Improvements in or relating to lighting devices

Abstract

A critical illumination device is provided for uniform illumination of an object in a reflected light microscope, the critical illumination device comprising: a coherent or partially coherent light source; an optical fiber coupled to the light source, the fiber having a core with a diameter of at least 15 microns and a numerical aperture of 0.1 to 0.5; and at least one collimator lens positioned at a focal distance from a tip of the optical fiber, the collimator lens configured to match the numerical aperture of the optical fiber to couple light out of the optical fiber.

Description

[Technical Field]

[0001] The present invention relates to illumination devices, and in particular to a critical illumination device for providing uniform illumination of an object in a reflected light microscope. A reflected light microscope and method for illuminating an object is also provided. [Background technology]

[0002] Illumination devices provide light in a variety of devices and may alternatively be described as light sources. In microscopes, it is essential to have adequate illumination for both viewing and imaging. A variety of light sources and means of illumination are known in the art.

[0003] Critical illumination is a commonly used method of sample illumination for transmitted and reflected light. Simply put, critical illumination focuses the image of the light source onto the sample for bright illumination. To date, a drawback of this type of illumination is that the field of view (FoV) is not uniformly illuminated.

[0004] Another limitation of critical illumination is the formation of an image of the light source in the sample image plane. Kohler illumination addresses this limitation by ensuring that the image of the light source is not visible in the sample plane. In addition, Kohler illumination can also provide uniform illumination of the sample across the field of view, thus reducing image artifacts and resulting in high sample contrast. To date, Kohler illumination is the preeminent technique for sample illumination in light microscopes.

[0005] Optical microscopy is a type of imaging technique that has been widely used to investigate biological and / or chemical structures and their dynamic behavior at microscopic and / or nanoscopic length scales. Optical microscopy essentially relies on an illumination device or light source. Optical microscopy techniques include, but are not limited to, dark-field illumination, wide-field, or fast beam scanning microscopy to illuminate the sample.

[0006] Molecular mass spectrometers are a type of optical microscope that utilizes wide-field scanning microscopy. They provide an efficient and effective method for measuring the mass of single molecules within a sample. This technique measures the mass of individual molecules by capturing the light scattered by each molecule at an interface. Extracting the scattering contrast provides an indirect measurement of mass. Molecular mass spectrometers are based on the principles of interference reflection microscopy and single-particle interferometric scattering microscopy. In molecular mass spectrometers, the illumination light can be spatially and temporally coherent, for example, using a laser as the light source. To date, critical illumination devices have not been suitable for use in mass spectrometers because the illumination is not uniformly equal.

[0007] The illumination device of the present invention is suitable for use in reflected light microscopes. There are many techniques and devices in the art that rely on illumination devices. Given that the technology can vary with respect to the illumination requirements of the illumination device, including illumination power and stability, devices suitable for medical / veterinary imaging will necessarily differ from those for microscopy.

[0008] Imaging techniques have been used to investigate biological and / or chemical structures and their dynamic behavior. Imaging techniques such as tomographic imaging, e.g., PET or CT scans, require optical scanners to provide excitation light from fiber-coupled lasers with small spot sizes. High-power lasers are often used for tomographic imaging because they allow sufficient power to be delivered deep within thick samples to excite fluorescent species located within the sample. However, tomographic imaging utilizes multiple excitation sources and multiple elements, such as galvanometer optical scanners, to perform multispectral imaging. This requires large amounts of power and is prone to damage. Scanning elements are expensive and require precise alignment. This can result in a time-consuming, expensive, and inefficient setup process for users. Furthermore, focused beams result in high peak power densities at the sample, which can damage biological objects within the sample. Such illumination devices are not suitable for use in reflected light microscopes.

[0009] Digital holography involves a collimated light source, such as from the collimated output of a single-mode fiber, to provide illumination for capturing a holographic image. Because digital holography allows for the a priori reconstruction of the electric field at a given plane, it does not require a microscope but relies on a computer to perform multiple reconstructions. The computer performs multiple reconstructions in which the optical wavefront information arising from the object is digitally recorded as a hologram, from which the computer then calculates the object image by using multiple reconstruction algorithms. This can often be a complex and time-consuming process for users. Furthermore, the use of single-mode fiber in holography often results in high spatial coherence, which leads to significant speckle when using lasers and therefore affects the overall image quality.

[0010] Dark-field illumination is a technique in optical microscopy that removes the illuminating light from light scattered by the specimen. This results in an image with a dark background surrounding the specimen, essentially the opposite of bright-field illumination. Because dark-field microscopy removes most of the bright light, this form of illumination often requires the use of expensive, highly sensitive image detectors to detect weakly scattering objects. In general, dark-field illumination is not well suited to critical illumination.

[0011] Wide-field microscopy illuminates a sample of interest with a uniform, collimated light beam, and the reflected light is captured and analyzed to determine the mass of any molecule within the sample. A uniform, collimated light beam that induces some level of coherence is used. Images of samples acquired from these surfaces by coherent imaging systems, such as lasers, suffer from a commonly observed interference phenomenon called speckle. These speckles can obscure or obscure the imaged sample and therefore adversely affect the final mass measurement in techniques such as molecular spectrophotometry. Wide-field illumination can also result in a loss of spatial resolution due to an extended point spread function (PSF), a result of the interference of scattered light with the illuminating light.

[0012] Alternatively, rapid beam scanning techniques use a focused beam and a scanning element, such as an acousto-optic deflector (AOD), to move the beam across the sample. This is scanned over a specific field of view (FoV) encompassing the sample. Because the final image is composed of the sum of different spots on the sample illuminated at different moments within the detector exposure time, the final image is speckle-free. However, scanning elements require a large amount of power and are prone to breakdown. Scanning elements are expensive and require precise alignment. Therefore, this can be time-consuming, expensive, and inefficient for the user. Furthermore, a focused beam results in a high peak power density at the sample, which can damage biological matter in the sample.

[0013] Important factors to consider when selecting a microscopy method are the desired illumination field (i.e., FoV) and illumination numerical aperture (NA) of the light used. A suitable illumination field allows the sample to be imaged in its entirety and ensures that the sample is fully illuminated. Critical illumination is a method of sample illumination used for transmitted and reflected light (trans- and epi-illumination) optical microscopy. Critical illumination focuses the image of the light source onto the sample for bright illumination. However, because the image of the illumination source, e.g., a halogen lamp filament, is often visible in the resulting image, critical illumination generally suffers from illumination uniformity issues. Therefore, ensuring a high degree of illumination uniformity is important to enable improvements in identifying and quantifying image features. Using light with a higher illumination numerical aperture is also important, as this can improve the spatial resolution of the final image.

[0014] Therefore, there is a need to overcome the various problems discussed above to provide devices and methods for enabling critical illumination with high illumination uniformity and for reducing speckle contrast.

