Method and apparatus for optimized coherent scattering microscopy - Patents.com

Abstract

The present application discloses a method and apparatus for imaging a sample by coherent scattering microscopy, the method comprising the steps of illuminating a sample with at least one coherent light source, the sample being held at a sample position including an interface having a refractive index change, illuminating the sample with illumination radiation to generate a counter-propagating signal from the sample including light reflected at the interface and light scattered by the sample; splitting the counter-propagating signal into a first signal and a second signal; modifying the second signal using a modifying element so that the second signal differs from the first signal; directing the first signal and the second signal to a first detector and a second detector to generate a first image and a second image, respectively; and comparing the first image and the second image by a processor to determine one or more properties of the sample.

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for optimized interferometric scattering microscopy (herein referred to as iSCAT). [Background technology]

[0002] iSCAT has emerged as a powerful approach for both single particle tracking with unique spatiotemporal resolution and label-free sensitivity down to the single molecule level. iSCAT has been disclosed, for example, in Kukura et al., "High-speed nanoscopic tracking of the position and orientation of a single virus," Nature Methods 2009 6:923-935, and Ortega Arroyo et al., "Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy," Physical Chemistry Chemical Physics 2012 14:15625-15636.

[0003] Despite its considerable potential, the widespread application of iSCAT is limited by the requirement for custom microscopes, unconventional cameras and complex sample illumination, limiting iSCAT's ability to reliably and accurately detect, image and characterize objects as small as single molecules.

[0004] In our previous patent WO2018 / 011591, we disclosed an interferometric scattering microscope that includes a novel contrast-enhancing spatial mask configured to improve the relative amplitudes of the reference and scattered light fields. The microscope described therein can achieve similar sensitivity to conventional iSCAT techniques, but with dramatically reduced implementation complexity and cost, to the extent that conventional microscopes can be configured to perform iSCAT through simple modification and inclusion of a spatial mask.

[0005] However, a number of limitations to the measurement sensitivity achievable using that approach have become apparent to the present inventors. First, background variations in illumination, due for example to laser noise or other interference phenomena, often cannot be removed, as these variations may carry information about the sample under examination. Such background signatures limit the detection sensitivity.

[0006] Second, the intensity of light that can illuminate the sample under investigation is limited by detector characteristics such as full-well capacity and readout speed. Thus, when the illumination intensity is high, strong spatial masks must be used to reduce the amount of light reaching the detector, limiting the mass range and making the instrument more sensitive to external influences.

[0007] Third, such microscopes measure sample interactions or events only once at the same instant in time, making it impossible to draw correlations with particle detection algorithms for single molecule events.

[0008] Finally, in applications where the device is used for mass detection of samples (mass spectrometry), the dynamic range of the measurement is limited by the selected intensity of the spatial mask, meaning that it is necessary to optimize the device for either high or low mass detection.

[0009] The present invention provides an optimized method and apparatus for iSCAT technology that addresses each of the limitations described above. The method disclosed herein approaches these limitations by manipulating the image signal at the detection side of the apparatus after a portion of the illuminating light has been scattered by the sample. Summary of the Invention [Means for solving the problem]

[0010] According to one aspect of the invention, there is provided a method for imaging a sample by coherent scattering microscopy. The method includes the steps of illuminating a sample with at least one coherent light source, where the sample is held at a sample position including an interface having a refractive index change, illuminating the sample with illumination radiation to generate a counter-propagating signal from the sample including light reflected from the interface and light scattered by the sample; splitting the counter-propagating signal into a first signal and a second signal; modifying the second signal using a modifying element such that the second signal differs from the first signal; directing the first signal and the second signal to a first detector and a second detector to generate a first image and a second image, respectively; and comparing, by a processor, the first image and the second image to determine one or more properties of the sample.

[0011] The optional spatial filter is configured to provide an attenuation of the intensity of the incident radiation, where the attenuation of the intensity is greater within a given numerical aperture. Thus, the spatial filter exploits the mismatch between the numerical aperture of the reflected illumination light and the numerical aperture of the light scattered from objects within the sample at the sample location to selectively reduce the intensity of the illumination light over the scattered light. In this way, the spatial filter exploits the different directionality of these two light sources. The reflected illumination light typically has a relatively small numerical aperture, while objects of sub-diffraction size near the surface of the sample scatter light preferentially to high numerical apertures. Thus, the attenuation of intensity by the spatial filter at low numerical apertures mainly affects the illumination light and only minimally affects the scattered light, thereby maximizing image contrast.

[0012] By splitting the counterpropagating signals, two images of the sample can be acquired from the same instant in time, which has many advantages, including the ability to probe the sample without time-dependent intensity variations, such as misalignment of illumination sources, introduced by sequential image acquisition.

[0013] It is also possible to characterize a sample, such as its mass in a mass spectrometer experiment, by independently modifying one of the two split signals before it reaches the detector. This can also be done to remove background noise signatures and improve measurement sensitivity.

[0014] In some embodiments, the at least one coherent light source includes a first laser and a second laser, and the beams are combined prior to illumination of the sample. Providing multiple light sources increases the photon flux at the sample, making it more practical to beam-split the reflected signal into two signals, each of which contains enough information to independently form an image of the sample.

[0015] In some embodiments, splitting the signal includes splitting the counterpropagating signal into two signals having orthogonal polarizations. By separating the image channels into two orthogonal polarizations, birefringence properties of the sample can be investigated.

