COMMON-PATH INTERFEROMETRIC SCATTERING IMAGING SYSTEM AND A METHOD OF USING COMMON-PATH INTERFEROMETRIC SCATTERING IMAGING TO DETECT AN OBJECT

Abstract

The present invention relates to a common-path interferometric scattering imaging system and a method of using such a system, where the system includes an illuminating unit for emitting an illumination beam; a light collecting arrangement for collecting through a common collection optical path a scattered beam provided by the light scattering on an object of the illumination beam and a reference beam provided by the reflection on or transmission through an interface of the illumination beam; an image sensor (D) for receiving and sensing the collected scattered and reference beams interfering thereon as an interferometric light signal; an attenuation mechanism arranged in the common collection optical path for attenuating the reference beam before it arrives at the image sensor; and a processor to process data corresponding to the interferometric light signal.

Claims

1. A common-path interferometric scattering imaging system, comprising: an illuminating unit comprising a light source configured and arranged for emitting an illumination beam along an illumination optical path including at least two different phases of matter; a light collecting arrangement configured and arranged for simultaneously at least partially collecting through a common collection optical path: a scattered beam provided by the light scattering by an object of a portion of said illumination beam, wherein said object is placed in at least one of said two different phases of matter; and a reference beam provided by the reflection on or transmission through an interface of another portion of said illumination beam (L.sub.0), wherein said interface is a surface forming a common boundary among said two different phases of matter; an image sensor configured and arranged for receiving and sensing the collected scattered and reference beams interfering thereon as an interferometric light signal; a processor connected to said image sensor (D) to receive data corresponding to said interferometric light signal, and configured to process said received data to at least detect said object; and an attenuation mechanism arranged in said common collection optical path for attenuating said reference beam before it arrives at said image sensor, and in that said illumination optical path and said common collection path are configured and arranged such that the reference and scattered beams are generated at such closer positions that ensure a phase-locked relationship between the reference and scattered beams, the system being absent of any phase varying mechanism for said reference and scattered beams.

2. The system of claim 1, wherein said attenuation mechanism comprises: a partially transmissive mask having a semi-transmissive region arranged in a corresponding region of the common collection path through which the reference beam travels, such that the reference beam is attenuated on transmission before reaching the image sensor; or a partially reflective mask having a semi-reflective region arranged in a corresponding region of the common collection path through which the reference beam travels, such that the reference beam is attenuated on reflection before reaching the image sensor.

3. The system of claim 2, wherein said semi-transmissive or semi-reflective region is a first region of said partially transmissive or partially reflective mask, the mask comprising a second region arranged in a corresponding region of the common collection path through which part of the scattering beam travels, such that said part of the scattering beam traverses said second region or is reflected thereon before reaching the image sensor, by transmission or by reflection, wherein said first and said second regions have different transmissive or reflective properties and said partially transmissive or partially reflective mask maintains the coherence relationship between the reference and scattered beams.

4. The system of claim 3, wherein said second region is a fully or substantially fully transmissive or reflective region.

5. The system of claim 3, wherein said first region has a circular or cylindrical shape and said second region has an annular or tubular shape with an inner diameter larger than the diameter of said first region and being arranged concentrically with respect thereto.

6. The system of claim 2, wherein said mask is arranged symmetrically and inline with the reference and scattered beams, to obtain reliable and symmetric interference patterns on the image sensor.

7. The system of claim 1, comprising a coverslip for said object, wherein said interface is the common boundary surface among said coverslip and a medium into which said object is placed, the material of which said coverslip is made being non-index matched with said medium.

8. The system of claim 1, wherein said light collecting arrangement is configured and arranged for collecting said reference beam provided by the reflection on said interface of said another portion of the illumination beam, wherein the system comprises an objective lens which forms part of both the illuminating unit and the light collecting arrangement and which is configured and arranged in both the illumination and the collection optical paths to, respectively: focus the illumination beam into the back-focal plane of said objective lens to produce illumination out of the front aperture of the objective lens, such that a portion thereof will be reflected by the interface generating the reference beam and the rest will pass through the interface up to the object generating the scattering beam; and receive and at least partially collect both the reference beam and the scattering beam.