[0015] It is against this background that the present invention arose. Summary of the Invention [Means for solving the problem]

[0016] According to an aspect of the present invention, there is provided a critical illumination device for providing uniform illumination of an object in a reflected light microscope, comprising: a coherent or partially coherent light source; an optical fiber coupled to a light source, the fiber having a core with a diameter of at least 15 microns and a numerical aperture of 0.1 to 0.5; at least one collimator lens positioned at a focal distance from the tip of the optical fiber, the collimator lens configured to match the numerical aperture of the optical fiber to couple light out of the optical fiber; A critical illumination device is provided, comprising:

[0017] According to another aspect of the present invention, there is provided a critical illumination device for providing uniform illumination of an object in a reflected light microscope, comprising: a coherent or partially coherent light source; an optical fiber coupled to a light source, the fiber having a core with a diameter of at least 15 microns and a numerical aperture of 0.1 to 0.5; at least one collimator lens positioned at a focal distance from the tip of the optical fiber, the collimator lens configured to match the numerical aperture of the optical fiber for coupling light into and / or out of the optical fiber; An illumination device is provided, comprising:

[0018] In some embodiments, a critical illumination device for providing uniform illumination of an object in a reflected light microscope is provided, the illumination device comprising: a coherent or partially coherent light source; an optical fiber coupled to the light source, the fiber having a core with a diameter of at least 15 microns and a numerical aperture of 0.1 to 0.5; and at least one collimator lens or focusing element, such as a curved mirror, positioned at its focal distance from the tip of the optical fiber, the collimator lens configured to match the numerical aperture of the optical fiber to couple light into the optical fiber.

[0019] A critical illumination device as disclosed herein can provide critical illumination with high illumination uniformity of an object. The critical illumination device comprises a coherent or partially coherent light source to provide bright illumination to form an image of the object.

[0020] The term "focal length," as disclosed herein, unless otherwise specified, refers to the axial distance between a lens or focusing element and the point in space where parallel light rays incident on the lens or focusing element converge to a focal point.

[0021] The entity may be, but is not limited to, a biomolecule such as a nucleic acid, a protein, a protein complex, a lipoprotein, a polypeptide, an antibody or antibody fragment thereof, an enzyme, a virus or viral vector such as an adenovirus and / or a lentivirus, a particle, a nanoparticle, a compound, a molecule, an ion, or a quantum dot.

[0022] The entity may be present in a sample. The entity may be suspected of being present in a sample. The sample may be any suitable sample, including a biological sample (e.g., blood, serum, blood fraction, saliva, tears, sweat, urine, semen, amniotic fluid, cerebrospinal fluid, bile, or interstitial fluid). The sample may be an environmental sample, such as water, or a waste product, such as sewage. The sample may be an industrial sample, such as from a manufacturing process. The manufacturing process may be any suitable process, including food manufacturing, biological or chemical manufacturing. The sample may be from a process for cell manufacturing or gene therapy, and may optionally be from a laboratory.

[0023] An example of quantitative imaging of an object using the devices and methods of the present invention may include, but is not limited to, resolving the population of viral vectors, such as adenovirus, adeno-associated virus (AAV), and / or lentivirus, by examining their genome content based on light scattering contrast, i.e., determining whether the virus is empty or full.

[0024] The optical fiber is a multimode fiber that can be coupled with a light source to enable critical illumination with high illumination uniformity as well as to enable speckle contrast reduction. Single-mode fiber does not provide high illumination uniformity in this case because the output has a non-uniform Gaussian distribution when used for critical illumination.

[0025] Therefore, it is an object of the present invention to provide a critical illumination device such that a large range of illumination angles for higher spatial resolution can be achieved with little loss of contrast. The use of a multimode optical fiber coupled to a light source enables critical illumination with high illumination uniformity of an object. Imaging can occur at the end of the multimode optical fiber. As disclosed herein, the optical fiber can be provided with a minimum length, and longer fibers, i.e., fibers greater than 2 meters, minimize speckle contrast. Suitable alignment of the optical fiber along the optical axis helps avoid aberrant point spread functions (PSFs) across the field of view. Consequently, the device of the present invention can be used to obtain high-spatial-resolution, high-contrast microscopy with uniform illumination capable of quantitative imaging of objects.

[0026] Quantitative imaging of objects can be broadly in the 1-500 nm range. In some embodiments, objects can be greater than 1, 50, 100, 150, 200, 250, 300, 350, 400, or 450 nm. In some embodiments, objects can be less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 nm. In some embodiments, objects can be in the 20-200 nm size range. In some embodiments, objects can be greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 nm. In some embodiments, the objects may be less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 nm. In some embodiments, the objects may be less than 20, 18, 16, 14, 12, 10, 8, 6, 4, or 2 nm in size. In some embodiments, the objects may be greater than 2, 4, 6, 8, 10, 12, 14, 16, or 18 nm.

[0027] In some embodiments, the optical fiber can have a numerical aperture (NA) greater than 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, or 0.24. In some embodiments, the optical fiber can have a numerical aperture (NA) less than 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, or 0.13. In some embodiments, the optical fiber can have a numerical aperture (NA) greater than 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, or 0.39. In some embodiments, the optical fiber can have a numerical aperture (NA) less than 0.40, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34, 0.33, 0.32, 0.31, 0.30, 0.29, 0.28, 0.27, 0.26, or 0.25.

[0028] The microscope may be a reflected light microscope or a reflection mode microscope with illumination provided by an SLD or modulated laser diode coupled into a multimode optical fiber. In some embodiments, the reflected light microscope may be an interferometric scattering microscope.

[0029] In some embodiments, the output of the fiber imaged onto the sample (critical illumination) can provide uniform illumination with a high illumination NA. Speckle contrast can be further reduced by increasing the fiber length, for example, by using a fiber longer than 10 meters, or by moving the fiber rapidly compared to the acquisition speed.

[0030] In some embodiments, the illumination device further comprises an optical element. Speckle contrast can be reduced by rapidly changing the direction or divergence of the input light into the fiber using optical elements such as deformable mirrors and / or optical deflection elements.

[0031] As used herein, and unless otherwise stated, the terms "coupled," "coupling," or "coupled with" refer to optical communication, and more specifically, the process of transmitting light between optical components. For example, optical coupling may occur between an optical fiber and a light source. Optical components such as an optical fiber and a light source may be directly or indirectly connected. Any suitable coupling technique may be used, for example, waveguide coupling.

[0032] In some embodiments, a collimator lens may be provided to couple the light into the optical fiber. The collimator lens is configured to match the numerical aperture of the optical fiber to ensure that no light is lost (focusing all of the light into the fiber acceptance angle) and that all modes of the optical fiber are efficiently used. This helps achieve a uniform output at one end of the fiber, resulting in a more uniform illumination pattern.

[0033] To provide the desired uniform illumination, it is important to have a collimator lens positioned at one end of the fiber, i.e., one focal length away from the exit face, to collect and collimate the output of the fiber, which is then imaged onto the sample plane when used with an optical system. The collimator lens can be of a numerical aperture greater than or equal to that of the fiber to ensure that no light is lost. The focal length of this collimating lens relative to the rest of the optical system controls the magnification (demagnification) of the fiber exit face onto the sample plane, thereby controlling the field of view.

[0034] In some embodiments, the coherent or partially coherent light source can have a bandwidth of 1 to 10 nm. In some embodiments, the bandwidth can be greater than 1, 2, 3, 4, 5, 6, 7, 8, or 9 nm. In some embodiments, the bandwidth can be less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nm.