[0016] In some embodiments, splitting the signal includes splitting the counterpropagating signal into two signals having different optical powers. By separating the image channel into two signals having different optical powers, a different balance between the reference beam and the scattered light from the sample can be achieved.

[0017] In some embodiments, passing at least one of the first signal and the second signal through a spatial filter comprises passing the first signal through a first spatial filter, and modifying the second signal such that the second signal differs from the first signal comprises passing the second signal through a second spatial filter, and the first spatial filter and the second spatial filter are each configured to provide an attenuation of the intensity of the incident radiation, the attenuation of the intensity being greater within a predetermined numerical aperture, the first signal having a greater optical power than the second signal, and the second spatial filter exerting a greater intensity attenuation on the incident radiation than the first spatial filter. The first spatial filter and the second spatial filter may each be configured to provide an attenuation of the intensity of the incident radiation, the attenuation of the intensity being greater within a predetermined numerical aperture. The first signal may have a greater optical power than the second signal, and the second spatial filter may exert a greater intensity attenuation on the incident radiation than the first spatial filter.

[0018] In conventional setups, the use of a single "catch-all" spatial filter limits the dynamic range of the measurement, forcing the instrument to be optimized for either high or low mass. A high-intensity mask optimizes image capture for particles with low molecular weight, while a weaker mask or no mask is sufficient for particles with high molecular weight. Splitting the reflected signal into two signals of different intensity (e.g. 90:10) and applying spatial filters of appropriate intensity (e.g. 0.1% and 1%) to each signal allows a broader mass spectrum to be interrogated, with one split arm optimized for low mass and another for high mass.

[0019] In some embodiments, directing the first and second signals to the first and second detectors includes adjusting the phase of the second signal relative to the first signal. Adjusting the phase of the second signal relative to the first signal may include passing the second signal through a phase shift mask and / or passing the second signal through an imaging lens at an appropriate location along the optical path of the second signal. The second signal may be adjusted by half a wavelength of the illumination radiation relative to the first signal.

[0020] Some variations in sample illumination (e.g., system reflectivity changes, buffer contributions, secondary reflections, laser noise and other interference phenomena) could not be removed in conventional setups. Such background signatures limit the ability to remove noise from the setup, since it is not known whether the variations are due to noise or to light scattered from the sample.

[0021] By independently adjusting the phase of the first or second signal and comparing two simultaneously generated images (advantageous as defined in relation to claim 1), it is possible to highlight uncorrelated variations in the two images and classify them as not originating from the image plane.

[0022] Doing so provides a second measurement with which to correlate images of single-molecule events, but with conventional setups they could only process with a single measurement per instant in time, meaning that deriving correlations would be difficult or impossible.

[0023] In some embodiments, the step of modifying the second signal comprises modifying the second signal by: Second In some embodiments, the method includes passing the first signal through an optical element configured to apply asymmetric magnification to the second signal along a first dimension corresponding to an x-dimension of a pixel grid of the detector. In some embodiments, the first signal may be passed through an optical element configured to apply asymmetric magnification to the first signal along a second dimension, the second direction being orthogonal to the first direction and corresponding to a y-dimension of the pixel grid of the second detector.

[0024] The amount of light available in conventional setups is limited by the readout of the digital camera, necessitating the use of stronger spatial filters, which result in greater sensitivity to vibrations and a wider mass distribution recorded in mass spectrometry. By splitting the signal in two and performing one-dimensional magnification processing on each signal in the x and y dimensions, the camera can receive the same number of photons with the same or more pixels and, more importantly, can be positioned in a way that optimizes the readout speed. This equates to the ability to process a higher flux of detected photons without increasing the readout speed of the digital camera.

[0025] In some embodiments, the at least one coherent light source is configured to provide illumination light having at least two different inspection wavelengths, and splitting the signal includes splitting the signal by wavelength such that light having a first inspection wavelength is directed to a first detector and light having a second inspection wavelength is directed to a second detector.

[0026] By measuring the same sample at two different probing wavelengths, it is possible to identify measurement variations caused by imperfect components in the optical system, or due to the sample absorbing at one wavelength but not another (e.g. due to a fluorescent tag or another dye added to the sample that affects scattering, or due to absorption at one wavelength on a coating on the sample carrier).

[0027] In some embodiments, the predetermined numerical aperture is the same as or similar to the numerical aperture of the illumination light reflected from the sample location in the counterpropagating signal, thereby maximizing the image contrast effect provided by the spatial filter.

[0028] In some embodiments, modifying the second signal comprises phase shifting, magnification in the first direction, or spatial filtering with a greater strength than the spatial filtering applied to the first signal.

[0029] According to another aspect of the invention, there is provided an interferometric scattering microscope configured to perform the method of any preceding claim, the microscope including a sample location including a reflective surface, at least one coherent light source configured to illuminate the sample location, a first detector and a second detector, a beam splitter configured to split a counter-propagating signal from the sample location into a first signal and a second signal, and a modifying element configured to modify the second signal such that the second signal differs from the first signal, the system configured to direct the first signal and the second signal to the first detector and the second detector, respectively.

[0030] In some embodiments, the coherent scattering microscope further includes at least one spatial filter positioned to filter at least one of the first signal and the second signal, the spatial filter configured to provide greater attenuation of the intensity of the incident radiation within a given numerical aperture.

