Methods and apparatus for optimized interferometric scattering microscopy

Abstract

The present application discloses a method and apparatus for imaging a sample using an interferometric scattering microscope, the method comprising irradiating the sample with at least one coherent light source, the sample being held at a sample position including an interface, the interface having a refractive index change, irradiating the sample with irradiating radiation to generate a reverse propagating signal from the sample, the reverse propagating signal including light reflected at the interface and light scattered by the sample, dividing the reverse propagating signal into a first signal and a second signal, modifying the second signal using a modifying element so that the second signal is different 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 characteristics of the sample.

Description

Technical Field

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

[0002] iSCAT has emerged as a powerful method for single-particle tracking with unique spatiotemporal resolution and label-free sensitivity down to the single-molecule level.

[0003] iSCAT is 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 in Ortega Arroyo et al., “Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy,” Physical Chemistry Chemical Physics, 2012, 14: 15625-15636.

[0004] Despite its great potential, the widespread application of iSCAT is limited by the requirements for custom microscopes, unconventional cameras, and complex sample illumination, thereby restricting iSCAT's ability to robustly and accurately detect, image, and characterize objects as small as single molecules.

[0005] In our previous patent application WO 2018 / 011591, we disclosed an interferometric scattering microscope that includes a novel, contrast-enhancing spatial mask configured to increase the relative amplitudes of the reference and scattered light fields. The microscope described therein achieves similar sensitivity to conventional iSCAT technology, but with significantly reduced complexity and expense, enabling conventional microscopes to function as iSCAT with simple modifications and the inclusion of a spatial mask.

[0006] However, it has become apparent to the inventors of the present application that there are a number of limitations to the measurement sensitivity that can be obtained using this approach.

[0007] First, background fluctuations in the illumination, for example due to laser noise or other interfering phenomena, cannot usually be removed, as these fluctuations may carry information about the sample being interrogated. This background signature limits detection sensitivity.

[0008] Second, the intensity of light that can illuminate the interrogated sample is limited by detector characteristics, including full well capacity and readout speed. Therefore, at higher illumination intensities, a stronger spatial mask must be used to reduce the amount of light reaching the detector, thereby limiting the mass range and making the instrument more sensitive to external influences.

[0009] Third, only a single measurement of a sample interaction or event can be obtained using this microscope at the same time, making it impossible to derive correlations using particle detection algorithms for single molecular events.

[0010] Finally, in applications where the device is used for sample mass detection (mass photometry), the dynamic range of the measurement is limited by the intensity of the chosen spatial mask. This means that the device needs to be optimized for either high-mass or low-mass detection.

[0011] The present invention provides methods and apparatus for optimized iSCAT technology that address each of the above limitations.The methods disclosed herein address these limitations by manipulating the image signal on the detection side of the apparatus after a portion of the illumination light has been scattered by the sample. Summary of the Invention

[0012] According to one aspect of the present invention, a method of imaging a sample by interferometric scattering microscopy is provided.

[0013] The method includes: illuminating a sample with at least one coherent light source, the sample being held at a sample position including an interface, the interface having a refractive index change, illuminating the sample with illuminating radiation to generate a backpropagating signal from the sample, the backpropagating signal including light reflected at the interface and light scattered by the sample; separating the backpropagating signal into a first signal and a second signal; modifying the second signal using a modifying element so that the second signal is different 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 characteristics of the sample.

[0014] The spatial filter is configured to produce an intensity reduction effect on the incident radiation, which intensity reduction is greater within a predetermined numerical aperture. Thus, the spatial filter exploits 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, selectively reducing the intensity of the illumination light compared to the scattered light. Thus, the spatial filter exploits the different directionality of the two light sources. The 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 regions with a high numerical aperture. Consequently, at low numerical apertures, the intensity reduction caused by the spatial filter primarily affects the illumination light and has minimal effect on the scattered light, thereby maximizing imaging contrast.

[0015] Many advantages are provided by separating the counter-propagating signals, which enables two images of the sample to be captured at the same moment in time, allowing the sample to be studied without time-dependent intensity fluctuations (e.g. due to inconsistencies in the illumination light source caused by sequential image acquisition).

[0016] The properties of a sample (e.g. its mass in mass photometry experiments) can also be studied by independently modifying one of the two separate signals before it reaches the detector. This also eliminates background noise signatures and increases measurement sensitivity.

[0017] In some embodiments, the at least one coherent light source includes a first laser and a second laser, and the beams of the first laser and the second laser are combined before irradiating the sample. Providing multiple light sources results in a higher photon flux at the sample, thereby making it more feasible to split the reflected signal into two signals, each of which contains sufficient information to independently form an image of the sample.

[0018] In some embodiments, splitting the signal comprises splitting the counter-propagating signal into two signals having orthogonal polarizations.Splitting the image channel into two orthogonal polarizations enables investigation of birefringence properties of the sample.

[0019] In some embodiments, splitting the signal comprises splitting the counter-propagating signal into two signals having different optical powers.Splitting the image channel into two signals having different optical powers enables achieving different balances between the reference beam and scattered light from the sample.