[0035] Coherent or partially coherent light sources can be configured to provide bright illumination to a sample. When coherent or partially coherent light is delivered to a sample through a multimode fiber in this manner, mutual interference of different modes can cause speckle patterns that reduce the uniformity of the illumination, and speckle contrast decreases with reduced coherence (increased bandwidth) of the light source. Therefore, additional measures to reduce speckle contrast, such as increasing the length of the fiber, mechanically stirring the fiber, or perturbing the input light into the fiber by, for example, rapidly modulating the pointing or divergence with a deformable mirror, are necessary to achieve uniform illumination.

[0036] In some embodiments, a critical illumination device is provided in which the optical fiber core can be circular. Alternatively, the optical fiber core can be non-circular, such as square or rectangular, or elliptical, hexagonal, octagonal, or any suitable polygonal shape. A non-circular optical fiber core can more efficiently use light by illuminating an area shaped for camera readout, while promoting a more uniform output for a given length of fiber. In addition, a non-circular optical fiber core, such as a square or rectangular optical fiber core, can improve scrambling because there are more angles available for the light to utilize as it travels through the optical fiber.

[0037] In some embodiments, critical illumination devices are provided in which the fiber length can be at least 2 meters. Providing a minimum of 2 meters of fiber can help maintain uniform illumination. If the optical fiber is too short, i.e., less than 1 meter, mode mixing can become inefficient. In some embodiments, it can be at least 5 meters. In some embodiments, the fiber length is at least 10 meters. In some embodiments, the fiber length can be between 2 and 500 meters. In some embodiments, the fiber length can be greater than 2, 3, 4, 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, or 450 meters. In some embodiments, the fiber length can be less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 10, 5, 4, or 3 meters. In some embodiments, the fiber length can be greater than 1 km.

[0038] In some embodiments, the length of the optical fiber is at least 10 meters. Increasing the length of the fiber can be advantageous because it reduces speckle contrast. However, the longer the fiber, the greater the potential for power loss. Therefore, it may be desirable to use only the length of fiber necessary to reduce speckle while still providing sufficient power for sample illumination.

[0039] In some embodiments, fiber lengths of more than 10 meters with modulated broadband lasers can be provided, resulting in output light that is more uniform and spatially incoherent with reduced speckle contrast.

[0040] In some embodiments, a 30 meter length of optical fiber may provide optimally low speckle noise without excessive power loss. In some embodiments, a partial or coherent light source according to any aspect of the present invention, combined with an optical fiber having a length of at least 10 meters, allows for critical illumination with high illumination uniformity over an object, as well as minimized speckle contrast, resulting in a high spatial resolution, high contrast microscope with reasonably uniform illumination capable of quantitative imaging of objects such as nanoparticles.

[0041] In some embodiments, the fiber core can have a diameter of 15 microns to 400 microns, hi some embodiments, the fiber core can have a diameter of greater than 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300, 315, 330, 345, 360, or 385 microns. In some embodiments, the fiber core can have a diameter of less than 400, 385, 360, 345, 330, 315, 300, 285, 270, 255, 240, 225, 210, 200, 195, 190, 180, 165, 150, 135, 120, 105, 90, 75, 60, 45, or 40 microns.

[0042] The core and length of the optical fiber are important considerations for providing uniform and spatially incoherent output light with reduced speckle contrast. In some embodiments, the optical fiber may include a 30 μm core size with a numerical aperture (NA) of 0.15. A higher fiber NA results in better speckle reduction, but using a shorter collimator, e.g., f=6.5 mm, maintains a similar FoV and adequate beam size.

[0043] In one example, the optical fiber may have a core size of 15 μm with a fiber NA of 0.25 in combination with a collimator lens (f) having a size of 3.3 mm. The parameters in this example are suitable for an iSCAT microscope with a small field of view of the sample, where a spatial filter is provided at the back focal plane.

[0044] In some embodiments, a useful range of optical fiber parameters may range from approximately 50 μm core, 0.12 NA to 15 μm core, 0.25 NA. Higher NA fibers may provide better reduction in speckle contrast, but require smaller core sizes to maintain a desired field of view (FoV).

[0045] In some cases, increasing the fiber NA can be advantageous because increased NA is better at reducing speckle contrast. In addition, the collimator focal length and fiber core size can be reduced to maintain the target beam diameter and FoV. For example, the fiber core can range from a 150 μm diameter at 0.39 NA to a 200 μm diameter at 0.22 NA. In another example, the fiber core diameter can be up to 400 μm, and a 0.22 NA and 9 mm collimator can be used with a larger (73 μm) FoV.

[0046] Those skilled in the art will appreciate that the selection of fiber and collimator core numerical aperture and / or core diameter also depends on the desired FoV and whether a spatial filter or mask is used. Typically, a higher NA is preferable for speckle reduction, but requires a corresponding reduction in core size and / or reduction in the focal length of the collimating lens to maintain a relevant FoV and illumination NA.

[0047] Other possible combinations of fiber core size and NA may be used, for example, allowing a larger core size and / or higher NA to be used while still keeping the beam diameter smaller than the mask diameter, if used, with a larger illumination area corresponding to either lower sensitivity or higher power requirements.

[0048] In some embodiments, the critical illumination device further comprises a vibration module directly or indirectly connected to the optical fiber such that the vibration module is configured to move the fiber.

[0049] A vibration module, such as a vibration motor or voice coil, is used to vibrate or shake the optical fiber to further reduce speckle. Alternatively or additionally, the length of the optical fiber can be significantly increased, up to a length of 500 meters, to assist in reducing speckle contrast.

[0050] In some embodiments, additional / alternative speckle reduction techniques are required, such as physically moving / bending the optical fiber to change the speckle pattern at the output or tip of the fiber. Moving the fiber at a speed much faster than the camera integration time can reduce speckle contrast by averaging out different speckle patterns within the exposure time. These techniques can be used with shorter fiber lengths to achieve the same speckle reduction as longer fiber lengths, without the increased risk of power loss.

[0051] In some embodiments, speckle contrast can be reduced by rapidly changing the direction or divergence of the input light into the fiber using optical elements such as deformable mirrors and / or optical deflection elements.

[0052] In some embodiments, the light source is a laser or an SLD. A superluminescent diode (SLD) can be used as the light source, which does not require the high power achievable by a laser. A superluminescent diode may not require modulation.

[0053] In some embodiments, the laser can be modulated to induce a wavelength shift. This is particularly advantageous because it reduces coherence within the light source. In some embodiments, at least 10 meters of fiber can be provided with a low-coherence light source, such as a modulated laser diode. The laser can be a modulated broadband laser. A modulated broadband laser can be used to reduce speckle contrast.

[0054] Imperfect temporal coherence, i.e., the emission of a certain range of wavelengths, leads to multiple fiber modes being used for efficient scrambling within the fiber. Modulation of diode lasers leads to a shift of their central wavelengths by approximately 1 nm. High-speed modulation (faster than the camera readout) can therefore increase the bandwidth used; as the speckle pattern at the end of the fiber changes due to the modulation, it averages over the camera frame readout.

