Method and apparatus for optimized coherent scattering microscopy - Patent Application 20070122999

Abstract

A method for imaging a sample by coherent scattering microscopy, comprising the steps of: illuminating the sample with at least one light source, the sample being held at a sample position including a reflective surface such that a reflected signal is formed, the reflected signal including light from the light source and light scattered by the sample; detecting output light for a first frame N1 over a first time window; detecting output light for a second frame N2 over a second time window; calculating a ratiometric signal R as the ratio of N1 to N2 minus 1; and calculating a ratiometric motion signature S=(S x ,S y ) where S is the step of estimating the motion vector m=(m x ,m y ), estimating m as the most consistent vector such that R is approximated using S and m, and calculating the corrected ratiometric contrast image R* from R, S, and m.

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 been embodied as a powerful approach for both single particle tracking with unique spatiotemporal resolution and label-free sensitivity down to the single molecule level, as well as for single particle mass determination by mass spectrometry and mass imaging.

[0003] 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.

[0004] Despite its considerable potential, the widespread application of IScat is limited by the requirement for custom-built 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.

[0005] 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.

[0006] However, a number of limitations to the measurement sensitivity achievable using that approach have become apparent to the present inventors. In IScat microscopy, the ratio between adjacent frames is often used to detect signals generated by particles bound to the boundary. In this way, a ratiometric contrast image is calculated, displaying the relative change in pixel intensity from two advancing frames before and after a given point in time.

[0007] However, problems can arise if the sample moves between adjacent frames. Sample movement is also associated with changes in pixel intensity, which are also detected in ratiometric images, creating problems for particle detection and mass determination from the data. In fact, the technique is so sensitive that even a movement of less than one pixel can drown out the signal from the particle due to the movement of the sample carrier. Movements of even 1% of the pixel size can result in a signal with the same amplitude as a single protein. Thus, even very small movements can produce very erroneous signals.

[0008] Currently, methods for correcting sub-pixel-level motion include iterative image registration, Fourier transform, optical flow, etc. These methods cannot be directly applied to ratiometric images, nor can they be performed accurately enough in real time at kHz frame rates.

[0009] The present invention provides a method and apparatus for an optimized IScat technique that corrects for sample motion. The method disclosed herein addresses this by detecting motion and subtracting it from the signal. Summary of the Invention [Means for solving the problem]

[0010] According to one aspect of the present invention, there is provided a method for imaging a sample by coherent scattering microscopy. The method includes a method for imaging a sample by coherent scattering microscopy, the method comprising the steps of illuminating the sample with at least one light source, the sample being held at a sample position including a reflective surface such that a reflected signal is formed, the reflected signal including light from the light source and light scattered by the sample; detecting output light over a first time window for a first frame N1; detecting output light over a second time window for a second frame N2; and calculating a ratiometric signal R as the ratio of N1 to N2 minus 1; Ne From native camera frames N1 and N2 , given a motion vector m=(m x ,m y ) is a ratiometric image measured from a stationary sample moving along x and y relative to Ratiometric motion signature S = (S x ,S y ), estimating m as the most consistent vector such that R is approximated using S and m, and calculating the corrected ratiometric contrast image R* from R, S, and m.

[0011] By removing the motion signature from the ratiometric signal in this way, rather than traditional image registration, image processing can still be performed in real time at kHz rates.

[0012] Horizontal and vertical ratiometric motion signature S at pixel location (x,y) x and S y is obtained from the native image N, S x [x,y]=(N[x+1,y]-N[x-1,y]) / (2N[x,y]) S y [x,y]=(N[x,y+1]-N[x,y-1]) / (2N[x,y]) It is estimated as follows.

[0013] m is R=m x S x +m y S y is estimated as the most consistent vector such that The corrected ratiometric contrast image frame R* is given by R*=R-(m x S x +m y S y ) is calculated as

[0014] Based on the calculated motion vector m, the sample can be moved or stabilized with high-precision actuators to compensate for the movement, drift, or vibration.

[0015] Additionally or alternatively, if the detected motion exceeds a predetermined level, an alert may be triggered to warn the user that the detected image may not be reliable. The method of any one of the preceding claims, further comprising the step of moving the sample to compensate for the motion.

[0016] The first frame may include an average over a first plurality of native frames, and the second frame may include an average over a second plurality of native frames. The method may further include passing at least one of the reflected signals through a spatial filter, the spatial filter being configured to attenuate the intensity of the incident radiation, the attenuation being greater within a given numerical aperture. Using a spatial filter can remove most of the reflected light. However, scattered light is generally not removed by the spatial filter due to its high numerical aperture. Therefore, using this spatial filter can result in a higher-contrast image.