[0020] 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 so that the second signal is different from the first signal comprises passing the second signal through a second spatial filter, wherein the first spatial filter and the second spatial filter are each configured to produce an intensity reduction effect on incident radiation that is greater within a predetermined numerical aperture, wherein the first signal has greater optical power than the second signal, and wherein the second spatial filter applies a greater intensity reduction to the incident radiation than the first spatial filter. The first spatial filter and the second spatial filter can each be configured to produce an intensity reduction effect on the incident radiation that is greater within a predetermined numerical aperture. The first signal can have greater optical power than the second signal, and the second spatial filter can apply a greater intensity reduction to the incident radiation than the first spatial filter.

[0021] With the previous setup, using a single "catch-all" spatial filter limits the dynamic range of the measurement, forcing the equipment to be optimized for either high or low masses. A high-intensity mask optimizes image capture of particles with low molecular weight, while for particles with high molecular weight, a weak mask or even no mask may be sufficient. Splitting the reflected signal into two signals of different intensities (e.g., 90:10) and applying spatial filters of appropriate intensities (e.g., 0.1% and 1%) to each signal enables interrogation of a wider mass spectrum by optimizing one split arm for low masses and the other split arm for high masses.

[0022] In some embodiments, directing the first and second signals onto 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 includes 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 can be adjusted relative to the first signal by half a wavelength of the illuminating radiation.

[0023] Some fluctuations in sample illumination (e.g., due to reflectivity variations, buffering effects, secondary reflections, laser noise, and other interfering phenomena in the system) could not be eliminated in previous setups. This background signature limited the ability to remove noise from the setup because it was unknown whether the fluctuations were noise or due to scattered light from the sample.

[0024] By independently adjusting the phase of the first or second signal and comparing the two simultaneously generated images, uncorrelated fluctuations in the two images can be highlighted and classified as not originating from the image plane.

[0025] Doing so provides a second measurement for a correlated image of single molecular events, which in previous setups could only be processed using a single measurement at each instant, meaning correlations were difficult or impossible to deduce.

[0026] In some embodiments, modifying the second signal includes passing the second signal through an optical element configured to asymmetrically amplify the second signal along a first dimension, the first dimension corresponding to an x-dimension of the pixel grid of the first detector. In some embodiments, the first signal may pass through an optical element configured to asymmetrically amplify 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.

[0027] In previous setups, the amount of light that could be used was limited by the digital camera's readout, necessitating the use of stronger spatial filters. This, in turn, led to increased sensitivity to vibrations and, in mass photometry, a widening of the recorded mass distribution. By splitting the signal into two and performing one-dimensional amplification on each signal in each of the x and y dimensions, the camera receives the same number of photons in the same or a greater number of pixels and, more importantly, can be arranged in a manner that optimizes readout speed. This translates to the ability to process a higher throughput of detected photons without increasing the digital camera's readout speed.

[0028] In some embodiments, the at least one coherent light source is configured to provide illumination light having at least two different interrogation wavelengths; and separating the signal comprises separating the signal by wavelength such that light having a first interrogation wavelength is directed to the first detector and light having a second interrogation wavelength is directed to the second detector.

[0029] Measuring the same sample with two different detection wavelengths can help identify measurement variations caused by imperfect parts of the optical system, or because the sample absorbs at one wavelength but not another (for example, due to different dyes or fluorescent labels added to the sample that affect scattering, or because a coating on the sample support absorbs one wavelength).

[0030] 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 back-propagating signal. This maximizes the image contrast effect provided by the spatial filter.

[0031] In some embodiments, modifying the second signal includes phase shifting, amplifying in a first direction, or spatially filtering at a greater intensity than the spatial filtering applied to the first signal.

[0032] According to another aspect of the present invention, an interferometric scattering microscope is provided, which is configured to perform the aforementioned method of imaging a sample by an interferometric scattering microscope. The interferometric scattering microscope includes: a sample position including a reflective surface; at least one coherent light source, the at least one coherent light source configured to illuminate the sample position; a first detector and a second detector; a beam splitter, the beam splitter configured to split a counter-propagating signal from the sample position into a first signal and a second signal; and a modifying element, the modifying element configured to modify the second signal so that the second signal is different from the first signal; wherein the system is configured to direct the first signal and the second signal to the first detector and the second detector, respectively.

[0033] In some embodiments, the interferometric scattering microscope includes at least one spatial filter, which is arranged to filter at least one of the first signal and the second signal, wherein the spatial filter is configured to produce an intensity reduction effect on the incident radiation, and the intensity reduction is greater within a predetermined numerical aperture.

[0034] The present invention can be advantageously applied to samples comprising objects having a scattering cross section of 10 -15 m 2 or less than 10 -15 m 2 Typically, the scattering cross section of such an object relative to the illumination light can also be 10 -26 m 2 or greater than 10 -26 m 2 , that is, in 10 -15 m 2 to 10 -26 m 2 Examples of objects that can be studied include proteins or small aggregates of proteins and metallic, organic or inorganic nanoparticles.