[0031] The present invention relates to -15 m 2 This can be advantageously applied to samples containing objects with a scattering cross section for the illumination light of 10 -26 m 2 Greater than or equal to 10 -15 m 2 From 10 -26 m 2 The scattering cross section for the illumination light may also be in the range of 100 to 150. Examples of objects that can be studied include proteins or small aggregates thereof, as well as metallic, organic or inorganic nanoparticles.

[0032] To image objects that are very weak scatterers, spatial filters are used to measure the intensity within a given numerical aperture to within 10 times the incident intensity. -2 The spatial filter is arranged to pass output light attenuated by, for example, 10 times the incident intensity. -2 From 10 -4 In the range of -4 The optical fiber 10 may be arranged to pass more or less attenuated output light.

[0033] For a better understanding, embodiments of the invention will now be described, by way of non-limiting example, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 is a schematic diagram of a prior art iSCAT microscope. [Diagram 2] FIG. 1 is a schematic diagram of an improved version of a prior art iSCAT microscope. [Figure 3a] FIG. 1 illustrates an exemplary embodiment of an iSCAT microscope according to the present invention. [Figure 3b] FIG. 1 illustrates an exemplary embodiment of an iSCAT microscope according to the present invention. [Figure 4] FIG. 1 illustrates an exemplary embodiment of an iSCAT microscope according to the present invention. [Diagram 5] FIG. 1 illustrates an exemplary embodiment of an iSCAT microscope according to the present invention. [Figure 6A] 5A-5C show experimental results obtained using the method and apparatus according to the invention. [Figure 6B] 5A-5C show experimental results obtained using the method and apparatus according to the invention. [Figure 6C] 5A-5C show experimental results obtained using the method and apparatus according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] In the systems and methods described herein, the light used may be ultraviolet (which may be defined herein as having a wavelength in the range of 10 nm to 380 nm), visible light (which may be defined herein as having a wavelength in the range of 380 nm to 740 nm), or infrared (which may be defined herein as having a wavelength in the range of 740 nm to 300 μm). The light may be a mixture of wavelengths. As used herein, the terms "optical" and "optical" are used generally to refer to the light to which the methods are applied.

[0036] 1 and 2 show a diagram of an iSCAT microscope configuration disclosed in WO2018 / 011591, which has many structural features and functionality in common with the devices and methods of the present invention.

[0037] The disclosure of WO2018 / 011591 is incorporated herein by reference, but for the sake of completeness, the following description sets out the configuration and functionality of the iSCAT microscope of the present invention which is common to that of WO2018 / 011591 and shown in Figures 1 and 2, and then describes various improvements of said configuration brought about by the present disclosure and provides example embodiments thereof.

[0038] Thus, with reference to FIG. 1, the microscope 1 includes a sample holder 2 for holding a sample 3 at a sample position. The sample 3 may be a liquid sample containing an object to be imaged, which will be described in more detail below. The sample holder 2 may take any form suitable for holding the sample 3. Typically, the sample holder 2 holds the sample 3 on a surface that forms an interface between the sample holder 2 and the sample 3. For example, the sample holder 2 may be a cover slip and / or may be made from glass. The sample 3 may be provided on the sample holder 2 in a simple manner, for example by means of a micropipette.

[0039] The microscope 1 further comprises an illumination source 4 and a detector 5. The illumination source 4 is arranged to provide illumination light. The illumination light may be coherent light. For example, the illumination source 4 may be a laser. The wavelength of the illumination light may be selected depending on the nature of the sample 3 and / or the nature to be examined. In one example, the illumination light has a wavelength of 405 nm.

[0040] Optionally, the illumination light may be spatially modulated to eliminate speckle patterns arising from the coherent nature of the illumination and laser noise, as detailed, for example, in Kukura et al., "High-speed nanoscopic tracking of the position and orientation of a single virus," Nature Methods 2009 6:923-935.

[0041] A detector 5 receives the output light upon reflection from the sample location. The illumination light that reaches the detector is primarily reflected from the surface of the sample, typically the interface between the sample and the sample holder, resulting in interference with objects in the sample close to that surface.

[0042] In the example where a glass-water interface is used, a relatively small amount of the illumination light is reflected (typically only 0.5%), but a very large amount (typically over 90%) is scattered back towards the illumination light by nanoscopic objects at the interface. This essentially improves the ratio of scattered to reflected light by more than 1000 times compared to a transmission geometry, thus increasing the contrast of the interferometer. As a result, for a given scatterer, illumination intensity, and exposure time, the number of photons that need to be detected to achieve the same nominal S / N is three orders of magnitude lower than in a transmission setup.

[0043] Typically, the microscope 1 may operate in a wide-field mode, in which case the detector 5 may be an image sensor that captures an image of the sample 3. The microscope 1 may alternatively operate in a confocal mode, in which case the detector 5 may be an image sensor or a point-like detector such as a photodiode, in which case a scanning arrangement may be used to scan an area of ​​the sample 3 to build up an image. Examples of image sensors that may be employed as the detector 5 include CMOS (complementary metal oxide semiconductor) image sensors or CCDs (charge-coupled devices), etc.

[0044] The microscope 1 further comprises an optical system 10 disposed between the sample holder 2, the illumination source 4 and the detector 5. The optical system 10 is arranged to direct illumination light to the sample position to illuminate the sample 3, to collect output light upon reflection from the sample position and to direct the output light to the detector 5 as follows:

[0045] The optical system 10 includes an objective lens 11, which is a lens system arranged in front of the sample holder 2. The optical system 10 also includes a condenser lens 12 and a tube lens 13. The condenser lens 12 condenses illumination light from a light source 11 (shown by a continuous line in FIG. 1) through the objective lens 11 onto a sample 3 at the sample position.