9. The system of claim 3, wherein said light collecting arrangement is configured and arranged for collecting said reference beam provided by the reflection on said interface of said another portion of the illumination beam, wherein the system comprises an objective lens which forms part of both the illuminating unit and the light collecting arrangement and which is configured and arranged in both the illumination and the collection optical paths to, respectively: focus the illumination beam into the back-focal plane of said objective lens to produce illumination out of the front aperture of the objective lens, such that a portion thereof will be reflected by the interface generating the reference beam and the rest will pass through the interface up to the object generating the scattering beam; and receive and at least partially collect both the reference beam and the scattering beam; and wherein said objective lens is configured and arranged such that the reference beam exits the objective lens as a diverging beam from the centre of the objective lens, and passes through or is reflected on the first region of the partially transmissive or partially reflective mask, and the scattering beam leaves the objective lens as a plane wave across a full back-aperture of the objective lens, when it entered as a spherical wave, and passes through or is reflected on both the first and the second regions of the partially transmissive or partially reflective mask.

10. The system of claim 9, wherein said first region of the partially transmissive or partially reflective mask is also placed in the illumination optical path and is configured and arranged to reflect the illumination beam coming from the light source towards the back-focal plane of the objective lens.

11. The system of claim 1, wherein said light collecting arrangement is configured and arranged for collecting said reference beam provided by the transmission through said interface of said another portion of the illumination beam, wherein: the illuminating unit comprises an illumination objective lens configured and arranged to focus the illumination beam into the back-focal plane of said illumination objective lens to produce plane-illumination out of the front aperture of the illumination objective lens, such that a portion thereof will be scattered by the object generating the scattering beam which will be transmitted through the interface, and another portion will be directly transmitted through the interface generating the reference beam; and the light collecting arrangement comprises a collection objective lens configured and arranged to receive and at least partially collect both the reference beam and the scattering beam.

12. The system of claim 3, wherein said light collecting arrangement configured and arranged for collecting said reference beam provided by the transmission through said interface of said another portion of the illumination beam, wherein: the illuminating unit comprises an illumination objective lens configured and arranged to focus the illumination beam into the back-focal plane of said illumination objective lens to produce plane-illumination out of the front aperture of the illumination objective lens, such that a portion thereof will be scattered by the object generating the scattering beam which will be transmitted through the interface, and another portion will be directly transmitted through the interface generating the reference beam; and the light collecting arrangement comprise a collection objective lens configured and arranged to receive and at least partially collect both the reference beam and the scattering beam; and wherein said collection objective lens is configured and arranged such that the reference beam exits the collection objective lens as a diverging beam from the centre of the collection objective lens, when it entered as a plane wave, and passes through or is reflected on the first region of the partially transmissive or partially reflective mask, and the scattering beam leaves the collection objective lens as a plane wave across a full back-aperture of the collection objective lens, when it entered as a spherical wave, and passes through or is reflected on both the first and the second regions of the partially transmissive or partially reflective mask.

13. The system of claim 8, wherein the first region of the partially transmissive or partially reflective mask is configured to highly attenuate the reference beam so that its beam intensity is reduced below 1%.

14. The system of claim 13, wherein the first region of the partially transmissive or partially reflective mask is configured to highly attenuate the reference beam so that its beam intensity is reduced below 0.1%.

15. The system of claim 12, wherein the first region of the partially transmissive or partially reflective mask is configured to highly attenuate the reference beam so that its beam intensity is reduced below 1%.

16. The system of claim 15, wherein the first region of the partially transmissive or partially reflective mask is configured to highly attenuate the reference beam so that its beam intensity is reduced below 0.1%.

17. The system of claim 1, wherein said processor implements an algorithm to process the received data according to the following equation: $I_{total} = I_{0} .Math. {\frac{r^{2}}{^{2}} + s^{2} + \frac{2 .Math. rs}{} .Math. \cos .Math. .Math.}$ where r is the normalised reference beam amplitude, s is the normalised scattering beam amplitude, is the phase difference between the reference and scattering beams, is the attenuation amplitude defined as the reciprocal transmission amplitude, I.sub.total is the total intensity of the light on the image sensor caused by the two interfering reference and scattering beams, and I.sub.0 is an initial light intensity on the image sensor, wherein said attenuation mechanism have a degree of attenuation for said reference beam calculated with the purpose of maximizing the term 2rs cos with respect to the term r.sup.2 of the above equation to enhance detection sensitivity.