[0035] In order to image an object that is a very weak scatterer, the spatial filter is arranged to pass the output light in such a way that the intensity within a predetermined numerical aperture is reduced to 10 of the incident intensity. -2 or less than 10 -2 Typically, the spatial filter may be arranged to pass the output light in such a way that the intensity within a predetermined numerical aperture is reduced to 10 of the incident intensity. -4 or greater than 10 -4 , for example, at an incident intensity of 10 -2 to 10 -4 within the range. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to enable a better understanding, embodiments of the invention will now be described by way of non-limiting examples with reference to the accompanying drawings, in which:

[0037] FIG1 is a schematic diagram of an iSCAT microscope of the prior art;

[0038] FIG2 is a schematic diagram of an improved iSCAT microscope of the prior art;

[0039] Figure 3a 、 Figure 3b 、 Figure 4 as well as Figure 5 An exemplary embodiment of an iSCAT microscope according to the present invention is shown;

[0040] Figure 6A 、 Figure 6B as well as Figure 6C Experimental results obtained using the method and apparatus according to the present invention are shown. DETAILED DESCRIPTION

[0041] In the systems and methods described herein, the light used can be: ultraviolet light (ultraviolet light can be defined herein as having a wavelength in the range of 10 nm to 380 nm); visible light (visible light can be defined herein as having a wavelength in the range of 380 nm to 740 nm); infrared light (infrared light can be defined herein as having a wavelength in the range of 740 nm to 300 μm). The light can be a mixture of multiple wavelengths. Herein, the terms "optical" and "optical" are generally used to refer to the light to which these methods are applied.

[0042] Figures 1 and 2 show the iSCAT microscope configuration disclosed in WO 2018 / 011591, which has many of the same structural features and functions as the apparatus and method of the present invention.

[0043] The disclosure of WO 2018 / 011591 is incorporated herein by reference, however, for the sake of completeness, the following description will set forth the components and functions of the iSCAT microscope of the present invention that are identical to those of the microscope of WO 2018 / 011591 and are illustrated in Figures 1 and 2 , and then describe various improvements to the configuration provided by the present disclosure and provide example embodiments of the improvements.

[0044] Thus, with reference to Figure 1, the microscope 1 includes a sample holder 2 for holding a sample 3 in a sample position. The sample 3 can be a liquid sample including an object to be imaged, as will be described in more detail below. The sample holder 2 can 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 can be a coverslip and / or can be made of glass. The sample 3 can be arranged on the sample holder 2 in a simple manner (e.g., using a micropipette).

[0045] Microscope 1 further includes an illumination source 4 and a detector 5. Illumination source 4 is configured to provide illumination light. The illumination light may be coherent light. For example, illumination source 4 may be a laser. The wavelength of the illumination light may be selected based on the properties of sample 3 and / or the characteristics to be detected. In one example, the wavelength of the illumination light is 405 nm.

[0046] Optionally, the illumination light can be spatially modulated to eliminate speckle patterns caused by the coherent nature of the illumination and laser noise, as described in detail in Kukura et al., "High-speed nanoscopic tracking of the position and orientation of a single virus," Nature Methods 2009 6:923-935.

[0047] Output light reflected from the sample location is received by a detector 5. The illumination light reaching the detector is primarily reflected from the surface of the sample (typically the interface between the sample and the sample holder), providing interference with objects in the sample close to the surface.

[0048] In the example using a glass-water interface, a relatively small amount (typically only 0.5%) of the illumination light is reflected, while a significantly higher amount (typically greater than 90%) of the light scattered by nanoscopic objects at the interface is scattered back toward the illumination direction. Compared to a transmission-type geometry, this essentially increases the ratio between the scattered and reflected light fields by more than a factor of 1000, resulting in greater interference contrast. Therefore, given a specific scatterer, illumination intensity, and exposure time, to achieve the same nominal signal-to-noise ratio, three orders of magnitude fewer photons need to be detected compared to a transmission-type setup.

[0049] Typically, the microscope 1 can be operated in widefield mode, in which case the detector 5 can be an image sensor that captures an image of the sample 3. Alternatively, the microscope 1 can be operated in confocal mode, in which case the detector 5 can be an image sensor or can be a point detector, such as a photodiode, in which case a scanning device can be used to scan an area of ​​the sample 3 to construct an image. Examples of image sensors that can be used as the detector 5 include CMOS (complementary metal-oxide semiconductor) image sensors or CCD (charge-coupled device).

[0050] The microscope 1 further comprises an optical system 10 arranged between the sample holder 2, the illumination source 4 and the detector 5. The optical system 10 is arranged in a manner as described below to direct illumination light onto the sample position for illuminating the sample 3, and to collect output light reflected from the sample position and direct the output light to the detector 5.

[0051] The optical system 10 includes an objective lens 11, which is a lens system arranged in front of the sample holding portion 2. The optical system 10 also includes a condenser lens 12 and a tube lens 13. The condenser lens 12 causes the irradiation light from the light source 4 (indicated by a solid line in FIG. 1 ) to pass through the objective lens 11 and condense onto the sample 3 at the sample position.

[0052] The objective lens 11 collects output light, which includes (a) illumination light reflected from the sample position (shown by a solid line in FIG1 ) and (b) light scattered from the sample 3 at the sample position (shown by a dotted line in FIG1 ). The reflected light is mainly reflected from the interface between the sample holder 2 and the sample 3.

[0053] Typically, this is a relatively weak reflection, such as a glass-water reflection. For example, the intensity of the reflected illumination light may be about 0.5% of the intensity of the incident illumination light. The scattered light is scattered by objects in the sample 3. The scattered light from objects at or near the sample surface constructively interferes with the reflected light and is therefore visible in the image captured by the detector 5.