[0055] In some embodiments, the critical illumination device includes a collimator lens that is an achromatic collimator lens, an aspheric collimator lens, or a reflective collimator lens, such as a curved mirror. The collimator lens (f) can be 2 mm to 10 mm to obtain a suitable FoV and illumination NA. In some embodiments, the collimator lens (f) can be greater than 3, 4, 5, 6, 7, 8, 9, 10, or 11 mm. In some embodiments, the collimator lens (f) can be less than 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 mm. In some embodiments, an aspheric collimator (f=9.6 mm) can be adjusted to image the fiber end at infinity.

[0056] In some embodiments, the critical illumination device further comprises an objective lens. In another aspect of the invention, there is provided a reflected light microscope comprising: a sample holder for holding the sample at the sample position; a critical illumination device according to any one of the previous aspects of the invention, the illumination device being configured to provide uniform illumination of the sample; A detector; an optical system arranged to direct the illumination light to the sample location, to collect reflected output light including both light scattered from the sample location and the illumination light reflected from the sample location, and to direct the output light to a detector; A reflected light microscope is provided, comprising:

[0057] The use of multimode optical fibers can significantly simplify microscope equipment because fewer components are required. This minimizes component costs and setup time. An illumination device including a coherent or partially coherent light source and an optical (multimode) fiber as disclosed herein, in conjunction with an optical system, can be configured to form an image of the tip of the fiber at the sample plane. This is important to enable critical illumination with highly uniform illumination of the sample while reducing speckle contrast in the image.

[0058] The sample may be a liquid sample containing the object to be imaged. The sample holder may take any form suitable for holding a sample. Typically, the sample holder holds the sample on a surface that forms an interface between the sample holder and the sample. For example, the sample holder may be a coverslip and / or may be made of glass. The sample may be provided on the sample holder in a simple manner, for example, using a micropipette. The sample position may be described as the surface on which the sample is disposed.

[0059] An optical fiber coupled to a light source, such as a laser module, can provide coherent or partially coherent light for bright illumination. By way of example only, a 1.5W 525nm laser can be provided, with approximately 250mW of power provided at the sample. Alternatively, a 3.5W 465nm laser can also be used as the light source.

[0060] In addition, the critical illumination device allows for a larger range of illumination angles for high spatial resolution than may typically be achievable in coherent imaging with little loss of contrast. A multimode optical fiber acting as the light source enables critical illumination with high illumination uniformity. By providing critical illumination in conjunction with an optical fiber, high spatial resolution, high contrast microscopy with excellent uniform illumination can be achieved for quantitative imaging of samples. Without a multimode optical fiber acting as a highly uniform light source as disclosed herein, there would be non-uniform illumination due to the image of the light source.

[0061] The reflected light microscope may be an interferometric scattering or iSCAT microscope. In some embodiments, a reflected light microscope as described herein can be used for quantitative imaging of objects present in a sample ranging in size from 20 to 200 nm. In some embodiments, a reflected light microscope as described herein can be used for quantitative imaging of samples larger than 10, 20, 40, 60, 80, 100, 120, 140, 160, or 180 nm. In some embodiments, a reflected light microscope as described herein can be used for quantitative imaging of samples smaller than 200, 180, 160, 140, 120, 100, 80, 60, 40, or 20 nm.

[0062] The object present in the sample may be a viral vector, such as, but not limited to, adenovirus, AAV, and / or lentivirus. A reflectance microscope, which may be a single particle interferometric scattering microscope, may be used to analyze the genome content (i.e., empty / full) of the sample based on light scattering contrast. Such is critical for the quality assurance process of novel therapeutics based on such viral vectors, such as gene therapy.

[0063] Additionally or alternatively, the entity in the sample can be, but is not limited to, a biomolecule such as a nucleic acid, a protein, a protein complex, a lipoprotein, a polypeptide, an antibody or antibody fragment thereof, an enzyme, a virus or viral vector such as adenovirus, AAV, and / or lentivirus, a particle, a nanoparticle, a compound, a molecule, an ion, or a quantum dot. Alternatively, the entity can be a nanotube, a nanowire, a dendrimer, a liposome, an ethosome, or an aquasome, a polymersome, a niosome, or a cubosome.

[0064] In some embodiments, the reflected light microscope may further comprise a spatial filter (or mask) positioned to filter the output light, the spatial filter being arranged to pass the output light but with a greater reduction in intensity within a predetermined numerical aperture than at larger numerical apertures.

[0065] The spatial filter or mask may be positioned as close as possible to the back of the objective lens. The spatial filter passes the output light but with a greater reduction in intensity within a predetermined numerical aperture than at larger numerical apertures.

[0066] As a result, spatial filters selectively reduce the intensity of illumination light over scattered light by exploiting the mismatch between the numerical aperture of the reflected illumination light and the numerical aperture of light scattered from objects in the sample at the sample location. Thus, spatial filters exploit the different directionality of these two sources of light. Reflected illumination light typically has a relatively small numerical aperture, while sub-diffraction-sized objects near the surface of the sample preferentially scatter light into the high numerical aperture. Therefore, intensity reduction by spatial filters at low numerical apertures preferentially affects illumination light and has minimal effect on scattered light, thereby maximizing imaging contrast.

[0067] This effect can be maximized by positioning the spatial filter so that its predetermined numerical aperture is the same as or similar to the numerical aperture of the illumination light reflected from the sample location. In some embodiments, the predetermined numerical aperture can be the numerical aperture of the illumination light reflected from the sample location that is included in the output beam.

[0068] In some embodiments, the spatial filter is 10 -1 It may be arranged to pass output light with a reduction in intensity within said predetermined numerical aperture to an incident intensity of: In some embodiments, the spatial filter is 10 -2It may be arranged to pass output light with a reduction in intensity within said predetermined numerical aperture to an incident intensity of:

[0069] In some embodiments, the optical system may include an objective lens. In some embodiments, the optical system may include an aperture or a series of apertures, which may be positioned within the optical system such that it optimizes stray light.

[0070] In some embodiments, an aspheric collimator f = 9.6 mm can be adjusted to image the fiber end at infinity. The aperture A1 is 1.3 mm diameter in the etched steel for an illumination NA of approximately 0.3 at the sample. A larger A1 would have had too much stray light with the mask farther from the back focal plane.

[0071] In some embodiments, at least one aperture of the optical system has a diameter of 1 to 5 mm. In some embodiments, at least one aperture has a diameter of more than 1, 2, 3, or 4 mm. In some embodiments, at least one aperture has a diameter of less than 5, 4, 3, or 2 mm. The sequence of apertures in the optical system, e.g., A1, A2, A3, can be arranged to optimize any stray light. This is advantageous because it ensures that any light loss is minimized.

[0072] In some embodiments, the optical system may further comprise a beam splitter configured to separate the reflected output light from the sample position from the illumination light, i.e., the beam splitter may be configured to separate the signal to be detected from the sample position from the illumination light.

[0073] In some embodiments, the reflected light microscope may further comprise a second detector. The detector can be a camera such as a FLIR or Ximea or a (high-speed) complementary metal-oxide semiconductor (CMOS) camera. The camera can provide magnification as an approximately square FoV to match the circular optical fiber core. By way of example only, the camera's FoV can be 650 x 400 pixels at 350 frames per second (fps).