18. The system of claim 17, wherein <0.1.

19. The system of claim 18, wherein is around 0.03.

20. The system of claim 1, wherein said light source is a coherent or substantially coherent light source.

21. The system of claim 1, further comprising an interference reduction arrangement for reducing spurious out of plane interferences at the imaging plane where the image sensor is placed for receiving and sensing the collected scattered and reference beams interfering thereon as an interferometric light signal.

22. The system of claim 21, wherein said interference reduction arrangement comprises a modulation unit to temporarily modulate said light source at a rate from 1 KHz to 1000 MHz, to reduce spurious interference fringes at the imaging plane, through destabilised modes and/or broadened bandwidth, thus reducing coherence length of the light source.

23. The system of claim 21, wherein said interference reduction arrangement comprises using, as said light source, a variable band width or broadband laser, with selected spectral region, of wavelength range between 0.1 nm and 1000 nm, to reduce spurious interference effects at the imaging plane.

24. The system of claim 21, wherein said interference reduction arrangement comprises, as said light source, a light-source with significantly reduced temporal coherence length compared to a light-emitting diode (LED) of high intensity, to reduce spurious interference fringing and related effects at the imaging plane.

25. The system of claim 21, wherein said interference reduction arrangement comprises a mechanical mechanism configured and arranged to modulate the illumination beam in free-space or within an optical fibre, to distort the mode profile and/or blur the spatial distribution of the illumination beam on the imaging plane to reduce spurious interferences.

26. The system of claim 1, wherein said light source is a continuous light source.

27. The system of claim 1, wherein said light source is a pulsed light source configured and arranged for emitting a pulsed illumination beam of temporal width in a picosecond order or femtosecond order.

28. The system of claim 1, wherein said light source is a white-light broadband light source.

29. The system of claim 1, wherein said interface is one of a glass/water interface and an glass/air interface.

30. A method of using common-path interferometric scattering imaging to detect an object, comprising: emitting an illumination beam along an illumination optical path including at least two different phases of matter; simultaneously at least partially collecting through a common collection optical path: a scattered beam provided by the light scattering on an object of a portion of said illumination beam, wherein said object is placed in at least one of said two different phases of matter; and a reference beam provided by the reflection on or transmission through an interface of another portion of said illumination beam, wherein said interface is a surface forming a common boundary among said two different phases of matter; receiving and sensing, on an image sensor, the collected scattered and reference beams interfering thereon as an interferometric light signal; receiving and processing data corresponding to said interferometric light signal to at least detect said object; and attenuating said reference beam in said common collection optical path before it arrives at said image sensor, and in that the method comprises configuring and arranging said illumination optical path and said common collection path such that the reference and scattered beams are generated at such closer positions that ensure a phase-locked relationship between the reference and scattered beams, the method being absent of any phase varying step caused by any phase varying mechanism for said reference and scattered beams.

31. A method of claim 30, wherein said object is a dielectric nanoparticle with a size of substantially 10 kDa or below.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0105] In the following some preferred embodiments of the invention will be described with reference to the enclosed figures. They are provided only for illustration purposes without however limiting the scope of the invention.

[0106] FIG. 1 shows two plots taken from iSCAT paper by Piliarik et al. (2014) showing iSCAT contrast achieved for different molecular weight proteins (a) and the signal noise as a function of frame averaging (b) when the camera is run at 3000 fps, indicating the shot-noise limit. Wavelength employed: 405 nm, 10 mW at 4.54.5 m field of view=approx. 50 kW/cm.sup.2.

[0107] FIG. 2 shows equivalent measurements to the iSCAT measurements by Piliarik et al. (2014) performed using the system of the first aspect of the present invention. Mean contrast is plotted against protein weight (a) and signal noise as a function of equivalent camera frame rate (b). Wavelength employed: 520 nm, 33 mW at 1010 m field of view=approx. 35 kW/cm.sup.2.

[0108] FIG. 3 schematically shows the system of the first aspect of the invention for an embodiment implementing a reflective mode arrangement, where the reference beam is reflected on an interface.