[0054] As shown in FIG1 , the reflected illumination light and the scattered light have different directionalities. In particular, the numerical aperture of the reflected illumination light is generated by the geometric shape of the light beam output by the optical system 10 and the light source 4. The scattered light scatters over a large angle range and therefore fills a larger numerical aperture than the reflected illumination light. The tube lens 13 focuses the output light from the objective lens 11 onto the detector 5.

[0055] The optical system 10 further comprises a beam splitter 14 arranged to split the optical paths of illumination light from the light source 4 and output light directed to the detector 5. The beam splitter 14 may be of a conventional construction providing partial reflection and partial transmission of light incident thereon.

[0056] In the example of the present disclosure, the light source 4 is deviated from the optical path of the objective lens 11, so that the illumination light from the light source 4 is reflected by the beam splitter 14 into the objective lens 11. Conversely, the detector 5 is aligned with the optical path of the objective lens 11, so that the output light from the sample position is transmitted through the beam splitter 14 toward the detector 5.

[0057] In addition to the components described above, which may be of conventional construction, the microscope 1 includes a spatial mask or filter 20. In the example of FIG1 , the spatial filter 20 is formed on the beam splitter 14 and is therefore arranged behind the rear aperture of the objective lens 11 and therefore directly behind the rear focal plane 15 of the objective lens 11, however, the spatial filter 20 may be placed at other points along the optical path of the iSCAT microscope to achieve the same effect as described below.

[0058] The spatial filter 20 is positioned to filter the counter-propagating output light passed from the sample holder interface to the detector 5. Thus, in the disclosed example where the detector 5 is aligned with the optical path of the objective lens 11, the spatial filter 20 is transmissive.

[0059] Spatial filter 20 is partially transmissive and therefore passes output light through, but at a reduced intensity, including reflected illumination light. Spatial filter 20 is also aligned with the optical axis and has a predetermined aperture such that the spatial filter provides intensity reduction within a predetermined numerical aperture. Numerical aperture is conventionally defined as a dimensionless quantity that characterizes the angular range relative to the sample position from which the output light originates.

[0060] 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 (eg, the material of the components of the optical system 10 ) through which the output light passes.

[0061] Spatial filter 20 can be formed in any suitable manner, and spatial filter generally comprises a layer of deposited material. The material may be, for example, a metal, such as silver. In some embodiments, spatial filter may comprise one or more dielectric coatings. In some embodiments, spatial filter may be shaped to partially reflect incident radiation within a given angular range. Deposition may be performed using any suitable technique.

[0062] Because sub-diffraction-sized objects near the interface preferentially scatter light into larger numerical apertures than 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, at low numerical apertures, the intensity reduction caused by spatial filter 20 primarily affects the reflected illumination light and minimally affects the scattered light, thereby maximizing contrast in the captured image. The enhanced imaging contrast enables high-contrast detection of objects that are weak scatterers.

[0063] Contrast enhancement can be understood as follows. When the spatial filter 20 allows a portion of the output light within a predetermined numerical aperture to pass through (i.e., partially transmits in this example), for a sufficiently coherent illumination source, a portion of the illumination light field and the scattered light field reach the detector and interfere. Therefore, the light intensity I reaching the detector is det is given by the following equation:

[0064] I det =|E inc | 2 {r 2 t 2 +|s| 2 +2rt|s|cosΦ},

[0065] Among them, E inc is the incident light field, r 2 is the reflectivity 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.

[0066] The additional filtering provided by the spatial filter 20 enables the amplitude of the reference field to be directly measured by selecting the transmittance t of the spatial filter 20. 2 Instead of being fixed by the reflectivity of the glass-water interface as in standard ISCAT. In the case where the spatial filter 20 is a layer of deposited material, the transmittance t 2 This can be selected by choosing the material and / or the thickness of the layer.Such an adjustment can be made, for example, depending on the scattering object of interest, the camera full well capacity, and the magnification.

[0067] Bright field illumination ensures that the strongest unwanted back reflections, typically from the objective, are directed away from the detector 5, minimizing the imaging background and enabling a large field of view without the need for complex scanning of the beam of illumination light.

[0068] In order to image an object that is a relatively weak scatterer, the spatial filter 20 may be arranged to pass the reflected illumination light with a reduced intensity, the intensity being reduced to 10 times the incident intensity (in this context, the intensity of the output light incident on the spatial filter 20) within a predetermined numerical aperture. -2 to 10 -4 intensity within the range.

[0069] For example, a sample comprising an object having a mass of 5000 kDa or less can be imaged. In general, the disclosed techniques can be applied to samples comprising an object having a mass of 10 kDa or greater, such as objects having a mass in the range of 10 kDa to 5000 kDa, and / or to samples comprising an object having a scattering cross section of 10 kDa relative to the illuminating light. -12 m 2 or less than 10 - 12 m 2 (or preferably a scattering cross section of 10 -17 m 2 or less than 10 -17 m 2 Typically, the scattering cross section of such an object relative to the illumination light can also be, for example, in the range of 10 -17 m 2 to 10 -26 m 2 Examples of objects for which the disclosed technology can be applied for imaging include proteins or small aggregates of proteins, or their binding partners.

[0070] In order to image stronger scatterers simultaneously, the transmittance of the second filter can be set between 1 and 10, depending on the desired detection range. -2 Any value in between.

[0071] Referring to Figure 2, there is shown a second example configuration of the microscope 1. The configuration of Figure 2 is also disclosed in WO 2018 / 011591 and is similarly suitable for optimization by applying the techniques of the present invention.

