microscopy with resolution as low as 15 to 20nm or even lower such as 1.5nm resolution are desirable, particularly for drug discovery, as it would allow for particularly effective single molecule imaging. Understanding how drugs bind to their receptors is the most fundamental purpose of pharmacological research that enables the effective development of novel therapeutics. Monitoring kinetic properties is critical, but often based on limited macroscopic measurements. Currently, deterministic measurements are made using methods that yield ensemble averages (e.g. radioligand binding, surface plasmon resonance (SPR), X-ray crystallography). Techniques such as single-channel patch clamp go some way towards monitoring the properties of single molecules, but are composite measurements of both ligand binding and a functional response, and can only be used for receptors that are functionally coupled to ion channels. Current pharmokinetic models for drug metabolism are also based entirely