[0109] FIG. 4 schematically shows the system of the first aspect of the invention for an embodiment implementing a transmissive mode arrangement, where the reference beam is transmitted through an interface.

[0110] FIG. 5 shows further measurements performed using the system of the first aspect of the present invention. Detection limit is plotted against camera frame rate and compared to existing iSCAT system based on extracted published data.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0111] FIGS. 3 and 4 show two alternative implementations of the system of the first aspect of the invention, particularly the above mentioned reflective mode implementation (FIG. 3) and transmissive mode implementation (FIG. 4), both of which relate to a common-path interferometric scattering imaging system, comprising:

[0112] illuminating means comprising a light source S configured and arranged for emitting an illumination beam L.sub.0 along an illumination optical path including two different phases of matter, one of which is constituted by the material from which the coverslip C is made (generally glass) and the other one by the medium W (in this case water) into which the objects T (in this case nanoparticles, such as proteins) are placed;

[0113] light collecting means configured and arranged for simultaneously at least partially collecting through a common collection optical path: [0114] a scattered beam L.sub.s provided by the light scattering on several objects T of a portion of the illumination beam L.sub.0; and [0115] a reference beam L.sub.r provided by the reflection on or transmission through an interface I of another portion of said illumination beam L.sub.0, wherein the interface I is a surface forming a common boundary surface among the coverslip C and medium W;

[0116] attenuation means comprising a partially transmissive mask M arranged in the common collection optical path for attenuating said reference beam L.sub.r before it arrives at image sensing means D;

[0117] image sensing means D (generally including an imaging lens and a camera) configured and arranged for receiving and sensing the collected scattered L.sub.s beam and the reference L.sub.r beam, once attenuated by the mask M, interfering thereon as an interferometric light signal; and

[0118] processing means P connected to the image sensing means D to receive data corresponding to the interferometric light signal, and configured to process the received data to at least detect the objects T.

[0119] For both arrangements, of FIGS. 3 and 4, the partially transmissive mask M has a semi-transmissive first region M1 arranged in a corresponding region of the common collection path through which the reference beam L.sub.r travels, such that the reference beam L.sub.r is attenuated before reaching the image sensing means D, and a fully or substantially fully transmissive second region M2 arranged in a corresponding region of the common collection path through which part of the scattering beam L.sub.s travels, such that it is traversed thereby before reaching the image sensing means D.

[0120] As shown in FIG. 3, for the implementation there illustrated, the light collecting means are configured and arranged for collecting the reference beam L.sub.r provided by the reflection on the interface I of the above mentioned another portion of the illumination beam L.sub.0, and the system comprises an objective lens OL which forms part of both the illuminating means and the light collecting means and which is configured and arranged in both the illumination and the collection optical paths to, respectively:

[0121] focus the illumination beam L.sub.0 into the back-focal plane of the objective lens OL to produce plane-illumination out of the front aperture of the objective lens OL, such that a portion thereof will be reflected by the interface I generating the reference beam L.sub.r and the rest will pass through the interface I up to the objects T generating the scattering beam; and

[0122] receive and at least partially collect both the reference beam L.sub.r and the scattering beam L.sub.s.

[0123] The objective lens OL is configured and arranged such that the reference beam L.sub.r exits the objective lens OL as a diverging beam from the centre of the objective lens OL, when it entered as a plane wave, and impinges on the first region M1 of the partially transmissive mask M which highly attenuates it letting pass through there only a small percentage (preferably below 1%, and more preferably around 0.1% in terms of beam intensity, or equivalently relative to field amplitude with an attenuation factor preferably below =0.1 or more preferably around =0.03) of the reference beam L.sub.r, while the scattering beam L.sub.s leaves the objective lens OL as a plane wave across a full back-aperture of the objective lens OL, when it entered as a spherical wave, and passes mostly through the fully or substantially fully transmissive second region M2 of the partially transmissive mask M, although a central part of the scattering beam L.sub.r passes through the first region M1 of the mask M and is thus attenuated thereby.

[0124] As shown FIG. 3, the first region M1 of the partially transmissive mask M is also placed in the illumination optical path and is configured and arranged to reflect the illumination beam L.sub.0 coming from the light source S towards the back-focal plane of the objective lens OL.