[0072] 2 arranges the spatial filter 20 at a conjugate focal plane 21 of the rear focal plane of the objective lens 11, rather than behind the rear aperture of the objective lens 11. The conjugate focal plane 21 of the rear focal plane 15 of the objective lens 11 is formed between a pair of telescope lenses 22 and 23, which are arranged behind the tube lens 13.

[0073] An acousto-optic deflector 32 is arranged after the light source 4 to provide scanning of the illumination light. As mentioned above, the acousto-optic deflector 32 can be operated to scan an area of ​​the sample 3 to construct an image and / or provide spatial modulation to eliminate speckle patterns caused by the coherent nature of the illumination and laser noise.

[0074] The condenser lens 12 is replaced by a pair of telecentric lenses 30 and 31 which perform the function of imaging any modification of the beam path at the acousto-optical deflector 32 into the back focal plane of the imaging objective.

[0075] 1 , the positions of the light source 4 and the detector 5 are opposite, so that the illumination light from the light source 4 is transmitted into the objective lens 11 through the beam splitter 14 , and conversely, the output light from the sample position is reflected by the beam splitter 14 toward the detector 5 .

[0076] The beam splitter 14 is a polarization beam splitter, and a quarter wave plate 33 is arranged between the beam splitter 14 and the sample 3 so that the beam splitter 14 splits the light. The mirror 34 is arranged to deflect the output light reflected by the beam splitter 14.

[0077] Now, referring to FIG. 3 to FIG. Figure 5 , the following description focuses on various improvements provided by the present invention to the construction of the iSCAT microscope 1 described above.

[0078] The use of the spatial filter 20 does not fundamentally change the sensitivity limit or SNR (signal-to-noise ratio) achievable for a given object, incident light intensity and exposure time, however, the sensitivity can be increased by manipulating the back-propagating signal on the detection side of the device after a portion of the illuminating light has been scattered by the object.

[0079] In Figures 3 to Figure 5 In the example configuration shown in FIG, the counter-propagating signal is manipulated by passing it through a second beam splitter 36, which is configured to split the counter-propagating signal into a first signal and a second signal, which are then directed to a first detector 5 and a second detector 6, respectively. In this way, two separate image signals can be captured at the same time for a given object, and the two separate image signals are processed through separate image channels. This, in turn, enables the study of light scattered by the sample through image comparison without introducing time-dependent intensity fluctuations caused by changes in the optical system 10 or the illumination source.

[0080] Furthermore, by independently modifying at least one of the two separate signals from the second beam splitter 36 before they reach the detector, additional characteristics of the sample can be investigated and the sensitivity of the measurement can be increased. For example, as will be described below, background noise characteristics can be removed from the illumination, and the first and second signals can be adjusted relative to each other and combined to provide more information about the sample.

[0081] The second beam splitter 36 is configured to split the counter-propagating signal in such a way that the characteristics of the sample / counter-propagating signal are under investigation.

[0082] For example, in some embodiments where it is desired to separate the signal by optical power, the second beam splitter 36 may include a plate provided with a film, which may be metallic or dielectric, and arranged at a 45° angle to the optical path. Alternatively, the second beam splitter 36 may be a cube beam splitter formed from a pair of matched prisms with a partially reflective film at the interface between the prisms.

[0083] In some embodiments, the second beam splitter 36 is a polarization beam splitter that is used in combination with a quarter-wave plate between the second beam splitter 36 and the sample 3 and is configured to split the counter-propagating signal into a first signal and a second signal that are orthogonally polarized. This configuration enables the birefringence characteristics of the sample to be analyzed by the first detector 5 and the second detector 6.

[0084] In some embodiments, the illumination light includes at least two different interrogation wavelengths for illuminating the sample, and the second beam splitter 36 is configured to separate the counter-propagating signals according to wavelength, so that light having the first interrogation wavelength is directed to the first detector 5 and light having the second interrogation wavelength is directed to the second detector 6.

[0085] Measuring the same sample at two different probe wavelengths can help identify intensity variations caused by wavelength-dependent components in the optical system. This configuration can also identify intensity variations caused by samples that absorb light at one wavelength but not others (e.g., due to different dyes or fluorescent markers added to the sample that would affect scattering, or due to a coating on the sample support that absorbs one wavelength).

[0086] The optimization methods described herein are complementary and can be used alone or in combination with each other. Thus, in some embodiments, the second beam splitter 36 can include multiple beam splitters, each of which is configured to split the signal in a different or the same manner, such that the counter-propagating signal is split into three or more signals, each of which can then be directed to a separate detector.

[0087] Reference Figure 3a , shows an example optimized configuration of the microscope 1 based on the optical configuration of FIG. 1 .

[0088] In addition to the optical components of Figure 1, Figure 3a The structure includes a second detector 6 and a second beam splitter 36, which is arranged along the optical path of the counter-propagating signal between the tube lens 13 and the first detector 5 and the second detector 6, and is configured to split the counter-propagating signal into a first signal and a second signal.

[0089] In the example shown, the first signal is passed directly to the first detector 5, while the second signal is guided through the signal modifying element 40 via the reflective element 38 before impinging on the second detector 6. The signal modifying element 40 is selected depending on the type of image signal optimization desired.