[0017] The light source may be a coherent light source. According to the present invention there is provided an interference scattering microscope comprising a sample holder for holding a sample at a sample position, an illumination source arranged to provide illumination light, a detector, optics arranged to direct the illumination light towards the sample position and arranged to collect reflected output light, the output light comprising both scattered light from the sample position and reflected illumination light from the sample position, and arranged to direct the output light towards the detector, a spatial filter arranged to filter the output light, the spatial filter arranged to pass the output light but to attenuate intensities within a given numerical aperture more than larger numerical apertures, and computer program means configured to direct the apparatus to perform the above steps.

[0018] The coherent scattering microscope further includes an actuator for stabilizing the sample in response to the estimated motion vector m. The actuator may be an electric actuator.

[0019] The present invention is -15 m 2 This can be advantageously applied to samples containing objects with a scattering cross section for the illuminating light of: -26 m 2 or more, i.e. 10 -15 m 2 From 10 -26 m 2The scattering cross section for the illuminating 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.

[0020] 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 Typically, the spatial filter is positioned 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 may be arranged to pass more or less attenuated output light.

[0021] 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 explanation of the drawings]

[0022] [Figure 1] 1 is a schematic diagram of a prior art IScat microscope. [Figure 2] 1 is a schematic diagram of an improved version of a prior art IScat microscope. [Figure 3] FIG. 1 shows images from an IScat microscope with no motion correction applied. [Figure 4] FIG. 1 shows an image from an IScat microscope to which motion correction according to the present invention has been applied. DETAILED DESCRIPTION OF THE INVENTION

[0023] In the systems and methods described herein, the light used can be ultraviolet light (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 light (which may be defined herein as having a wavelength in the range of 740 nm to 300 μm). The light may also be a mixture of wavelengths. The terms "optical" and "optical" are used herein to generally refer to the light to which the methods are applied.

[0024] 1 and 2 show the configuration of the IScat microscope disclosed in WO2018 / 011591, which has many structural features and functionality in common with the apparatus and methods of the present invention.

[0025] The disclosure of WO2018 / 011591 is incorporated herein by reference, but for the sake of completeness, the following description will set 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 describe various improvements to said configuration brought about by the present disclosure and provide example embodiments thereof.

[0026] Thus, with reference to FIG. 1 , microscope 1 includes sample holder 2 for holding sample 3 at a sample position. Sample 3 may be a liquid sample containing an object to be imaged, as described in more detail below. Sample holder 2 may take any form suitable for holding sample 3. Typically, sample holder 2 holds sample 3 on a surface that forms an interface between sample holder 2 and sample 3. For example, sample holder 2 may be a coverslip and / or may be made of glass. Sample 3 may be provided on sample holder 2 in a simple manner, for example, using a micropipette.

[0027] 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.

[0028] 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.

[0029] A detector 5 receives the output light upon reflection from the sample location. 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, thereby causing interference with objects in the sample close to that surface.

[0030] 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 toward the illumination light by nanoscopic objects at the interface. This essentially improves the ratio of scattered to reflected light by over 1000 times compared to transmission geometries, thereby 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.

[0031] 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 construct an image. Examples of image sensors that may be employed as the detector 5 include a CMOS (complementary metal-oxide semiconductor) image sensor or a CCD (charge-coupled device), etc.

[0032] 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 at the sample position to illuminate the sample 3, collect output light upon reflection from the sample position and direct the output light to the detector 5 as follows:

[0033] 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 the sample 3 at the sample position.

[0034] The objective lens 11 collects the output light, which includes both (a) the illumination light reflected from the sample position (shown by the continuous line in FIG. 1) and (b) the scattered light from the sample 3 at the sample position (shown by the dotted line in FIG. 1). The reflected light is mainly reflected from the interface between the sample holder 2 and the sample 3.

[0035] Typically, this is a relatively weak reflection, such as 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 near the surface of the sample constructively interferes with the reflected light and is therefore visible in the image captured by detector 5.

[0036] As shown in Figure 1, the reflected illumination light and the scattered light have different directional properties. 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 is scattered over a larger range of angles, so it satisfies a larger numerical aperture than the reflected illumination light. Tube lens 13 focuses the output light from objective lens 11 onto detector 5.

[0037] 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.

[0038] In the embodiment of the present disclosure, the light source 4 is offset from the optical path of the objective lens 11 so 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 so that output light from the sample position is transmitted through the beam splitter 14 towards the detector 5.

[0039] In addition to the above-described 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, thereby placing it behind the back aperture of objective lens 11 and therefore just behind the back focal plane 15 of objective lens 11, although spatial filter 20 may be placed at other points along the optical path of an IScat microscope to achieve the same effect as described below.

[0040] Spatial filter 20 is positioned to filter the counter-propagating output light passing from the sample holder interface to detector 5. In embodiments of the present disclosure where detector 5 is aligned with the optical path of objective lens 11, spatial filter 20 is therefore transmissive.