[0125] Specifically, for the reflective mode implementation of FIG. 3, the system of the present invention constitutes a stand-alone microscope imaging system based on reflection scattering as described above and in more detail as follows: [0126] 1. Light of short temporal-coherence length is created by modulating the supply current of a standard laser-diode (light source S) at high frequency (>1 MHz) which is commonly implemented in consumer laser systems such as Blu-ray players. This decreases the laser coherence length to reduce interference of objects outside of the range of interest. This is preferably as short as possible, but long enough to keep coherence between the source of the reflection as the reference beam L.sub.r, for the illustrated embodiment, the glass-water interface between the coverslip C, and the particle T positioned on top of this. A super-bright LED or similar short coherence length light source could be used instead of modulated laser. [0127] 2. Light is focused into the back-focal plane of the objective lens OL to produce plane-illumination out of the front aperture of the objective lens OL. The interference mask M is used here as a mirror to simplify coupling of the incident beam L.sub.0 into the objective lens OL (although this can be accomplished in other ways). [0128] 3. The plane illumination beam is partially reflected from the non-index matched glass-water interface I of the objective lens OL generating the reference beam L.sub.r. The rest of the illumination beam L.sub.0 passes through the interface I interacting with the sample T in the water W. This light principally generates Rayleigh scattering (in small particles) in-phase with the transmitted light. The scattered light can be approximated as a point-source of light emitting spherical waves propagating in all directions and this is the sample or scattering beam L.sub.s. The phase of the scattered beam L.sub.s is shifted relative to the incoming beam L.sub.0 due to a Gouy phase shift of pi/2. [0129] 4. The reference L.sub.r and scattered sample beam L.sub.s are partially collected by the same objective lens OL. The sample beam L.sub.s leaves the objective lens OL as a plane wave across the full back-aperture of the objective lens OL since it entered as a spherical wave. The reference beam L.sub.r (entering as a plane wave) exits the objective lens as a diverging beam from the centre of the objective lens OL. [0130] 5. The sample beam L.sub.s impinges on the interference mask M and is mostly transmitted in the transparent regions M2 surrounding the centre, with the central part M1 of the mask M blocking only a small percentage of this beam [as in dark-field microscopy]. The reference beam L.sub.r hits the centre region M1 of the interference mask M and is almost completely attenuated. However, importantly the centre region M1 of the mask M leaks some of the reference beam L.sub.r through to allow for interference on the detector, i.e. on the image sensing means D (which includes at least an imaging lens and a camera). [0131] 6. The sample plane is then imaged onto a camera of the image sensing means D where the two beams L.sub.r, L.sub.s interfere providing the contrast to measure the particles T.

[0132] In contrast to the implementation of FIG. 3, for the transmissive mode implementation of the system of the first aspect of the invention illustrated in FIG. 4, the light collecting means are configured and arranged for collecting the reference beam L.sub.r provided by the transmission through the interface I of the above mentioned other portion of the illumination beam L.sub.0, wherein:

[0133] the illuminating means comprises an illumination objective lens OLi configured and arranged to focus the illumination beam L.sub.0 into the back-focal plane of the illumination objective lens OLi to produce plane-illumination out of the front aperture of the illumination objective lens OLi, such that a portion thereof will be scattered by the objects T generating the scattering beam L.sub.s which will be transmitted through the interface I, and another portion will be directly transmitted through the interface I generating the reference beam L.sub.r; and

[0134] the light collecting means comprise a collection objective lens OLc configured and arranged to receive and at least partially collect both the reference beam L.sub.r and the scattering beam L.sub.s.

[0135] The collection objective lens OLc is configured and arranged such that the reference beam L.sub.r exits the collection objective lens OLc as a diverging beam from the centre of the collection objective lens OLc, when it entered as a plane wave, and passes through the first region M1 of the partially transmissive mask M which highly attenuates it letting pass there through only a small percentage (preferably below 1%, and more preferably around 0.1% in terms of beam intensity, or equivalently relative to field amplitude with an attenuation factor preferably below =0.1 or more preferably around =0.03) of the reference beam L.sub.r, while the scattering beam L.sub.s leaves the collection objective lens OLc as a plane wave across a full back-aperture of the collection objective lens OLc, when it entered as a spherical wave, and passes mostly through the fully or substantially fully transmissive second region M2 of the partially transmissive mask M, although a central part of the scattering beam L.sub.r passes through the first region M1 of the mask M and is thus attenuated thereby.