[0090] In one exemplary optimization method, second beam splitter 36 splits the counter-propagating signal into a first signal and a second signal having equal optical power, and modifying element 40 includes a delay mask configured to cause light scattered by an object held at the sample position to produce a phase shift of π / 2 in the second signal without affecting the phase of the illuminating light. In doing so, the image of the sample in the second signal is inverted, causing scattered light that would otherwise interfere constructively to interfere destructively.

[0091] Because the scattered light in the first signal is not phase-shifted, it constructively interferes, as in conventional iSCAT. Therefore, when comparing the images acquired by the first detector 5 and the second detector 6, intensity fluctuations in the images caused by scattering from the sample will be correlated, and uncorrelated intensity fluctuations between the two images can be identified and attributed to background features, such as imperfections in the illumination light.

[0092] The identification of such background features in the iSCAT image, obtained by independent “phase adjustment” of the scattered light in the separate signals, enables the features to be removed and thus improves the sensitivity of the microscope 1 .

[0093] In an alternative example, the phase of the scattered light is instead inverted by having the signal-modifying element 40 include an imaging lens, and moving the 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. This configuration enables the same "modulation" effect on the phase of the scattered light as provided by a phase-shift 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 sample 3. This effect can be understood as follows.

[0094] If a collimated beam of light is focused to a single point in space by an ideal lens / focusing element, the phase of the beam measured on the other side of the focus at a distance equal to the focal length of the focusing element will be shifted by π. Exactly at the focus, the phase shift will be π / 2. This phase shift is called the Guy phase shift and is approximately linearly dependent on the distance from the focus.

[0095] In practice, no focusing element can focus a beam to a single point in space, and for a given light beam, the intensity of the phase variation depends on the distance based on how focused the beam is. Weakly focused light, such as the illumination light from illumination source 4, is barely affected by Guy phase shifts. In contrast, single molecules or very small samples, such as those interrogated by the iSCAT microscope of the present disclosure, have sizes below the diffraction limit of the illumination light and therefore act as near-ideal focusing elements. Consequently, the scattered light in the counter-propagating signal will have a high Guy phase dependence along its optical path, whereas the illumination light has no Guy phase dependence.

[0096] In the case of iSCAT microscopy, the scattered light and the reflected illumination light undergo an additional phase shift due to the refractive index variation at the sample position interface / cover glass before passing through the second beam splitter 36 and being imaged onto the first and second detectors 5, 6. In this optical setup, changing the position of the imaging lens (in this example, the modifying element 40) enables the Guy phase shift of the scattered light to be adjusted without affecting the weakly focused illumination light, simulating a change in distance from the focal point.

[0097] Thus, the phase of the scattered light can be changed without changing the phase of the illumination light, and the image of the sample received by the second detector 6 can be inverted as described with respect to the phase shift mask.

[0098] Figure 3a An arrangement is shown with two lasers 4a and 4b, which are combined via a beam splitter 4c to form a more powerful illumination beam.

[0099] Although this embodiment is shown and described as an optimization of the configuration of FIG. 1 , this embodiment may also be implemented as an optimization of the configuration of FIG. 2 .

[0100] Figure 3b Shown Figure 3a An alternative arrangement of , except without the spatial filter 20. The beam is still split, with a modifying element 40 being arranged in the path of the second split signal. The modifying element may amplify, phase shift or even spatially filter the second split signal to increase the sensitivity of the device.

[0101] Reference Figure 4 , shows an example optimized configuration of the microscope 1 based on the optical configuration of FIG. 2 .

[0102] In addition to the optical components of Figure 2, Figure 4 The configuration includes a second detector 6 and a second beam splitter 36, which is arranged along the optical path of the counter-propagating signal between the tube lens 13 and the first detector 5 and the second detector 6, and is configured to split the counter-propagating signal into a first signal and a second signal. In this configuration, one or more spatial filters 20 are arranged between the second beam splitter 36 and the first detector 5 and the second detector 6.

[0103] In the example shown, a second beam splitter 36 asymmetrically splits the counter-propagating signal into a first signal and a second signal having different optical powers. The first signal passes through a first spatial filter 20 to reach a first detector 5, and the second signal is guided through a signal-modifying element 40 via a reflective element 38 before striking a second detector 6. The signal-modifying element 40 is a second spatial filter having a different strength than the first spatial filter.

[0104] In mass photometry experiments (in which the iSCAT microscope is used to measure the mass of a sub-diffraction-limited object held in place at the sample), the image signal quality typically has to be optimized for a given mass range because weak scatterers require higher optical power for illumination than strong scatterers, and therefore more of the illumination light is attenuated by the spatial filter 20.

[0105] Asymmetrically splitting the counter-propagating signals as described above and passing the first and second signals through spatial filters of different strengths enables the generation of first and second signals, each of which has a different balance between the illumination light (reference beam) and the light scattered from the sample in the image plane (image signal). Thus, first and second images can be acquired by first and second detectors 5, 6, each of which is optimized for either high-quality object measurement or low-quality object measurement. This increases the dynamic mass range that can be measured using microscope 1.