[0074] In some embodiments, the optical system can be configured to direct the first and second signals to first and second detectors, respectively. In a further aspect of the invention, there is provided a method of illuminating an object, comprising the steps of: providing a critical illumination device, the critical illumination device comprising: a coherent or partially coherent light source; an optical fiber having a core with a diameter of at least 15 microns with a numerical aperture of 0.1 to 0.5; at least one collimator lens positioned at a focal distance from the tip of the optical fiber, the collimator lens configured to match the numerical aperture of the optical fiber to couple light out of the optical fiber; and transmitting light from a light source into a fiber core to provide uniform illumination of the object; A method is provided, comprising:

[0075] In a further aspect of the invention, there is provided a method of illuminating an object, comprising the steps of: providing a critical illumination device, the critical illumination device comprising: a coherent or partially coherent light source; an optical fiber having a core with a diameter of at least 15 microns with a numerical aperture of 0.1 to 0.5; at least one collimator lens positioned at a focal distance from the tip of the optical fiber, the collimator lens configured to match the numerical aperture of the optical fiber for coupling light into and / or out of the optical fiber; and transmitting light from a light source into a fiber core to provide uniform illumination of the object; A method is provided, comprising:

[0076] In some embodiments, the method according to any one of the aspects of the present invention further comprises providing a coherent or partially coherent light source having a bandwidth of 1 to 10 nm. In some embodiments, the bandwidth is greater than 1, 2, 3, 4, 5, 6, 7, 8, or 9 nm. In some embodiments, the bandwidth is less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nm.

[0077] The light source may be a laser. The method may further include modulating the laser to induce a wavelength shift. In some embodiments, the method may further include vibrating the optical fiber. The vibration module is directly or indirectly connected to the optical fiber such that, during use, the vibration module vibrates the optical fiber. The vibration module, such as a vibration motor, is used to vibrate or rock the optical fiber to further reduce speckle.

[0078] In some embodiments, the method may further include providing an optical fiber having a length of at least 10 meters. Increasing the length of the fiber may be advantageous because it reduces speckle contrast of the object being imaged, which improves contrast resolution.

[0079] There are several techniques to reduce speckle contrast and improve the contrast resolution of objects in an image. Speckle contrast reduction can include, but is not limited to, one or more of the following: using a longer optical fiber, at least 10 meters in length, to reduce the coherence of the light source; using a square-core fiber over a given length, i.e., at least 10 meters in length; rapid shaking / bending of the fiber using one or more motors or a Fiberguide® speckle reduction system. This can be particularly important in low contrast, i.e., contrast from about 1% of the coverslip surface, where the speckle contrast in the illumination is significant relative to the surface roughness image.

[0080] In some embodiments, the method may further include imaging the object using a detection module. The detection module may be a camera that can be used to provide an image of the object at the tip of the optical fiber. The detector may be a CMOS camera, such as a FLIR or Ximea (high-speed) camera. The camera may provide magnification as an approximately square FoV to match the circular optical fiber core. The camera's FoV may be 650 x 400 pixels at 350 fps.

[0081] The invention will now be further and more particularly described, by way of example only, and with reference to the accompanying drawings in which: [Brief explanation of the drawings]

[0082] [Figure 1] 1 is a schematic diagram of a fiber-coupled laser module according to the present invention; [Figure 2] 2 is a schematic diagram of a fiber-coupled laser module according to FIG. 1 used in conjunction with a reflected light microscope. [Figure 3A] 1 shows experimental data of objects detected by the apparatus and method of the present invention. [Figure 3B]1 shows experimental data of objects detected by the apparatus and method of the present invention. [Figure 3C] 1 shows experimental data of objects detected by the apparatus and method of the present invention. [Figure 3D] 1 shows experimental data of objects detected by the apparatus and method of the present invention. [Figure 4] Schematic diagram of an iSCAT microscope. [Figure 5] Figure 5A shows an image captured by a reflected light microscope. Figure 5B shows an image captured by a reflected light microscope. [Figure 6] FIG. 1 is a schematic diagram of an illumination device coupled to a reflected light microscope. [Figure 7] 1 is a histogram of measured scattering contrast for the detection of adenovirus particles. DETAILED DESCRIPTION OF THE INVENTION

[0083] Referring to FIG. 1 , a critical illumination device 10 is provided that includes a light source 12, a modulator such as a shutter 14, a first collimator lens 16, and an optical fiber 18. The light source 12 provides light that is focused by the first collimator lens 16 onto the optical fiber 18. The generated light can be coherent or partially coherent. The optical fiber 18 can be a multimode fiber. The partially coherent light source 12 is advantageous because, when coupled with the optical fiber 18, it utilizes multiple fiber modes, thereby efficiently scrambling the light, thereby enabling critical illumination with high illumination uniformity of the object.

[0084] An optical fiber 18 having multiple modes can help reduce speckle in the image captured by the detector 26. Additionally, increasing the fiber length of the optical fiber 18 further reduces speckle.

[0085] The first collimator lens 16 is configured to match the numerical aperture of the optical fiber 18 to ensure that no light is lost and that all modes of the optical fiber 18 are efficiently used.

[0086] The light coupled into the optical fiber 18 is then recollimated in the second collimator lens 21, which may then be output to the reflected light microscope 20. The first and / or second collimator lenses 16, 21 are positioned at a focal distance from the tip of the optical fiber.

[0087] A shutter 14 is provided in the critical illumination device 10 to modulate the space between the optical fiber 18 , the first collimator lens 16 , and the light source 12 . Referring to FIG. 2 , a reflected light microscope 20 is provided. The reflected light microscope 20 includes a sample holder 22 for holding one or more objects, such as a sample 24, at a sample position. The sample 24 may be a liquid sample containing one or more particles to be imaged. The sample holder 22 may take any form suitable for holding the sample 24. Typically, the sample holder 22 holds the sample 24 on a surface that forms an interface between the sample holder 22 and the sample 24. For example, the sample 24 may be provided on the sample holder 22 in a simple manner, such as using a micropipette.

[0088] The reflected light microscope further comprises a critical illumination device 10 and a detector 26. The critical illumination device 10 is positioned to provide illumination light to a sample 24. Detector 26 receives reflected output light from the sample location. The reflected light microscope 20 may be deployed in a wide field mode, in which case detector 26 may be an image sensor that captures an image of sample 24. The detector may be a FLIR (Forward Looking Infrared), Ximea, or (high speed) CMOS camera.

[0089] The reflected light microscope 20 further comprises an optical system 30 disposed between the sample holder 22, the critical illumination device 10, and the detector 26. The optical system 30 is positioned to direct illumination light to a sample location to illuminate the sample 24, and to collect output light reflected from the sample location and direct the output light to the detector 26, as follows:

[0090] The optical system 30 includes an objective lens 31, which is a lens system disposed in front of the sample holder 22. The optical system 30 also includes a first aperture 32 configured to reduce the maximum diameter of the light so that the beam is no larger than a spatial filter 33 formed on a window 34. A second aperture 35 is configured to reduce the maximum diameter of the light beam so that it is no larger than a first lens 36, and a third aperture 37 is configured to reduce the maximum diameter of the light beam so that it is no larger than a second lens 38. The optical system 30 further includes a mirror 39 configured to reflect the output light so that it is incident on the detector 26. The output light incident on the detector 26 generates an image that is used to perform mass measurements and determine the mass of any objects in the sample.