[0046] The objective lens 11 collects the output light, which includes both (a) the illumination light reflected from the sample position (shown by a continuous line in FIG. 1) and (b) the light scattered from the sample 3 at the sample position (shown by a dotted line in FIG. 1). The reflected light is mainly reflected from the interface between the sample holder 2 and the sample 3.

[0047] Typically, this is a relatively weak reflection, e.g., 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. This scattered light is scattered by objects within the sample 3. The scattered light from objects at or close to the surface of the sample is visible in the image captured by detector 5, as it constructively interferes with the reflected light.

[0048] As shown in Figure 1, the reflected illumination light and the scattered light have different directivities. In particular, the reflected illumination light has a numerical aperture due to the geometry of the light beam output by the light source 4 and the optical system 6. The scattered light satisfies a larger numerical aperture than the reflected illumination light because it is scattered over a larger range of angles. The tube lens 13 focuses the output light from the objective lens 11 onto the detector 5.

[0049] Optical system 10 also includes a beam splitter 14 positioned to split the optical path between the illumination light from light source 4 and the output light directed to detector 5. Beam splitter 14 may have any conventional structure that provides partial reflection and partial transmission of light incident thereon.

[0050] In an embodiment of the present disclosure, the light source 4 is offset from the optical path of the objective lens 11 such that illumination light from the light source 4 is reflected by the beam splitter 14 to the objective lens 11, and conversely, the detector 5 is aligned with the optical path of the objective lens 11 such that output light from the sample position is transmitted through the beam splitter 14 towards the detector 5.

[0051] In addition to the above-mentioned components, which may be of conventional construction, microscope 1 includes a spatial mask or filter 20. In the example of Figure 1, spatial filter 20 is formed on beam splitter 14 and is thereby positioned behind the back aperture of objective lens 11 and thus just behind the back focal plane 15 of objective lens 11, although spatial filter 20 may be positioned at other points along the optical path of the iSCAT microscope to achieve the same effect as described below.

[0052] Spatial filter 20 is positioned to filter the counter-propagating output light passing from the sample holder interface to detector 5. In the embodiment of the present disclosure in which detector 5 is aligned with the optical path of objective lens 11, spatial filter 20 is therefore transmissive.

[0053] The spatial filter 20 is partially transmissive, allowing the output light, including the reflected illumination light, to pass, but at a reduced intensity, and is aligned with the optical axis and has a predetermined aperture, providing intensity reduction within a predetermined numerical aperture, where numerical aperture is defined in its usual manner as being a dimensionless quantity that characterizes the range of angles relative to the sample position from which the output light originates.

[0054] Specifically, the numerical aperture NA can be defined by the following equation: NA=n·sin(θ), where θ is the collection half angle and n is the refractive index of the material through which the output light passes (e.g., the material of a component of optical system 10).

[0055] The spatial filter 20 may be formed in any suitable manner and typically includes a layer of deposited material. The material may be, for example, a metal such as silver. In some embodiments, the spatial filter may include one or more dielectric coatings. In some embodiments, the spatial filter may be formed to be partially reflective to incident radiation within a predetermined range of angles. The deposition may be performed using any suitable technique.

[0056] Because sub-diffraction magnitude objects near an interface preferentially scatter light to larger numerical apertures than to the reflected illumination light, the intensity reduction provided by spatial filter 20 preferentially reduces the detected intensity of the reflected illumination light over the scattered light. Thus, the intensity reduction provided by spatial filter 20 at low numerical apertures primarily affects the reflected illumination light and minimally affects the scattered light, thereby maximizing contrast in the captured image. The increased imaging contrast allows objects that are weak scatterers to be detected with high contrast.

[0057] The contrast enhancement can be understood as follows: since the spatial filter 20 passes a portion of the output light for a given numerical aperture (i.e., is partially transmissive in this example), a fraction of the illumination light and scattered light fields reach the detector and interfere for a sufficiently coherent illumination source. The light intensity Idet reaching the detector is then given by:

[0058] Idet=│E inc │ 2 {r 2 t 2 +│s│ 2 +2rt│s│cosΦ} Here, E inc is the incident light field, r 2is the reflectance of the interface, t 2 is the transmittance of the spatial filter 20, s is the scattering amplitude of the object, and Φ is the phase difference between the transmitted illumination light and the scattered light.

[0059] The additional filtering provided by spatial filter 20 is not fixed by the reflectance of the glass-water interface as in standard iSCAT, but by the transmittance t 2 The choice of t makes it possible to directly adjust the amplitude of the reference field. If the spatial filter 20 is a layer of deposited material, the transmittance t 2 can be selected by the choice of material and / or thickness of the layer. Such adjustments can be made depending on, for example, the scatterers of interest, the full well capacity of the camera, and the magnification.

[0060] Bright field illumination redirects the strongest unwanted back reflections, which usually come from the objective lens, away from the detector 5, minimizing imaging background and allowing a large field of view without complex scanning of the illumination light beam.

[0061] To image objects that are relatively weak scatterers, spatial filter 20 may be configured to, within a given numerical aperture, filter 20 to filter out 10% of the incident intensity (in this context, the intensity of the output light incident on spatial filter 20). -2 From 10 -4 The illumination filter may be arranged to pass reflected illumination light having an intensity reduced to within a range of .