[0136] Specifically, for the transmissive mode implementation of FIG. 4, the system of the present invention constitutes a stand-alone microscope imaging system based on transmission scattering as described above and in more detail as follows.

[0137] In transmissive-mode, the microscope operates mainly the same as in reflective-mode. More optics (principally a second objective) are required as a new excitation path from above the sample is needed.

[0138] The principle remains the same as that in reflection with a reference beam L.sub.r and scattering signal beam L.sub.s generated by a single excitation light source S then interfere on a detector D after the reference beam L.sub.r is partially attenuated by a partially transmissive mask M or equivalent.

[0139] The main difference here is that the reference beam L.sub.r will be much stronger in intensity, as here it is almost 100% the intensity t.sub.0 of the incident beam L.sub.0, as most of the light is transmitted rather than reflected by the interface I. This differs in the reflective-mode case, as in that case the reference beam L.sub.r is generated by the reflection off the glass/water interface I, which reduces its intensity to around 0.5% of the initial beam L.sub.0.

[0140] In practice this means that in transmissive mode, the mask M must attenuate the reference beam L.sub.r by at least one order of magnitude more compared to the reflective mode case. This potentially complicates the production of the mask M. Along with simpler optics, this highlights the distinctive benefit of the reflective mode case where the reference beam L.sub.r is pre-attenuated by the glass/water interface I. However, given a suitable mask, both are equivalent.

Mask Construction:

[0141] Regarding the above described mask M, it was built for its inclusion in the system of the present invention, for an embodiment (for the arrangements of FIGS. 3 and 4), as follows: [0142] 1. The mask M consists of a semi-transmissive section and a transmissive section. [0143] 2. The semi-transmissive section M1 was created by depositing metal onto a sacrificial premask defining the semi-transmissive region on an optical flat. [0144] 3. A vinyl sticker was used as a pre-mask to define the area. [0145] 4. Metal was evaporated at the desired thickness to attenuate signal passing through, ensuring metal was evenly deposited.

[0146] The mask M itself can be constructed in many different forms and materials depending on availability and exact implementation needed. A well-formed mask with precise thickness is key to obtaining reliable and symmetric interference patterns on the detector.

[0147] Specifically, as stated in a previous section, for an embodiment (not shown), the collection of both the reference and the scattering beams is performed on reflecting from the mask, the latter having a semi-reflective section (almost transparent) for the reference beam (thus attenuated by reflection) and a reflective section for the scattering beam.

[0148] Also, for the manufacturing of the mask, instead of metallic coatings, dielectric anti-reflective/reflective Bragg type coatings can be used, for other embodiments.

Technical Advantage

[0149] The technique used in the system and method of the present invention significantly improves on the published conventional iSCAT technique (described in the Background section above) while allowing better contrast and sensitivity. Here the benefit of the technique of the present invention over iSCAT, the best implementation as yet of interferometric light scattering microscopy, is elaborated.

[0150] In general, in interference scattering microscopy, the signal imaged onto the detector has intensity:

I.sub.0=I.sub.0{r.sup.2+s.sup.2+2rs cos }

[0151] Where, as stated in a previous section, r is a co-efficient describing the amplitude of the reference beam, s is a co-efficient relating to the amplitude of the scattering signal, and is the phase difference between the two signals. For detecting small particles such as proteins the difference between r.sup.2 and s.sup.2 is many orders of magnitude (around 10.sup.7 for a 100 kDa protein) making it practically impossible to measure the scattering signal upon the background of the reference beam. Crucially the interference term, scales both with r and s, meaning there is much less difference between this and the r.sup.2 term, only around 10.sup.4 for the same 100 kDa protein. This then becomes possible to measure with the latest detectors and very stable light source coupled with low noise levels.

[0152] This key advantage of the system and method of the present invention, is the in-line suppression of the reference signal relative to the scattering signal in an in-line interference microscopy setup similar to iSCAT. This allows the optimisation of the contrast between the reference beam intensity and the interference cross-term. This enables the dramatic reduction of the unwanted reference beam intensity relative to the interference intensity and thus increase the sensitivity of the setup and reduce dependency on noise and stability of the excitation light source and overall setup. Since far fewer photons overall are now being detected, the camera used can be replaced with far cheaper versions as the huge dynamic range is no longer needed. It also means that a very cheap laser or LED light source with short coherence length can be used.

