METHOD AND APPARATUS FOR DETECTING NANOPARTICLES AND BIOLOGICAL MOLECULES

Abstract

Disclosed are an optical interferometry apparatus for detection of dielectric nanoparticles and a method for enhancing visibility of the nanoparticles. An imaging system for detection of dielectric nanoparticles includes at least one light source for illumination, a detector array or a camera for image capture, an objective lens, a sample substrate and a computing unit. The sample substrate is capable of carrying sub-wavelength particles smaller than the diffraction resolution limit of the imaging system, and the imaging system includes a movable means which moves the sample substrate in the axial direction such that depthwise different images are captured at different axial distances from the sample substrate to the objective lens. The computing unit computes a correlation image using the depth images wherein the sub-wavelength particles become resolvable and appear with higher contrast in the correlation image.

Claims

1. An imaging system for a detection of nanoparticles, comprising at least one light source for illumination, a camera for image capture, an objective lens, a sample substrate and a computing unit; wherein the sample substrate comprises a structure carrying sub-wavelength particles smaller than a diffraction resolution limit of the imaging system, a reference light is provided by a part of a light reflected from the sample substrate, and a scattered light is provided by a part of a light scattered on the sub-wavelength particles, the reference light and the scattered light are collectable by the objective lens to interfere at the camera, the imaging system further comprises a movable means, wherein the movable means moves the sample substrate in an axial direction to induce a change in an axial distance between the sample substrate and the objective lens such that depthwise different images are captured by the camera at different axial distances, a correlation image is computed by the computing unit using the depthwise different images, wherein the sub-wavelength particles become resolvable and appear with a higher contrast in the correlation image.

2. The imaging system according to claim 1, wherein the light source provides a nearly collimated illumination beam on the sample substrate such that a background illumination on the depthwise different images vary as the movable means is moved.

3. The imaging system according to claim 1, wherein the light source provides a collimated illumination beam on the sample substrate.

4. The imaging system according to claim 1, wherein the light source provides an uncollimated illumination beam on the sample substrate.

5. The imaging system according to claim 2, wherein the light source provides a converging beam on the sample substrate.

6. The imaging system according to claim 2, wherein the light source provides a diverging beam on the sample substrate.

7. The imaging system according to claim 1, wherein the depthwise different images are a collection of focused images and defocused images.

8. The imaging system according to claim 1, wherein the sample substrate is inside a fluidic chamber.

9. The imaging system according to claim 1, wherein the sub-wavelength particles are immobilized on a top of the sample substrate.

10. The imaging system according to claim 1, wherein the sample substrate is a flat surface.

11. The imaging system according to claim 1, wherein the sample substrate is a non-flat surface.

12. The imaging system according to claim 1, wherein the sub-wavelength particles are immobilized on top of other larger particles comprising biological molecules, and the reference light is created and interferes at the camera with the scattered light from the sub-wavelength particles.

13. The imaging system according to claim 1, wherein at least one light source is an array of LEDs comprising at least one LED.

14. The imaging system according to claim 13, wherein the LEDs are of the same or different wavelengths.

15. The imaging system according to claim 1, wherein the sample substrate comprises a dielectric layer.

16. The imaging system according to claim 1, wherein a dielectric layer thickness of the sample substrate is adjusted according to a wavelength of the illumination to provide a maximum interference signal.

17. The imaging system according to claim 1, wherein a sample comprising immobilized nanoparticles on a top of the sample substrate is illuminated by the light source in a wide field illumination configuration.

18. The imaging system according to claim 7, wherein the correlation image is calculated using the defocused images.

19. The imaging system according to claim 7, wherein the correlation image is calculated using the defocused images and the focused images.

20. The imaging system according to claim 1, wherein a visibility of the nanoparticles is improved by using a difference of a defocusing response of the sub-wavelength particles and a background region.

21. The imaging system according to claim 1, wherein the sub-wavelength particles are smaller than 100 nm.

22. The imaging system according to claim 1, wherein the sub-wavelength particles are smaller than 50 nm.

23. The imaging system according to claim 1, wherein the sub-wavelength particles are smaller than 100 nm and bigger than 10 nm.

24. The imaging system according to claim 1, wherein the sub-wavelength particles are immersed in a liquid.

25. The imaging system according to claim 1, wherein the sub-wavelength particles are extracellular vesicles comprising exosomes or viruses.

26. The imaging system according to claim 1, wherein the sub-wavelength particles are cells, viruses, or bacteria.

27. The imaging system according to claim 1, wherein the camera forms an image using a scanning detector.

28. The imaging system according to claim 1, wherein a microscope objective compensates for aberrations when the sub-wavelength particles are immersed in a liquid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Accompanying drawings are given solely for the purpose of exemplifying an apparatus for high-contrast imaging of sub-wavelength size particles, whose advantages over prior art were outlined above and will be explained in brief hereinafter.