[0062] For example, samples containing objects having a mass of 5000 kDa or less can be imaged. Typically, the disclosed techniques are capable of imaging objects having a mass of 10 kDa or more, such as objects having a mass in the range of 10 kDa to 5000 kDa, and / or objects having a mass of 10 kDa or more. -12 m 2 Less than 10, more preferably -17 m 2 The scattering cross section of the illumination light may be applied to a sample containing an object having a scattering cross section for the illumination light of, for example, 10 -17 m 2 ~10 -26 m 2The scattering cross section for the illuminating light may also be in the range of 0.01 to 0.05. Examples of objects that can be imaged using the techniques of the present disclosure include proteins or small aggregates thereof, or their binding partners.

[0063] To simultaneously image stronger scatterers, the transmittance of the second filter can be set to 1–10, depending on the desired detection range. -2 It can be set to any range between With reference to Figure 2, there is shown a second example configuration of the microscope 1. The configuration of Figure 2 is also disclosed in WO2018 / 011591 and is similarly suitable for optimization by application of the techniques of the present invention.

[0064] 2 positions the spatial filter 20 not behind the rear aperture of the objective lens 11, but at a conjugate focal plane 21 of the back focal plane 15 of the objective lens 11. The conjugate focal plane 21 of the back focal plane 15 of the objective lens 11 is formed between a pair of telescope lenses 22, 23 positioned behind the tube lens 13.

[0065] An acousto-optical deflector 32 is positioned after the light source 4 to scan the illumination light. The acousto-optical deflector 32 can be operated to scan an area of ​​the sample 3 to build up an image and / or to provide spatial modulation to remove speckle patterns resulting from the coherent nature of the illumination and laser noise as described above.

[0066] The collecting lens 12 is replaced by a pair of telecentric lenses 30 and 31 whose function is to image any beam path modifications at the acousto-optic deflector 32 into the back focal plane of the imaging objective.

[0067] The positions of the light source 4 and detector 5 are reversed with respect to the configuration of FIG. 1 such that illumination light from the light source 4 is transmitted through the beam splitter 14 to the objective lens 11 and conversely output light from the sample position is reflected by the beam splitter 14 towards the detector 5.

[0068] The beam splitter 14 is a polarizing beam splitter, and a quarter-wave plate 33 is disposed between the beam splitter 14 and the sample 3 so that the beam splitter 14 splits the light. Also, a mirror 34 is disposed to deflect the outgoing light reflected by the beam splitter 14.

[0069] 3-5, the following description focuses on various improvements provided by the present invention to the above-described configuration of iSCAT microscope 1. The use of spatial filter 20 does not fundamentally change the achievable sensitivity limit or SNR (signal-to-noise ratio) for a given object, incident light intensity, and exposure time, but it is possible to improve sensitivity by manipulating the back-propagating signal on the detection side of the device after some of the illuminating light has been scattered by the object.

[0070] In the example configuration shown in Figures 3-5, the counter-propagating signal is manipulated by passing it through a second beam splitter 36 configured to split the counter-propagating signal into first and second signals, which are then directed to first and second detectors 5 and 6, respectively. In this way, two separate image signals can be captured for a given object at the same instant in time and processed through separate image channels. This allows the light scattered by the sample to be investigated by image comparison without introducing time-dependent intensity variations from the illumination source or changes to the optical system 10.

[0071] Furthermore, by independently modifying at least one of the two split signals from beam splitter 36 before they reach the detector, additional properties of the sample can be probed, increasing the sensitivity of the measurement. For example, background noise signatures can be removed from the illumination, and the first and second signals can be adjusted relative to one another and combined as described below to provide more information about the sample.

[0072] Beam splitter 36 is configured to split the counterpropagating signal in a manner dependent on which characteristic of the sample / counterpropagating signal is under investigation. For example, in some embodiments where it is desired to split the signal by optical power, the beamsplitter 36 may include a plate with a membrane, which may be metallic or dielectric, positioned at 45° to the optical path, or alternatively, the beamsplitter 36 may be a cube beamsplitter formed by a matched pair of prisms with partially reflective membranes at the interfaces between the prisms.

[0073] In some embodiments, beam splitter 36 is a polarizing beam splitter used in combination with a quarter wave plate between beam splitter 36 and sample 3 and configured to split the counter-propagating signal into orthogonally polarized first and second signals. Such a configuration allows for analysis of the birefringence properties of the sample by detectors 5 and 6.

[0074] In some embodiments, the illumination light includes at least two different inspection wavelengths for illuminating the sample, and the beam splitter 36 is configured to split the counter-propagating signal by wavelength such that light having a first inspection wavelength is directed to a first detector 5 and light having a second inspection wavelength is directed to a second detector 6.

[0075] By measuring the same sample at two different probing wavelengths, it is possible to identify intensity changes due to wavelength-dependent components in the optical system. Such a setup can also identify intensity changes caused by a sample absorbing light at one wavelength but not the other, for example, by adding a fluorescent tag or another dye to the sample that affects scattering, or because one wavelength is absorbed by a coating on the sample support.

[0076] The optimization methods described herein are complementary and may be used separately or in combination with one another. Thus, in some embodiments, beam splitter 36 includes multiple beam splitters, each configured to split the signal in a different or the same manner, such that the counterpropagating signal is split into three or more signals, each of which may then be directed to a separate detector.