[0106] For example, the second beam splitter 36 can be configured so that the first signal has a greater optical power than the second signal, in which case the second spatial filter applies a greater intensity reduction to the incident radiation than the first spatial filter. The optical power ratio between the first and second signals can be, for example, 90:10, in which case spatial filters of appropriate strength (e.g., attenuated to 0.1% and 1%, respectively) will be used. This ratio can be adjusted depending on the desired detection range in either direction, for example, the ratio is 99:1, attenuated to 0.01% and 10%, respectively. Ideally, the combination of optical power ratio and attenuation is always selected so that the same light intensity is maintained in both imaging channels.

[0107] Figure 4 An optional acousto-optical deflector (AOD) 27 is shown in the path of the illumination beam. The AOD is arranged after the light source to provide scanning of the illumination light. As described above, the AOD 27 can be operated to scan an area of ​​the sample 3 to construct an image and / or provide spatial modulation to eliminate speckle patterns caused by the coherent nature of the illumination and laser noise.

[0108] Although this embodiment is shown with reference to a configuration based on the configuration of FIG. 2 , it should be understood that this optimization method can also be implemented based on the configuration of FIG. 1 .

[0109] Reference Figure 5 , shows another example embodiment based on the optical configuration of FIG. 1 .

[0110] The amount of light that could be used in previous setups was limited by the digital camera readout, so stronger spatial filters had to be used20, which in turn led to an increased sensitivity to vibrations and, in mass photometry, a broadening of the recorded mass distribution.

[0111] Similar to the structure of Figure 3, Figure 5 The second detector 6 and the second beam splitter 36 are arranged along the optical path of the counter-propagating signal between the tube lens 13 and the first detector 5 and the second detector 6, and are configured to split the counter-propagating signal into a first signal and a second signal.

[0112] However, in this embodiment, the first signal is not passed directly to the first detector 5, but passes through a first amplifying element 42, which is configured to amplify the first signal along the first axis.

[0113] exist Figure 5 In the embodiment of , the second signal is guided via the reflective element 38 through a signal modifying element, in this case a second amplifying element 43 configured to amplify the second signal along an axis perpendicular to the first axis.

[0114] The first detector 5 and the second detector 6 are oriented differently relative to 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 excitations are processed column by column on a grid. Therefore, the first detector 5 and the second detector 6 are oriented so that the direction in which the camera readout electronics can process pixel excitations fastest is aligned with the first axis of the first detector 5 and the second axis of the second detector 6.

[0115] Using the configuration described above, the rate at which photons scattered from the sample are processed can be increased without using expensive dedicated detectors by improving the matching of detected photons with digital camera readout operations. For example, the first detector 5 can read out only along a single X-axis column, in which the first amplification element 42 has amplified the photon flux from the first signal, while the second detector 6 can read out only along a single Y-axis column, along which the second amplification element 43 has amplified the photon flux from the second signal.

[0116] Thus, the pixels of the first detector 5 and the second detector 6 can be mapped to each other to appropriately assign XY positions to the two cameras so that the images acquired from the first detector 5 and the second detector 6 can be blended together. In some cases, the blended image needs to be corrected for distortion. However, in general, the method described above enables a conventional detector to increase the rate at which images are captured by a number proportional to the magnification of the first and second amplifying elements with little loss in image quality.

[0117] Thus, the described embodiment compensates for the reduction in the total number of photons in the first and second signals due to the spatial filter 20 by the above-listed I det As can be seen from the equation, the spatial filter enhances the scattering contrast in the acquired image at the expense of the total number of photons detected.

[0118] Although this embodiment is shown and described as an optimization of the configuration of FIG. 1 , this embodiment may also be implemented as an optimization of the configuration of FIG. 2 .

[0119] Additionally, in some implementations of the embodiment (not shown), only the first signal passes through the amplification element, while the second image channel is viewed with a normal spatial distribution. In such a configuration, the second signal can pass through a different mask or conventional intensity filter instead of the amplification element. In such an implementation, the second channel retains full spatial resolution and can be used for localization.

[0120] Above about Figure 3 to Figure 5 Each of the described optimization methods can be used in combination with one another or individually.

[0121] Furthermore, each of the described optimization methods can be facilitated by providing a higher intensity illumination source 4 when the counter-propagating signal is split into the first signal and the second signal by the second beam splitter 36 having a lower photon flux. As shown in FIG3 , in some embodiments, the higher intensity illumination source 4 is provided by combining the beams of the first laser and the second laser.

[0122] Reference Figure 6A, example experimental results are two channel mass photometric ratiometric images using one chip, which show two images of the same sample mixture obtained at the same time point using a beam splitting configuration similar to that described above Figure 5 The iSCAT microscope is constructed like the iSCAT microscope, but without the magnification component.

[0123] Reference Figure 6B , shows another example experimental result, which shows that using a method similar to Figure 5 An image obtained from another iSCAT microscope configuration comprising a magnifying element configured to stretch one of the separated beams along a single dimension. Figure 6B As can be seen in FIG. 1 , the image of the sample mixture is magnified / stretched along the X dimension.

[0124] Referring to FIG. 6c, it is shown that Figure 6B The same experimental results shown in , but using 1-D binning to recombine the pixels in the stretched dimension into one pixel, demonstrates that image content and resolution are not lost by this process, but rather the photons are simply spread over multiple pixels, which can then be recombined.

[0125] Microscope 1 can be used as iSCAT for a wide range of applications, including single-molecule detection. A particular application is label-free imaging of weak scatterers, where the object of interest must be detected unchanged against a large background, which reduces imaging contrast. Microscope 1 can be used for a wide range of studies and measurements, such as measuring any change in refractive index, including, for example: single-molecule binding / unbinding, phase transitions, clustering, assembly / disassembly, aggregation, protein / protein interactions, protein / small molecule interactions, and high-sensitivity label-free imaging.