[0021] The drawings are not meant to delimit the scope of protection as identified in the claims nor should they be referred to alone in an effort to interpret the scope identified in said claims without recourse to the technical disclosure in the description of the present invention.

[0022] FIG. 1 is an interferometric imaging system for implementing the method in accordance with this invention.

[0023] FIGS. 2A-2C show focused and defocused responses of dielectric nanoparticles in accordance with this invention. FIG. 2A shows orientation of the nanoparticles in the simulations.

[0024] FIG. 2B shows the nanoparticle contrast calculated. FIG. 2C shows the calculated images of the particle.

[0025] FIG. 3 shows calculated contrast values for different axial positions of the sample stage in accordance with this invention.

[0026] FIG. 4 shows depth scanning correlation enhancement procedure in accordance with this invention.

[0027] FIGS. 5A-5D show visibility enhancement for nanoparticles in accordance with this invention. FIGS. 5A and 5B show nanoparticles with a diameter of 50 nm that are immobilized on the substrate and imaged without depth scanning correlation (DSC) technique. FIGS. 5C and 5D show nanoparticles with a diameter of 50 nm that are immobilized on the substrate and imaged with depth scanning correlation (DSC) technique.

[0028] FIGS. 6A-6D show a comparison between correlation image and SEM image in accordance with this invention. FIG. 6A shows the correlation image, and FIGS. 6B-6D show the SEM images of the particles identified in the correlation image.

[0029] FIGS. 7A-7B show a comparison between correlation image and interferometric image, FIG. 7A shows a conventional image and FIG. 7B shows depth scanning correlation image in accordance with this invention.

[0030] FIGS. 8A-8H show intensity variation with respect to defocus for particle (black line) and background (dotted line) pixels in accordance with this invention. FIGS. 8B, 8D, 8F, and 8H respectively show the correlation image generated by the analysis using the axial ranges shown in FIGS. 8A, 8C, 8E, and 8G.

[0031] FIG. 9 shows the data acquisition and analysis flow chart in accordance with this invention.

REFERENCED PARTS LIST

[0032] 11 Light source [0033] 12 Beam splitter [0034] 13 Movable means [0035] 14 Camera [0036] 15 Sample substrate [0037] 16 Objective lens [0038] 17 Lens [0039] 18 Imaging system

[0040] Abbreviations used in the detailed description of the invention are listed below:

TABLE-US-00001 E.sub.inc) Incident field r) Reflection coefficient of the sample plate s) Scattering amplitude of the particle Ø) Phase difference between reference and scattering fields I.sub.t(x, y)) Pixel's intensity captured at time t R(t)) Actuation signal's level at time t σ.sub.I) Standard deviation of pixel intensity σ.sub.R) Standard deviation of actuation signal

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] FIG. 1 demonstrates an imaging system (18) for enhancing visibility of nanoparticles according to the present invention. The imaging system (18) comprises various lenses (17) and a processing unit. In a variation, the imaging system (18) can employ a Michelson-type interferometer configuration. It is illuminated by a light source (11), which in a preferred embodiment has, high spatial coherence (small emission area) and high temporal coherence (limited spectral width). Light emitting diodes (LED), VCSELs, and laser light sources can be used for the purpose. The imaging system (18) comprises a detector array, for instance a CCD or CMOS based camera (14), abeam splitter (12) that makes it possible to send into the interferometer the wave emitted by the light source (11) and an objective lens (16). A movable means (13) allows for an axial displacement of a sample substrate (15). The acquisition in depth in the sample is produced by an axial displacement of the sample relative to the camera (14). In a variation, the objective lens (16) is a zoom objective. Wide-field illumination can be provided through sample illuminated in Kohler configuration. In a variation, as a sample substrate (15), Si/SiO.sub.2 surface with 100 nm oxide thickness is used. The oxide thickness can be varied and optimized for the wavelengths used in the system to control the interference between the scattered light and the reference light. Scattered field from nanoparticles together with the reference field, reflected from layered substrate is imaged onto the camera (14) using the objective lens (16). Preferably, objective lens (16) is a 40× or higher magnification objective. Detected signal intensity can be written as:

I.sub.det=|E.sub.ref+E.sub.sca|.sup.2=r.sup.2E.sub.inc+s.sup.2E.sub.inc+2.Math.r.Math.s.Math.cos ϕE.sub.inc  Eq 1