[0077] With reference to FIG. 3a, an example optimized configuration of the microscope 1 based on the optical configuration of FIG. 1 is illustrated. In addition to the optical configuration of FIG. 1, the configuration of FIG. 3a includes a second detector 6 and a second beam splitter 36 disposed along the optical path of the counter-propagating signal between the tube lens 13 and the detectors 5 and 6 and configured to split the counter-propagating signal into first and second signals.

[0078] In the illustrated example, the first signal passes directly to detector 5, while the second signal is directed via reflective element 38 through signal modifying element 40 before impinging on second detector 6. The signal modifying element 40 is selected depending on the type of image signal optimization desired.

[0079] In one example optimization approach, the beam splitter 36 splits the counterpropagating signal into first and second signals with equal optical power, and the correction element 40 includes a retarding mask configured to phase-shift light scattered by an object held at the sample position in the second signal by π / 2 without affecting the phase of the illumination light, such that the image of the sample in the second signal is inverted and scattered light that would otherwise interfere constructively now interferes destructively.

[0080] Since the scattered light in the first signal is not out of phase, the scattered light constructively interferes as in conventional iSCAT. Thus, by comparing the images acquired with detectors 5 and 6, intensity variations in the images due to scattering from the sample will be correlated, and intensity variations that are uncorrelated between the two images can be identified as being due to background signatures, for example imperfections in the illumination light.

[0081] The identification of such background signatures in iSCAT images obtained by independent "phasing" of the scattered light in the split signals makes it possible to remove the signatures and thus improve the sensitivity of the microscope 1.

[0082] In the alternative, the signal-modifying element 40 includes an imaging lens, and by moving said imaging lens to an appropriate point along the optical path of the second split signal between the second beam splitter 36 and the second detector 6, the phase of the scattered light is instead inverted. Such a configuration can provide a "tuning" effect on the phase of the scattered light similar to that provided by a phase-shifting mask. This is due to the unfocused nature of the illumination light from the illumination source 4 and the highly focused nature of the light scattered by the sub-diffraction-limited sized sample 3. This effect can be understood as explained below.

[0083] If a parallel beam of light is focused to a point in space by a perfect lens / focusing element, the phase of the beam measured opposite the focus at a distance equal to the focal length of the focusing element will experience a phase shift of π. At the exact point of focus, the phase shift is π / 2. This phase shift is called the Guy phase shift, and has an approximately linear dependence on the distance from the focus.

[0084] In reality, there is no focusing element that can focus a beam to a single point in space, and the strength of the phase change versus distance dependence of a given light beam is based on how well focused the beam is. Weakly focused light, such as the illumination light from illumination source 4, is almost unaffected by Guy phase shift. In contrast, single molecules or very small samples, such as those inspected by the iSCAT microscope of the present disclosure, have a size below the diffraction limit of the illumination light and therefore act as nearly perfect focusing elements. Thus, the scattered light of the counterpropagating signal has a high Guy phase dependence along its optical path, whereas the illumination light does not.

[0085] In the case of the iSCAT microscope, both the scattered and reflected illumination light undergo an additional phase change due to the refractive index change at the sample position interface / coverslip before passing through beam splitter 36 and being imaged onto detectors 5 and 6. In this optical setup, adjustment of the Guy phase shift of the scattered light can be made without affecting the weakly focused illumination light, by changing the position of the imaging lens, in this example correction element 40, to emulate changes in distance from the focus.

[0086] Thus, the phase of the scattered light can be changed without changing the phase of the illuminating light, and the image of the sample received at the detector 6 can be inverted, as described in connection with the phase shift mask.

[0087] FIG. 3a depicts an arrangement in which there are two lasers 4a, 4b that are combined via a beam splitter 4c to form a more intense illumination beam. Although this embodiment is shown and described as an optimization of the configuration of FIG. 1, it may also be implemented as an optimization of the configuration of FIG.

[0088] Figure 3b shows an alternative arrangement to Figure 3a, except without the spatial filter 20. The beam is still split, and a modifying element 40 is placed in the path of the second split signal. The modifying element may magnify, phase shift, or spatially filter the second split signal to increase the sensitivity of the device.

[0089] Referring to FIG. 4, an example optimized configuration of the microscope 1 based on the optical configuration of FIG. 2 is shown. In addition to the optical components of Figure 2, the configuration of Figure 4 includes a second detector 6 and a second beam splitter 36 disposed along the optical path of the counter-propagating signal between the tube lens 13 and the detectors 5 and 6 and configured to split the counter-propagating signal into first and second signals. In this configuration, one or more spatial filters 20 are disposed between the beam splitter 36 and the detectors 5, 6.

[0090] In the illustrated example, the beam splitter 36 asymmetrically splits the counter-propagating signal into first and second signals having different optical powers: the first signal passes through the first spatial filter 20 to the detector 5, and the second signal is directed via a reflecting element 38 through a signal modifying element 40, which is a second spatial filter having a different strength than the first spatial filter, before impinging on the second detector 6.

[0091] In mass photometry experiments, where the iSCAT microscope is used to measure the mass of sub-diffraction-limited objects held at the sample position, the image signal quality typically needs to be optimized for a given mass range, since weak scatterers require higher optical power in the illumination and therefore a larger amount of illumination light is attenuated by the spatial filter 20 than strong scatterers.