[0091] First lens 36 focuses the output light towards a third aperture 37 and second lens 38. Second lens 38 focuses the output light towards mirror 39, which then reflects the output light towards detector 26. This is advantageous because it provides greater magnification over a shorter beam path than if a single lens were used.

[0092] The objective lens 31 collects output light that includes both (a) illumination light reflected from the sample location and (b) light scattered from the sample 24 at the sample location. The reflected light is preferentially reflected from the interface between the sample holder 22 and the sample 24. Typically, this is a relatively weak reflection, e.g., a glass-water reflection. For example, the intensity of the reflected illumination light may be on the order of 0.5% of the intensity of the incident illumination light. The scattered light is scattered by objects in the sample 24.

[0093] Light scattered from objects at or near the surface of sample 24 constructively interferes with the reflected light and is therefore visible in the image captured by detector 26 . 2, the reflected illumination light and the scattered light have different directionality. In particular, the reflected illumination light has a numerical aperture that results from the geometry of the beam of light output by the critical illumination device 10 and optical system 30. The scattered light is scattered over a larger range of angles and therefore satisfies a larger numerical aperture than the reflected illumination light.

[0094] Output light from objective lens 31 is incident on spatial filter 33, which is positioned to filter the output light through detector 26. In the example shown in Figure 2, where detector 26 is aligned with the optical path of objective lens 31, spatial filter 33 is therefore transmissive.

[0095] The spatial filter 33 is partially transmissive and therefore passes the output light, including the reflected illumination light, but with an intensity reduction. The spatial filter 33 is also aligned with the optical axis and has a predetermined aperture to provide the intensity reduction within a predetermined numerical aperture. Numerical aperture is defined herein in its usual manner as a dimensionless quantity that characterizes the range of angles relative to the sample position from which the output light originates. Specifically, the numerical aperture (NA) may be defined by Equation 1 as follows:

[0096] NA==n·sin(θ) (Equation 1) where θ is the collection half angle and n is the refractive index of the material through which the output light passes (eg, the material of the components of optical system 30).

[0097] 3A-3D, experimental data for detected objects using the presently claimed apparatus and method are provided. The graphs show the mass of objects measured by the present apparatus and method as disclosed herein. For all measurements, proteins were diluted to a 5 nM final concentration in a 20 μl droplet of PBS within a silicone gasket on a clean coverslip, and single particle contrast was recorded for 90 seconds.

[0098] To provide the desired uniform illumination, it is important to have a collimator lens positioned one focal length away from one end of the fiber, i.e., the exit face, to collect and collimate the fiber's output, which is then imaged onto the sample plane when used with an optical system. The collimator lens can have a numerical aperture greater than or equal to that of the fiber to ensure that no light is lost. The focal length of this collimating lens relative to the rest of the optical system controls the magnification (demagnification) of the fiber exit face onto the sample plane, thereby controlling the field of view. For example, in a setup such as that shown in Figure 2, the ratio of the focal lengths of the collimator and objective lens results in a magnification of the image of the fiber's end face at the sample plane.

[0099] Referring to Figure 3A, there is shown a plot of the number of counts versus mass in kDa for the results of Example 1 measuring a 90 kDa oligomerized protein. Referring to Figure 3B, a plot of the number of counts from the results of Example 1 measuring Protein A versus mass in kDa is shown.

[0100] Referring to FIG. 3C, a plot of the number of counts from the results of Example 1 measuring bovine serum albumin (BSA) versus mass in kDa is shown. Referring to Figure 3D, a plot of the number of counts from the results of Example 1 measuring pure PBS buffer against mass in kDa is shown.

[0101] Referring to FIG. 4 , an iSCAT microscope 40 is provided. The microscope 40 includes a sample holder 41 for holding a sample 42 at a sample position. The sample 42 may be a liquid sample containing an object to be imaged, as described in more detail below. The sample holder 41 may take any form suitable for holding the sample 42. Typically, the sample holder 41 holds the sample 42 on a surface that forms an interface between the sample holder 41 and the sample 42. For example, the sample holder 41 may be a coverslip and / or may be made of glass. The sample 42 may be provided on the sample holder 41 in a simple manner, for example, using a micropipette.

[0102] The microscope 40 further comprises a critical illumination device 10 and a detector 44. The critical illumination device 10 is positioned to provide illumination light to the sample 42. The detector 44 receives reflected output light from the sample location. Typically, the microscope 40 may operate in a wide field mode, in which case the detector 44 may be an image sensor that captures an image of the sample 42. Examples of image sensors that may be used as the detector 44 include a CMOS (complementary metal oxide semiconductor) image sensor or a CCD (charge coupled device).

[0103] The microscope 40 further comprises an optical system 50 disposed between the sample holder 41, the critical illumination device 10, and the detector 44. The optical system 50 is disposed to direct illumination light to the sample position to illuminate the sample 42, and to collect output light reflected from the sample position and direct the output light to the detector 44.

[0104] The optical system 50 includes an objective lens 51, which is a lens system disposed in front of the sample holder 41. The optical system 50 also includes a tube lens 52. The objective lens 51 collects output light that includes both (a) illumination light reflected from the sample position (indicated by the solid line in FIG. 4) and (b) light scattered from the sample 3 at the sample position (indicated by the dotted line in FIG. 4). The reflected light is preferentially reflected from the interface between the sample holder 41 and the sample 42. Typically, this is a relatively weak reflection, e.g., a glass-water reflection. For example, the intensity of the reflected illumination light may be on the order of 0.5% of the intensity of the incident illumination light. The scattered light is scattered by objects in the sample 43.

[0105] In a manner similar to conventional iSCAT, scattered light from objects at or near the surface of the sample interferes with the reflected light and is therefore visible in the image captured by detector 44. This effect differs from microscopes operating in transmission mode, where the illumination light reaching the detector is transmitted through the depth of the sample, leading to much smaller imaging contrast.

[0106] As shown in Figure 4, the reflected illumination light and scattered light have different directionality. In particular, the reflected illumination light has a numerical aperture that results from the geometry of the beam of light output by the objective illumination device 10 and optical system 50. The scattered light is scattered over a larger range of angles and therefore satisfies a larger numerical aperture than the reflected illumination light. Tube lens 52 focuses the output light from objective lens 51 onto detector 44.

[0107] The optical system 10 also includes a window 53 , a spatial filter 55 , and a dichroic mirror 56 that directs light towards the objective lens 51 . In addition to the components described above, microscope 40 includes spatial filter 55. In the example shown in FIG. 4 , spatial filter 55 is formed on window 53 and is thus positioned behind the back aperture of objective lens 51, and thus just behind the back focal plane 54 of objective lens 51. Thus, spatial filter 55 can be implemented without entering objective lens 51, as in phase-contrast microscopy. Placing the spatial filter just behind the entrance aperture of objective lens 51 rather than at a conjugate plane (e.g., as described below) has the obvious advantage of strongly suppressing back reflections arising from the multiple lenses in high-numerical-aperture microscope objectives. This, in turn, reduces imaging noise, attenuates incoherent measurement background, and reduces experimental complexity, the number of optical systems, and optical path lengths, resulting in increased stability of the optical setup and therefore image quality.