[0042] In contrast to purely scattering based techniques where particle signal is scaled with the square of the volume, in interferometric techniques interference term in Eq. (1) is scaled with s and thereby the volume of the particle. Interferometric detection is implemented by using a reflective sample substrate (15). In addition to interferometric enhancement, nanoparticles have unique defocusing response that can be used for further enhancement

[0043] FIGS. 2A-2C show simulation results of the defocusing response of dielectric nanoparticles on top of sample substrate (15). In the simulations, nanoparticles are modeled as point dipoles with an orientation determined by the illumination field (FIG. 2A). Due to spatial incoherence of the light source (11), for instance LED, illumination field is modeled as incoherent sum of plane waves covering the illumination angle defined by NA (numerical aperture) of the objective lens (16). For each of the plane wave image of the dipole is calculated using the PSF (point spread function) of the imaging system (FIG. 2B). In the final step, image of the particle is calculated by summing the individual dipole images (FIG. 2C).

[0044] FIG. 3 represents focusing response of polystyrene nanoparticles with 100 nm diameter. In this variation, to capture defocused particle images, sample is placed on a movable means (13) and axial position is modulated with intervals of 100 nm. Image of nanoparticle is highly sensitive to axial position of the sample. Particles can induce positive or negative contrast according to its axial position.

[0045] FIG. 4 shows discrimination particles from the background, movable means (13) is actuated towards the camera (14) with a sawtooth pattern and a cross-correlation analysis is performed between actuation signal and every pixel in the captured images. Pearson correlation coefficient ρ is calculated for every pixel location (x, y) as follows:

[00001]$\begin{array}{cc}{I}_{\mathrm{corr}}\left(x,y\right)={.Math.\rho \left(x,y\right).Math.}^2={.Math.\frac{1}{N-1}\underset{t=t_0}{\overset{t_{N-1}}{.Math.}}\left(\frac{I_t\left(x,y\right)-\stackrel{\_}{1\left(x,y\right)}}{{\sigma }_I}\right)\left(\frac{R\left(t\right)-\stackrel{\_}{R}}{{\sigma }_R}\right).Math.}^2& \mathrm{Eq}2\end{array}$

[0046] where I.sub.t(x,j) is the pixel's intensity captured at time t, R(t) actuation signal's level at t, σ.sub.I and σ.sub.R are the standard deviation of pixel intensity and actuation signal respectively; <I(x,y)> and <R> mean values of pixel intensity and actuation signal over one period. Correlation image is composed of square of the correlation values of each pixel. According to Eq. (2), ρ can get values between −1 and 1. Zero means highly uncorrelated signal, one means highly correlated signal. In order to obtain a highly correlated signal for the background signal to distinguish nanoparticles from the background noise level, illumination is slightly tuned to have a converging beam on the sample to vary the background with the movement of the movable means (13). Hence, inverse relation between background signal intensity and axial position of the sample is achieved (<%0.5 variation in background signal over one period). Final correlation image can represent highly correlated (background) pixels as white and uncorrelated (nanoparticle) pixels as black.

[0047] FIGS. 5A-5D show enhanced visibility of the nanoparticles with higher SNR. Particles with a diameter of 50 nm are immobilized on the substrate and imaged with and without depth scanning correlation (DSC) technique. After an initial rough focusing, the movable means (13) is actuated with 100 nm steps for a total of 2 μm in sawtooth pattern. DSC technique enhances the signal to noise ratio (SNR) of individual nanoparticles. SNR can be further increased, by increasing the number of periods in the analysis.

[0048] FIGS. 6A-6D demonstrate that depth-scanning correlation enhancement can also be used to detect even smaller nanoparticles. Developed optical system is capable of detecting samples (for example; polystyrene nanoparticles) as small as 33 nm in diameter with SNR of about 14, which is also verified by SEM imaging. Assuming the volume dependence of the signal, the detection of nanoparticles smaller than 20 nm nanoparticles seem possible.

[0049] FIGS. 7A-7B show the sensitivity capability of the system for biological nanoparticles. Single unlabeled exosomes with a diameter of <50 nm to sample substrate (15) are coated. In FIG. 7A, conventional interferometric image of the sample is shown. Implementation of the depth scanning correlation technique significantly enhanced the visibility of the particles and improved the sensitivity limit as it can be seen in FIG. 7B.

[0050] FIGS. 8A-8H show the effect of analysis range on correlation image. During analysis, optimum analysis range is selected to have highest signal to noise ratio (SNR).

[0051] FIG. 9 demonstrates data acquisition and analysis flow chart. First defocused image stack is captured, then correlation analysis is done to generate a correlation image. Once particles are detected and SNR for each particle is measured, defocusing range [z1, z2] is updated until the optimum SNR for the particle is calculated.