[0092] By splitting the counterpropagating signal asymmetrically and passing the first and second signals through spatial filters with different intensities as described above, first and second signals, respectively, can be generated that have different balances between the illumination light (reference beam) and the scattered light from the sample in the image plane (image signal). Thus, the first and second images can be acquired by detectors 5 and 6, respectively, optimized for either high-mass or low-mass object measurements. This expands the dynamic mass range measurable by microscope 1.

[0093] For example, the beam splitter 36 may be configured such that the first signal has a greater optical power than the second signal, in which case the second spatial filter applies a greater intensity attenuation to the incident radiation than the first spatial filter. The ratio of optical power between the first and second signals may be, for example, 90:10, in which case a spatial filter of appropriate strength would be used, for example a spatial filter with an attenuation of 0.1% and 1%, respectively. This ratio can be adjusted depending on the desired detection range in either direction, for example 99:1 with an attenuation of 0.01 and 10%, respectively. Ideally, the combination of optical power ratio and attenuation is selected to maintain the same optical intensity in both imaging channels at all times.

[0094] 4 depicts an optional acousto-optical deflector (AOD) 27 in the path of the illumination beam. The acousto-optical deflector is positioned after the light source to provide scanning of the illumination light. The acousto-optical deflector 27 can be operated to scan an area of ​​the sample 3 to build an image and / or to provide spatial modulation to remove speckle patterns resulting from the coherent nature of the illumination and laser noise as described above.

[0095] Although this embodiment is described with reference to a configuration based on the configuration of FIG. 2, it should be appreciated that this optimization method may equally be implemented based on the configuration of FIG. Referring to FIG. 5, another exemplary embodiment based on the optical configuration of FIG. 1 is illustrated.

[0096] The amount of light available in the conventional setup was limited by the digital camera readout, necessitating the use of stronger spatial filters 20, resulting in increased sensitivity to vibrations and a wider range of mass distributions recorded in mass photometry.

[0097] Similar to the configuration of FIG. 3, FIG. 5 includes a second detector 6 and a second beam splitter 36 disposed along the optical path of the counter-propagating signal between tube lens 13 and detectors 5 and 6 and configured to split the counter-propagating signal into first and second signals.

[0098] However, in this embodiment, instead of passing directly to the detector 5, the first signal passes through a first magnifying element 42 configured to magnify the first signal along a first axis.

[0099] In the embodiment of FIG. 5, the second signal is directed via the reflecting element 38 through a signal-modifying element, in this case a second magnifying element 43 configured to magnify the second signal along an axis perpendicular to the first axis.

[0100] Detectors 5 and 6 are also oriented differently from each other. Conventional pixel-based detectors tend to have one electronic readout direction that is faster than the other due to the fact that pixel excitation is processed column-by-column on a grid. Detectors 5 and 6 are therefore oriented such that the direction in which the camera readout electronics can process pixel excitation fastest is aligned with a first axis for detector 5 and a second axis for detector 6.

[0101] Using the above described configuration, it is possible to improve the speed at which photons scattered from the sample are processed by improving the matching of detected photons to the digital camera readout without using expensive specialized detectors. For example, detector 5 may read out only along one X-column where the first magnifying element 42 magnifies the photon flux from the first signal, and detector 6 may read out only along one Y-column where the second magnifying element 43 magnifies the photon flux from the second signal.

[0102] It is then possible to map the pixels of detectors 5 and 6 to each other and assign appropriate XY positions to both cameras so that the images acquired from detectors 5 and 6 can be blended together. In some cases, distortions in the blended image need to be corrected. Overall, however, the above method allows a conventional detector to increase the speed at which images can be captured by a number proportional to the magnification provided by the first and second magnifying elements, with little loss in image quality.

[0103] Thus, the described embodiment is det As can be seen from the equation, compensating for the reduction in the total number of photons in the first and second signals due to the spatial filter 20 which enhances the scattering contrast in the acquired image at the expense of the total number of detected photons.

[0104] Although this embodiment is shown and described as an optimization of the configuration of FIG. 1, it may also be implemented as an optimization of the configuration of FIG. Furthermore, in some implementations of the embodiment (not shown), only the first signal is passed through a magnification element, while the second image channel is viewed in its normal spatial distribution. In such configurations, the second signal may be passed through another mask or a general intensity filter instead of a magnification element. In such implementations, the second channel maintains full spatial resolution and can be used for localization.

[0105] The optimization methods described above in relation to Figures 3 to 5 may be used in combination with each other or independently. Additionally, since the counterpropagating signal is split into first and second signals by beam splitter 36 having a lower photon flux, each of the described optimization methods may be facilitated by the provision of a higher intensity illumination source 4. In some embodiments, the higher intensity illumination source 4 is provided by combining the beams of a first and second laser, as illustrated in FIG.

[0106] Referring to FIG. 6A, an exemplary experimental result, a two-channel mass spectrometry ratiometric image using one tip, shows two images of the same sample mixture taken at the same time using a beam splitting configuration similar to the iSCAT microscope of FIG. 5 described above, but without the magnification element.

[0107] Referring to Figure 6B, another example experimental result is displayed showing images obtained using another iSCAT microscope configuration similar to that of Figure 5, including a magnification element configured to elongate one of the split beams along a single dimension. As can be seen in Figure 6B, the image of the sample mixture has been magnified / stretched along the x dimension.

[0108] Referring to FIG. 6C, the same experimental results as shown in FIG. 6B are displayed, but the stretched dimension pixels are recombined into one using 1D binning, proving that image content and resolution are not lost by this procedure, but rather photons can simply be spread over multiple pixels and then recombined.