[0108] However, this location is not essential and a spatial filter with equivalent functionality may be provided elsewhere, as described below. Spatial filter 55 is thus positioned to filter the output light through detector 44. In the example shown in Figure 4, where detector 44 is aligned with the optical path of objective lens 51, spatial filter 55 is therefore transmissive.

[0109] The spatial filter 55 is partially transmissive, thus passing the output light, including the reflected illumination light, but with a reduction in intensity. The spatial filter 55 is also aligned with the optical axis and has a predetermined aperture to provide the intensity reduction within a predetermined numerical aperture. Numerical aperture is defined herein in its usual manner as a dimensionless quantity that characterizes the range of angles relative to the sample position from which the output light originates. Specifically, the numerical aperture (NA) may be defined by Equation 1:

[0110] Figures 5A and 5B show images of individual adenovirus particles captured by the device using critical illumination. Adenovirus particles were collected in PBS at 10 -9The virus particles were diluted to 1 / ml, and a 20 μl droplet was placed in a silicone gasket on a clean coverslip. The virus particles were allowed to settle on the glass surface for 2 minutes, and an image covering the entire illumination area was taken. The data in Figure 5A was taken on a microscope using critical illumination with a relatively low NA (i.e., angle) without a spatial filter. Figure 5A shows particles near the edge of the circular illumination area. As illustrated in Figure 5A, the image has relatively poor spatial resolution (seen in the distinct ringing from each particle). It also illustrates the effect of mispositioning the output of the optical fiber along the optical axis. As a result of the mispositioned output of the optical fiber, the output from the fiber is not properly imaged onto the sample. This causes an aberrant PSF at the edge of the illumination area.

[0111] Referring to Figure 5B, an image captured on a microscope using critical illumination with a multimode fiber at a high illumination NA (>1), close to the full aperture of the objective, is shown, which is only possible when no spatial filters are used. Figure 5B shows a particle near the center of the illumination field, thus demonstrating uniform illumination of the entire area. The high illumination NA results in improved spatial resolution; i.e., the "ringing" around each PSF is much less noticeable away from the center of the particle. As illustrated in Figure 5B, the positioning of the fiber's output was correct, resulting in round PSFs around the field of view.

[0112] Referring to FIG. 6, an alternative microscope apparatus 60 is shown. The alternative microscope apparatus 60 as illustrated in FIG. 6 does not include a spatial filter or mask. The microscope apparatus 60 includes a sample holder 61 for holding a sample 62 at a sample position. The sample 62 may be a liquid sample containing the object to be imaged. The sample holder 61 may take any suitable form for holding the sample 62. Typically, the sample holder 61 holds the sample 62 on a surface that forms an interface between the sample holder 61 and the sample 62. For example, the sample holder 61 may be a coverslip and / or may be made of glass. The sample 62 may be provided on the sample holder 61 in a simple manner, for example, using a micropipette.

[0113] The microscope apparatus 60 further comprises a critical illumination device 10 as disclosed herein, and a detector 64. The critical illumination device 10 is positioned to provide illumination light to the sample 62. The detector 64 receives output light reflected from the sample location. Typically, the microscope 60 may operate in a wide-field mode, in which case the detector 64 may be an image sensor that captures an image of the sample 62. Examples of image sensors that may be used as the detector 64 include a CMOS (complementary metal-oxide semiconductor) image sensor or a CCD (charge-coupled device). The detector 64 may also be a camera.

[0114] Microscope 60 further comprises an optical system 70 disposed between sample holder 61, critical illumination device 10, and detector 64. Optical system 70 is positioned to direct illumination light to a sample position for illuminating sample 62, and to collect output light reflected from the sample position. The output light is then directed to detector 64.

[0115] The optical system 70 includes an objective lens 71, which is a lens system disposed in front of the sample holder 61. The objective lens 71 collects output light that includes both (a) illumination light reflected from a sample position provided around the sample holder 61 and (b) light scattered from the sample at the sample position provided around the sample holder 61. The reflected light is preferentially reflected from the interface between the sample holder 61 and the sample 62. Typically, this is a relatively weak reflection, e.g., a glass-water reflection. For example, the intensity of the reflected illumination light may be on the order of 0.5% of the intensity of the incident illumination light. The scattered light is scattered by objects in the sample 62.

[0116] In a manner similar to conventional iSCAT, light scattered from objects at or near the surface of the sample interferes with the reflected light and is therefore visible in the image captured by detector 64. This effect differs from microscopes operating in transmission mode, where the illumination light reaching the detector is transmitted through the depth of the sample, leading to much smaller imaging contrast.

[0117] The light provided by the critical illumination device 10 is coupled into an optical fiber 18, which is then collimated in a collimator lens 21. This is then output to the microscope 60. The collimator lens 21 may be positioned at a focal distance from the tip of the optical fiber 18. The optical system further comprises a series of lenses 72, 74 for focusing the illumination light 67 from the critical illumination device 10.

[0118] The optical system also includes a beam splitter 66 positioned to split or separate the optical path for illumination light 67 from a light source (not shown in FIG. 6) provided within critical illumination device 10, with output light 68 directed to detector 64. Beam splitter 66 may have a conventional structure that provides partial reflection and partial transmission of light incident thereon.

[0119] The optical system 70 further comprises a mirror 76 configured to reflect the output light 68 so that it is incident on the detector 64. A tube lens 78 is provided to focus the output light 68 from the objective lens 71 onto the detector 64. The output light 68 incident on the detector 64 produces an image that is used to perform a mass measurement and determine the mass of any objects in the sample.

[0120] Referring to Figure 7, a histogram (number of counts versus scattering contrast in arbitrary units) is provided showing the measured scattering contrast of adenovirus particles using an iSCAT microscope as illustrated in Figure 6. The iSCAT microscope can be used to acquire images of adenovirus particles immobilized on a clean coverslip surface. Adenovirus particles are collected in PBS at approximately 10 -9 The solution can be diluted to 100 μl of particles / ml and 5 μl of this solution can then be added to 10 μl of PBS in a silicone gasket well placed on a clean coverslip as described in Example 1. The droplets can be mixed by aspirating with a pipette and the particles allowed to settle on the surface for approximately 1-2 minutes.

[0121] Several images of 15 different fields on the same coverslip can then be acquired with fixed adenovirus particles positioned in focus, i.e., at the point where the adenovirus exhibits maximum contrast, and the scattering contrast of each particle is measured by an automated spot detection routine and 2D Gaussian fitting. A histogram, such as that shown in Figure 7, is generated from all measured particle contrasts in the 15 acquired images.

[0122] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. "And / or," as used herein, shall be deemed to be a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" shall be deemed to be a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0123] Unless the context indicates otherwise, the feature descriptions and definitions set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0124] While the present invention has been described by way of example with reference to certain embodiments, it will be further understood by those skilled in the art that it is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

[0125] The present invention is used in the following non-limiting examples. [Example]

[0126] Borosilicate glass coverslips (No. 1.5, 24 × 50 mm, VWR) were cleaned by successive rinses with MilliQ water, followed by ethanol, and then MilliQ water again. They were then dried under a stream of dry nitrogen. A CultureWell silicone gasket (Grace Bio-Labs) was cut and placed onto the freshly cleaned coverslip.