[0041] Spatial filter 20 is partially transmissive, allowing output light, including reflected illumination, to pass through but at a reduced intensity. Spatial filter 20 is also 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 a dimensionless quantity that characterizes the range of angles relative to the sample position from which the output light originates.

[0042] 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 the components of optical system 10).

[0043] 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.

[0044] Because sub-diffraction-sized objects near an interface preferentially scatter light to larger numerical apertures than to reflected illumination, the intensity reduction provided by spatial filter 20 preferentially reduces the detected intensity of reflected illumination over scattered light. Therefore, intensity reduction provided by spatial filter 20 at low numerical apertures primarily affects reflected illumination and minimally affects scattered light, maximizing contrast in the captured image. The improved imaging contrast allows for high-contrast detection of weakly scattering objects.

[0045] The contrast enhancement can be understood as follows: Because the spatial filter 20 passes a portion of the output light of a given numerical aperture (i.e., is partially transmissive in this example), a fraction of the illumination light and scattered light field reaches the detector and interferes for a sufficiently coherent illumination source. Then, the light intensity I reaching the detectordet is given by the following equation:

[0046] I det =│E inc │ 2 {r 2 t 2 +│s│ 2 +2rt│s│cosΦ} where E inc is the incident light field, r 2 is 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.

[0047] 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 of spatial filter 20. 2 The choice of τ allows for direct adjustment of 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 layer material and / or thickness. Such adjustments can be made depending on, for example, the scatterers of interest, the full well capacity of the camera, and the magnification.

[0048] Bright field illumination directs the strongest unwanted back reflections, usually from the objective lens, away from the detector 5, minimizing imaging background and allowing a wide field of view without complex scanning of the illumination light beam.

[0049] To image objects that are relatively weak scatterers, spatial filter 20 may be configured to, within a given numerical aperture, 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 filter may be arranged to pass reflected illumination light at an intensity reduced to within a range of .gtoreq..times ...

[0050] For example, samples containing objects having a mass of 5000 kDa or less can be imaged. Typically, the disclosed techniques are suitable for 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 or equal to 10, more preferably -17 m 2 It may be applied to a sample containing an object with a scattering cross section for the illumination light of, for example, 10 -17 m 2 ~10 -26 m 2 The scattering cross section for the illuminating light may also be in the range of

[0043] . Examples of objects that can be imaged using the techniques of the present disclosure include proteins or small aggregates thereof, or their binding partners.

[0051] 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 Referring 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.

[0052] 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 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.

[0053] An acousto-optic deflector 32 is positioned after the light source 4 to scan the illumination light, and 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.

[0054] The collecting lens 12 is replaced by a pair of telecentric lenses 30 and 31 whose function is to image any beam path modifications in the acousto-optic deflector 32 into the back focal plane of the imaging objective.

[0055] The positions of the light source 4 and detector 5 are reversed relative to the configuration of FIG. 1 so 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.

[0056] 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. In addition, a mirror 34 is disposed to deflect the outgoing light reflected by the beam splitter 14.

[0057] The ratiometric frame R is calculated from the two native frames N1 and N2 such that for each pixel at location (x, y), the ratiometric signal is calculated as R[x, y] = N2[x, y] / N1[x, y]-1.

[0058] However, when a sample is shifted between frames N1 and N2, there will be contributions from neighboring pixels. For example, in the case of sub-pixel shifts, a pixel at location (x, y) can have contributions from any neighboring pixel, including (x+1, y), (x-1, y), (x, y+1), and (x, y-1).

[0059] Considering motion only in the x direction, the motion contribution m from pixel N1[x+1,y] of frame N1 to pixel N2[x,y] when N2 is acquired is x There may be a possibility that, to first order, this can be expressed as

[0060] N2[x,y]=m x N1[x+1,y]+(1-m x )N1[x,y] The contribution to the ratiometric image is given by Eq.

[0061]

number

[0062] In this way, if the ratio of adjacent pixels can be calculated, the best fit m x The contribution of the motion can be calculated as the value of Similarly, for motion in the y direction, the motion contribution m from pixel N1[x,y+1] in frame N1 to pixel N2[x,y] when N2 is acquired is y This gives the ratiometric image a contribution of Eq.

[0063]

number

[0064] In two dimensions, the motion vector between frames is m=(m x ,m y ) m is calculated from R and S as R=m x S x +m y S y The vector is estimated as the most consistent vector such that

[0065] Horizontal and vertical motion signature S at pixel index (x,y) x ,S y can be estimated from the native image N as follows: S x [x,y]=(N[x-1,y]-N[x+1,y]) / (2N[x,y]) S y [x,y]=(N[x,y-1]-N[x,y+1]) / (2N[x,y]) Then, the motion-corrected ratiometric image R* is R*=R-(m x S x +m y Sy ) can be calculated as

[0066] It uses linear interpolation between pixels, but more advanced interpolation techniques such as Lanczos resampling can be used to produce more refined motion estimates. Furthermore, this approach can be applied to the motion of more than one pixel by computing the gradient of the pixel across the frame.