[0109] The microscope 1 can be used to perform iSCAT for a wide range of applications, including single molecule detection. In particular, for label-free imaging applications of weak scatterers, the object of interest must necessarily be detected over a large background, resulting in poor imaging contrast. The microscope 1 can be used for a wide range of studies and measurements, for example, measurements of any change in refractive index, including single molecule binding / desorption, phase transitions, clustering, assembly / disassembly, aggregation, protein / protein interactions, protein / small molecule interactions, and high-sensitivity label-free imaging.

[0110] Thus, Microscope 1 has numerous applications, from fundamental research to industrial applications such as the pharmaceutical industry. For example, iSCAT is currently the world's most sensitive label-free single molecule imaging biosensor, which could have a major impact on the surface plasmon resonance sensing market, for example. As mentioned above, Microscope 1 can also be used for mass measurements, acting as an accurate, precise and high-resolution single molecule mass spectrometer in solution, which has many applications in research and industry.

Claims

1. 1. A method for imaging a sample by coherent scattering microscopy, comprising: illuminating a sample with at least one coherent light source, the sample being held at a sample position including an interface having a refractive index change, illuminating the sample with illumination radiation to generate a counter-propagating signal from the sample including light reflected from the interface and light scattered by the sample; splitting the counterpropagating signal into a first signal and a second signal, such that each of the first signal and the second signal includes at least a portion of the light scattered by the sample; modifying the second signal with a modifying element such that the second signal differs from the first signal, where modifying the second signal includes expanding the second signal along a first dimension or spatial filtering with a greater strength than the spatial filtering applied to the first signal; directing the first signal and the second signal to a first detector and a second detector to generate a first image and a second image, respectively; comparing, by a processor, the first image and the second image to determine one or more properties of the sample. A method comprising:

2. passing at least one of the first signal and the second signal through a spatial filter, the spatial filter configured to cause an attenuation of an intensity of incident radiation, the attenuation of intensity being greater within a predetermined numerical aperture. The method of claim 1.

3. the at least one coherent light source includes a first laser and a second laser; the beams of the first laser and the second laser are combined prior to illumination of the sample; The method according to claim 1 or 2.

4. splitting the signal includes splitting the counter-propagating signal into two signals having orthogonal polarizations.

4. The method according to any one of claims 1 to 3.

5. splitting the signal includes splitting the counter-propagating signal into two signals having different optical powers.

5. The method according to any one of claims 1 to 4.

6. passing at least one of the first signal and the second signal through a spatial filter includes passing the first signal through a first spatial filter; Modifying the second signal such that the second signal differs from the first signal includes passing the second signal through a second spatial filter; the first spatial filter and the second spatial filter are each configured to provide an attenuation of an intensity of incident radiation; said attenuation of intensity is greater within a given numerical aperture, the first signal has a greater optical power than the second signal; the second spatial filter exerts a greater intensity attenuation on incident radiation than the first spatial filter; The method according to claim 5.

7. modifying the second signal includes adjusting a phase of the second signal relative to the first signal.

7. The method according to any one of claims 1 to 6.

8. adjusting the phase of the second signal relative to the first signal includes passing the second signal through a phase shift mask. The method of claim 7.

9. conditioning the second signal includes passing the second signal through an imaging lens at an appropriate location along the optical path of the first signal.

9. The method according to claim 7 or 8.

10. adjusting the phase of the second signal relative to the first signal comprises adjusting the phase of the second signal relative to the first signal by half a wavelength of the illumination radiation.

10. The method according to any one of claims 7 to 9.

11. modifying the second signal includes passing the second signal through an optical element configured to magnify the second signal along the first dimension corresponding to an x-dimension of a pixel grid of the second detector.

11. The method according to any one of claims 1 to 10.

12. passing the first signal through an optical element configured to magnify the first signal along a second dimension, the second dimension being orthogonal to the first dimension and corresponding to a y dimension of a pixel grid of the second detector; Further comprising: The method of claim 11.

13. the at least one coherent light source is configured to provide illumination light having at least two different inspection wavelengths; splitting the signal includes splitting the signal by wavelength such that light having a first test wavelength is directed to the first detector and light having a second test wavelength is directed to the second detector.

13. The method according to any one of claims 1 to 12.

14. The method of claim 13, wherein the predetermined numerical aperture is the same as or similar to a numerical aperture of illumination light reflected from the sample location in the counterpropagating signal. The method of claim 2.

15. 15. An interference scattering microscope configured to carry out the method according to any one of claims 1 to 14, comprising: a sample position including a reflecting surface; at least one coherent light source configured to illuminate the sample location; a first detector and a second detector; a beam splitter configured to split a counter-propagating signal from the sample location into a first signal and a second signal, such that each of the first signal and the second signal includes at least a portion of the light scattered at the sample; a modifying element configured to modify the second signal such that the second signal differs from the first signal, where modifying the second signal comprises expanding the second signal along a first dimension or spatial filtering with a greater strength than spatial filtering applied to the first signal; Including, the system is configured to direct the first signal and the second signal to the first detector and the second detector, respectively; Interference scattering microscope.

16. further comprising at least one spatial filter arranged to filter at least one of the first signal and the second signal; the spatial filter is configured to provide an attenuation of the intensity of incident radiation; The attenuation of the intensity of the incident radiation is greater within a given numerical aperture, 16. The interference scattering microscope of claim 15.

Images