[0052] In a nutshell, the present invention proposes an imaging system (18) that works with much higher contrast and sensitivity compared to traditional microscopy instruments. With their unique defocusing response, a set of images are captured to gather more information about the requested pixels. Correlation image is provided through the correlation analysis. Correlation analysis involves actuation signal which moves the movable means (13), pixel intensity of nanoparticle and pixel intensity of background for all of the frames that are captured. It is experimented that the imaging system (18) can be used to direct detection of dielectric nanoparticles as small as 33 nm in diameter without using any optical or mechanical resonant behavior.

[0053] A particular feature, structure, or characteristic described herein in connection with one embodiment may be implemented within other embodiments without departing from the scope of the invention. In addition, it is to be noted that the location or arrangement of individual elements within each disclosed embodiment may be modified without departing from the scope of the invention. The detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims to be interpreted along with the full range of equivalents to which the claims are entitled.

[0054] In one embodiment of the present invention, an imaging system (18) for detection of dielectric nanoparticles comprises at least one light source (11) for illumination, a camera (14) for image capture, an objective lens (16), a sample substrate (15) and a computing unit.

[0055] In a further embodiment of the present invention, said sample substrate (15) is capable of carrying sub-wavelength particles smaller than the diffraction resolution limit of the imaging system.

[0056] In a further embodiment of the present invention, said imaging system (18) comprises a movable means (13) which moves the sample substrate (15) in the axial direction such that depthwise different images are captured at different axial distances from the sample substrate (15) to said objective lens (16).

[0057] In a further embodiment of the present invention, said computing unit computes a correlation image using the depth images wherein the sub-wavelength particles become resolvable and appear with higher contrast in the correlation image.

[0058] In a further embodiment of the present invention, said light source (11) provides a nearly collimated illumination beam on the sample plate such that the background illumination on the images vary as the movable means (13) is moved.

[0059] In a further embodiment of the present invention, said light source (11) provides a collimated illumination beam on the sample substrate (15).

[0060] In a further embodiment of the present invention, said light source (11) provides an uncollimated illumination beam on the sample substrate (15).

[0061] In a further embodiment of the present invention, said light source (11) provides a converging beam on the sample substrate (15).

[0062] In a further embodiment of the present invention, said light source (11) provides a diverging beam on the sample substrate (15).

[0063] In a further embodiment of the present invention, said objective lens (16) provides magnification.

[0064] In a further embodiment of the present invention, said depthwise images are a collection of focused and defocused images.

[0065] In a further embodiment of the present invention, said sample substrate (15) reflects some of the scattered light creating reference light.

[0066] In a further embodiment of the present invention, said sample substrate (15) is inside a fluidic chamber.

[0067] In a further embodiment of the present invention, the particles are immobilized on top of said sample substrate (15).

[0068] In a further embodiment of the present invention, said sample substrate (15) is a flat surface.

[0069] In a further embodiment of the present invention, said sample substrate (15) is a non-flat surface.

[0070] In a further embodiment of the present invention, said the particles are immobilized on other larger particles which are resolvable in captured images.

[0071] In a further embodiment of the present invention, at least one light source (11) is an array of LEDs.

[0072] In a further embodiment of the present invention, LEDs are of the same or different wavelengths.

[0073] In a further embodiment of the present invention, the sample substrate (15) comprises a dielectric layer.

[0074] In a further embodiment of the present invention, dielectric layer thickness of the sample substrate (15) is adjusted according to the wavelength of illumination to provide maximum interference signal.

[0075] In a further embodiment of the present invention, sample comprising the immobilized nanoparticles on top of sample substrate (15) is illuminated by the light source (11) in wide field illumination configuration.

[0076] In a further embodiment of the present invention, correlation image is calculated using defocused images.

[0077] In a further embodiment of the present invention, correlation image is calculated using defocused and focused images.

[0078] In a further embodiment of the present invention, visibility of particles is improved by using difference of defocusing response of particles and background region.

[0079] In a further embodiment of the present invention, said imaging system (18) detects particles that are smaller than 100 nm.

[0080] In a further embodiment of the present invention, said imaging system (18) detects particles that are smaller than 50 nm.

[0081] In a further embodiment of the present invention, said imaging system (18) detects particles that are smaller than 100 nm and bigger than 10 nm.

[0082] In a further embodiment of the present invention, said imaging system (18) detects particles that are smaller than 100 nm and bigger than 10 nm.

[0083] In a further embodiment of the present invention, the particles are immersed in liquid.

[0084] In a further embodiment of the present invention, imaging system (18) detects particles that are extracellular vesicles such as exosomes

[0085] In a further embodiment of the present invention, imaging system (18) detects particles that are cells, viruses, or bacteria.