[0067] Other applications of this method include the detection of motion parallel to the observation direction, which is detected as nonlinear motion (rather than linear translation as described above). Blurring in the image needs to be calibrated using the optical properties of the microscope.

[0068] Although the present description uses the individual native frames N1 and N2, it is also possible to use an average of the frames instead of the native frames. The IScat microscope may include an alarm or alert that is issued if the detected movement exceeds a predetermined value. The alarm may be an audio or visual alarm.

[0069] The IScat microscope may include an actuator for correcting or compensating for movement, drift, or vibration. The actuator may be a motorized actuator capable of moving the sample in three orthogonal directions.

[0070] Figures 3 and 4 show images from IScat microscopy of a sample with three distinct molecular species: monomer, dimer, and trimer. Figure 3 shows a (ratiometric) frame taken with the IScat microscope without motion suppression according to the present invention. Due to the motion, individual particles cannot be identified. The program detects the motion, as indicated by the exclamation point in the upper right corner of the application window. In the lower left panel, the amplitude of the motion is plotted against time. The right side of Figure 3 shows a histogram of the mass of the detected particles that landed on the observation slide. There are no clear peaks for the three different molecular species, as the motion noise interferes with the selection and identification process.

[0071] Figure 4 shows the same (ratiometric) frame taken with the IScat microscope, but this time with motion correction according to the present invention. Two particles are clearly visible as black circles, and the motion amplitude plot in the bottom left panel shows much lower values ​​than in Figure 3. The histogram calculated from the ratiometric frame shows three distinct peaks, corresponding to the masses of the three particle species in the sample.

[0072] This microscope 1 can be used to perform a wide range of IScat applications, including single-molecule detection. In particular, label-free imaging of weakly scattering objects requires that the object of interest be detected against a large background, resulting in reduced imaging contrast. The microscope 1 can be used for a wide range of research and measurements, including single-molecule binding / desorption, phase transitions, clustering, assembly / disassembly, aggregation, protein / protein interactions, protein / small molecule interactions, and measurements of any change in refractive index, including high-sensitivity label-free imaging.

[0073] As such, microscope 1 has numerous applications, ranging from basic 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. Furthermore, as mentioned above, microscope 1 can also be used for mass measurement, functioning 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 light source, the sample being held at a sample position including a reflective surface such that an output light is formed, the output light including illumination light reflected from the sample position and light scattered from the sample position; First Frame N １ detecting the output light over a first time window for Second Frame N ２ detecting the output light over a second time window for N １ and N ２ calculating a ratiometric signal R by subtracting 1 from the ratio of The first frame N １ and the second frame N ２ From a comparison of pixels in the x-axis and y-axis, a ratiometric motion signature S=(S ｘ , S ｙ ) and Estimating m as the most consistent vector such that R is approximated using S and m; calculating a corrected ratiometric contrast image R* from R, S, and m; Including, The corrected ratiometric contrast image R* is R*=R-(m ｘ S ｘ +m ｙ S ｙ ) is calculated as, method.

2. Horizontal and vertical ratiometric motion signature S at pixel location (x, y) ｘ and S ｙ is obtained from the native image N by S ｘ [x,y]=(N[x+1,y]-N[x-1,y]) / (2N[x,y]) S ｙ [x,y]=(N[x,y+1]-N[x,y-1]) / (2N[x,y]) It is estimated as The method of claim 1.

3. m is R = m ｘ S ｘ +m ｙ S ｙ is estimated as the most consistent vector such that The method according to claim 1 or claim 2.

4. Moving the sample to compensate for motion further comprising: The method according to any one of claims 1 to 3.

5. The output light is set to 10 of the incident intensity. －２ Passing the light through a spatial filter that attenuates the intensity within a given numerical aperture so that the intensity is attenuated to: further comprising: The method according to any one of claims 1 to 4.

6. the light source is a coherent light source; The method according to any one of claims 1 to 5.

7. a sample holder for holding the sample at the sample position; an illumination source arranged to provide illumination light; A detector; an optical system arranged to direct illumination light to the sample location and arranged to collect reflected output light, the output light including both scattered light from the sample location and reflected illumination light from the sample location, and arranged to direct the output light to the detector; a spatial filter positioned to filter the output light, said spatial filter filtering said output light at 10 times the intensity of the input light; －２ a spatial filter arranged to attenuate the intensity within a predetermined numerical aperture such that: computer program means adapted to instruct an apparatus to carry out the steps of any one of claims 1 to 6; Including, Interference scattering microscope.

8. an actuator for stabilizing the sample in response to the estimated motion vector m; further comprising: The interference scattering microscope of claim 7.

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