[0127] Various test samples were prepared from stock solutions. These included a 90 kDa oligomerized protein, Protein A, bovine serum albumin (BSA), and pure phosphate-buffered saline (PBS) buffer. For all measurements, the protein was diluted to a final concentration of 5 nM in a 20 μl droplet of PBS within a silicone gasket on a clean coverslip. Single particle contrast was recorded for 90 seconds.

[0128] Data acquisition and analysis were then performed by an iSCAT microscope in the presence of a spatial filter and using the illumination device described herein to image and record nonspecific binding of the object to the glass substrate. Images were acquired over a 90 second period.

[0129] Ratiometric image stacks were generated from the raw footage as previously described (e.g., Cole et al., ACS Photonics, 2017, 4(2), pp. 211-216). Objects dropped onto the glass surface were identified in the ratiometric images by an automated spot detection routine based on 2D Gaussian fitting of the point spread function. The resulting ratiometric images reveal the binding of objects to the cover glass surface.

[0130] The results are shown in Figure 3 (A-D), which provides plots of the signal contrast data acquired upon detection and assigns masses to the peaks observed. [Example]

[0131] An apparatus such as that illustrated in Figure 6 was used to acquire images of adenovirus particles immobilized on a clean coverslip surface. Adenovirus particles (stock solution) were diluted to 10 in PBS. -9 The virus was diluted to 1000particles / ml and a 20 μl droplet was placed in the silicone gasket on a clean cover clip. The virus particles were allowed to settle on the glass surface for approximately 2 minutes and an image was taken covering the entire illuminated area.

[0132] The results are shown in Figure 5. Figure 5A shows particles near the edge of the circular illuminated area, while Figure 5B illustrates the center of the illuminated area, showing uniform illumination of the entire area. The data in Figure 5A was taken on a microscope using critical illumination with a relatively low NA (i.e., angle) of illumination, without the presence of a spatial filter. Figure 5A shows particles near the edge of a circular illumination area. As illustrated in Figure 5A, the image has relatively poor spatial resolution (seen in the distinct ringing from each particle). It also illustrates the effect of mispositioning the output of the optical fiber along the optical axis. As a result of the mispositioned output of the optical fiber, the output from the fiber is not properly imaged onto the sample. This causes an aberrant point spread function (PSF) at the edge of the illumination area.

[0133] Referring to Figure 5B, an image captured on a microscope using critical illumination with a multimode fiber at a high illumination NA (>1), close to the full aperture of the objective, is shown, which is only possible when no spatial filters are used. Figure 5B shows a particle near the center of the illumination field, thus demonstrating uniform illumination of the entire area. The high illumination NA results in improved spatial resolution; i.e., the "ringing" around each PSF is much less noticeable away from the center of the particle. As illustrated in Figure 5B, the positioning of the fiber's output was correct, resulting in round PSFs around the field of view. [Example]

[0134] An apparatus such as that illustrated in Figure 6 was used to acquire images of adenovirus particles immobilized on a clean coverslip surface. Adenovirus particles were collected in PBS at approximately 10 -9 particles / ml, and 5 μl of this solution was added to 10 μl of PBS in a silicone gasket well placed on a clean coverslip as described in Example 1. The droplet was mixed by aspirating with a pipette, and the particles were allowed to settle on the surface for 1-2 minutes.

[0135] Several images of 15 different fields of view on the same coverslip were acquired with fixed adenovirus particles positioned in focus (i.e., at the point where the adenovirus exhibits maximum contrast), and the scattering contrast of each particle was measured by an automated spot detection routine and 2D Gaussian fitting. A histogram (Figure 7) was generated from all measured particle contrasts in the 15 acquired images.

Claims

1. 1. A critical illumination device for providing uniform illumination of an object in a reflected light microscope, comprising: a coherent or partially coherent light source; an optical fiber coupled to the light source, the optical fiber comprising a core having a diameter of at least 15 microns and a numerical aperture of 0.1 to 0.5; at least one collimator lens positioned at a focal distance from the tip of the optical fiber, the at least one collimator lens configured to match the numerical aperture of the optical fiber to couple light out of the optical fiber; A critical illumination device comprising:

2. the coherent or partially coherent light source has a bandwidth of 1 to 10 nm; 10. The critical illumination device of claim 1.

3. the fiber core is non-circular; 3. A critical illumination device according to claim 1 or 2.

4. The length of the fiber is at least 2 meters. A critical illumination device according to any one of claims 1 to 3.

5. the fiber core has a diameter of 15 microns to 400 microns; 5. A critical illumination device according to any one of claims 1 to 4.

6. further comprising a vibration module directly or indirectly connected to the optical fiber, the vibration module configured to move the fiber. A critical illumination device according to any one of claims 1 to 5.

7. Further comprising an optical element, A critical illumination device according to any one of claims 1 to 6.

8. The light source is a laser or an SLD. A critical illumination device according to any one of claims 1 to 7.

9. the laser is modulated to induce a wavelength shift; 9. The critical illumination device of claim 8.

10. The collimator lens is an achromatic collimator lens or an aspheric collimator lens.

10. A critical illumination device according to any one of claims 1 to 9.

11. 1. A reflected light microscope, comprising: a sample holder for holding the sample at the sample position; a critical illumination device according to claims 1 to 10 configured to provide uniform illumination of the sample; A detector; an optical system arranged to direct illumination light to the sample location, to collect reflected output light including both light scattered from the sample location and illumination light reflected from the sample location, and to direct the output light to the detector; A reflected light microscope comprising:

12. further comprising a spatial filter positioned to filter the output light; the spatial filter is positioned to pass output light with a greater reduction in intensity within a given numerical aperture than at larger numerical apertures; 12. The reflected light microscope of claim 11.

13. the predetermined numerical aperture is the numerical aperture of the illumination light reflected from the sample position and included in the output light; 13. The reflected light microscope of claim 12.

14. The spatial filter is 10 －１ arranged to pass output light with a reduction in intensity within said predetermined numerical aperture to an incident intensity of:

14. A reflected light microscope according to claim 12 or 13.

15. The optical system includes an objective lens. A reflected light microscope according to any one of claims 11 to 14.

16. the optical system comprises a series of apertures; A reflected light microscope according to any one of claims 11 to 15.

17. At least one opening has a diameter of 1 to 5 mm; A reflected light microscope according to any one of claims 11 to 16.

18. the optical system further comprising a beam splitter configured to separate reflected output light from the sample location from the illumination light.

18. A reflected light microscope according to claims 11 to 17.

19. 1. A method of illuminating an object, comprising: providing a critical illumination device, said critical illumination device comprising: a coherent or partially coherent light source; an optical fiber having a core with a diameter of at least 15 microns with a numerical aperture of 0.1 to 0.5; at least one collimator lens positioned at a focal distance from the tip of the optical fiber, the collimator lens configured to match the numerical aperture of the optical fiber to couple light out of the optical fiber; transmitting light from the light source into the fiber core to provide uniform illumination of the object; A method comprising:

20. further comprising modulating the laser to induce a wavelength shift.

20. The method of claim 19.

21. further comprising vibrating the optical fiber.

21. The method of claim 19 or 20.

22. further comprising imaging the object using a detection module.

22. The method of claims 19 to 21.

Images