[0126] Therefore, microscope 1 has numerous applications, ranging from basic research to industrial applications, such as in the pharmaceutical industry. For example, iSCAT is currently the world's most sensitive label-free single-molecule imaging biosensor, and iSCAT could have a significant impact on the surface plasmon resonance sensing market. Furthermore, microscope 1 described above can be used for mass measurement, serving as a precise, accurate, and high-resolution single-molecule mass spectrometer solution, with numerous applications in research and industry.

Claims

1. A method for imaging a sample by interferometric scattering microscopy, the method comprising: illuminating a sample with at least one coherent light source, the sample being held at a sample position comprising an interface, the interface having a refractive index variation, the sample being illuminated with illuminating radiation to produce a counter-propagating signal from the sample, the counter-propagating signal comprising light reflected at the interface and light scattered by the sample; splitting the counter-propagating signal into a first signal and a second signal such that the first signal and the second signal each include at least a portion of light scattered by the sample; modifying the second signal using a modifying element so that the second signal differs from the first signal, wherein modifying the second signal comprises phase shifting, amplifying along a first dimension, or spatially filtering at a greater intensity than spatial filtering applied to the first signal; directing the first signal and the second signal onto a first detector and a second detector to produce 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 characteristics of the sample, Therein, the first dimension corresponds to the x-dimension of the pixel grid of the second detector.

2. The method of claim 1 , further comprising passing at least one of the first signal and the second signal through a spatial filter configured to produce an intensity reduction effect on incident radiation, the intensity reduction becoming greater within a predetermined numerical aperture.

3. The method according to claim 1 or claim 2, wherein: The at least one coherent light source comprises a first laser and a second laser, the beams of the first laser and the second laser being combined prior to irradiating the sample.

4. The method according to claim 1 or claim 2, wherein: Splitting the counter-propagating signal into a first signal and a second signal includes splitting the counter-propagating signal into two signals having orthogonal polarizations.

5. The method according to claim 1 or claim 2, wherein: Splitting the counter-propagating signal into a first signal and a second signal includes splitting the counter-propagating signal into two signals having different optical powers.

6. The method according to claim 2, wherein: 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, and modifying the second signal so that the second signal is different from the first signal includes passing the second signal through a second spatial filter, wherein the first spatial filter and the second spatial filter are each configured to produce an intensity reduction on incident radiation, the intensity reduction becoming greater within a predetermined numerical aperture, wherein the first signal has greater optical power than the second signal, and wherein the second spatial filter implements a greater intensity reduction on the incident radiation than the first spatial filter.

7. The method according to claim 1 or claim 2, wherein: Modifying the second signal includes adjusting a phase of the second signal relative to the first signal.

8. The method according to claim 7, wherein: Adjusting the phase of the second signal relative to the first signal includes passing the second signal through a phase shift mask.

9. The method according to claim 7, wherein: Adjusting the phase of the second signal relative to the first signal includes passing the second signal through an imaging lens at an appropriate location along the optical path of the first signal.

10. The method according to claim 7, wherein: Adjusting the phase of the second signal relative to the first signal includes adjusting the phase of the second signal relative to the first signal by half a wavelength of the illumination radiation.

11. The method according to claim 1 or claim 2, wherein: Modifying the second signal includes: The second signal is passed through an optical element configured to asymmetrically amplify the second signal along a first dimension, the first dimension corresponding to an x-dimension of a pixel grid of the second detector.

12. The method according to claim 11, wherein The method further comprises: The first signal is passed through an optical element configured to asymmetrically amplify the first signal along a second dimension, the second dimension being orthogonal to the first dimension and corresponding to the y-dimension of the pixel grid of the second detector.

13. The method according to claim 1 or claim 2, wherein: The at least one coherent light source is configured to provide illumination light having at least two different interrogation wavelengths; and Wherein, separating the counter-propagating signal into a first signal and a second signal comprises separating the counter-propagating signal by wavelength such that light having a first interrogation wavelength is directed to the first detector and light having a second interrogation wavelength is directed to the second detector.

14. The method according to claim 2, wherein: The predetermined numerical aperture is the same as or similar to the numerical aperture of the illumination light reflected from the sample location.

15. An interferometric scattering microscope, configured to perform the method according to any one of claims 1 to 14, comprising: Sample location including reflective surfaces; 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 the first signal and the second signal each include at least a portion of light scattered by the sample; as well as a modifying element configured to modify the second signal such that the second signal differs from the first signal, wherein modifying the second signal comprises phase shifting, amplifying along a first dimension, or spatially filtering at a greater intensity than spatial filtering applied to the first signal; wherein the interferometric scattering microscope is configured to direct the first signal and the second signal onto the first detector and the second detector, respectively, and wherein the first dimension corresponds to the x-dimension of a pixel grid of the second detector.

16. The interferometric scattering microscope according to claim 15, further comprising at least one spatial filter, wherein the at least one spatial filter is configured to filter at least one of the first signal and the second signal, wherein: The spatial filter is configured to produce an intensity reduction on the incident radiation, said intensity reduction becoming greater within a predetermined numerical aperture.

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