Comparison to Conventional iSCAT:

[0153] With reference to FIGS. 1 and 2, the technique of the system and method of the present invention is directly compared to the best achieved in the literature taken from Piliarik et al. (2014). They use iSCAT to detect the binding of single proteins of various sizes from 65.5 kDa (BSA) to 340 kDa (fibrinogen). Using their noise statistics it is calculated how long they need to integrate for to detect their smallest protein (BSA), i.e. to detect the signal (iSCAT contrast) above the noise. For the BSA protein the molecular weight is 38 kDA and from FIG. 1(a), this corresponds to an iSCAT contrast of 310.sup.4. From the noise statistics, FIG. 1(b), they need to average over 600-700 frames. Since they run their camera at 3000 fps, this corresponds to 0.2 s integration or equivalently running at 5 fps, which is the minimum speed they can run and detect/track BSA protein.

[0154] The present inventors repeated similar experiments using the system of the present invention (FIG. 2), for the arrangement of FIG. 3. It can be seen that to detect BSA (see FIG. 2(a)) given the signal noise measured in the present experiment it can run at a higher rate of 60 fps (see FIG. 2(b)). This is more than an order of magnitude (12) improvement in sensitivity.

[0155] Further experiments were performed by the present inventors with the system of the first aspect of the invention, particularly for non-specific binding of a variety of single proteins to a coverglass in comparison to a control with buffer only. FIG. 5 shows the results of said further experiments, where detection limit is plotted as a function of frame averaging (dots) in comparison to shot-noise-limited behaviour (dashed line). The acquisition rate for the experiments was 400 FPS. The dotted line and triangles show a comparison to the detection limit extracted from Piliarik et al. (2014).

Key Advantages of the System and Method of the Present Invention:

[0156] a) Increased signal level [0157] Scattering intensity scales inversely with the fourth power of illuminating wavelength, and the interference cross term [2rs cos ] scales inversely with the square of illuminating wavelength. Since in Piliarik et al. they used a shorter wavelength and more powerful laser, actual gains of the system and method of the present invention are higher than an order of magnitude. If parameters identical to those reported previously (wavelength and intensity) were to be employed in the present invention, signal sensitivity would increase by another factor of 2.4. Thus with a total improvement in sensitivity of around 30.

[0158] b) Reduced sensitivity to reference beam instability [0159] The attenuation of the reference beam according to the present invention reduces the effect of instability in phase and intensity in this signal introduced throughout the beam path or from the laser and spatially across the field of view in the system/microscope. This allows to move to larger field of view on the system/microscope thus detecting more particle binding sites at once. No deterioration was noticed in signal moving from the 1010 m field of view shown in FIGS. 2, to 4040 m (16 bigger area). This is a large advantage over conventional iSCAT which struggles with even detection of gold nanoparticles in a wide-field setup (i.e. without using Galvometric scanning or other scanning techniques) [see Opt. Express 14, 405 (2006)], due to its extreme sensitivity to phase shifts from interfering back reflections.

[0160] c) Cost [0161] The increased signal, lower photon count on the detector and increased stability of the signal lead to a setup which for the same level of detection costs far less to implement and requires a simpler geometry. The detectors, light source and other optical elements in a conventional iSCAT setup, typically put the cost at >$150,000, while in the proposed setup for the system of the present invention, the purchased elements can easily be found for <$10,000. With most of this cost due to the objective lens. With further modifications it is feasible to imagine a system costing even less and at the cost of sensitivity objective could be massively simplified for systems in the sub-$2000 range.

[0162] These advantages clearly illustrate the unique nature of the system and method of the present invention and the large impact it could have in industry.

[0163] A person skilled in the art could introduce changes and modifications in the embodiments described without departing from the scope of the invention as it is defined in the attached claims.

COMMON-PATH INTERFEROMETRIC SCATTERING IMAGING SYSTEM AND A METHOD OF USING COMMON-PATH INTERFEROMETRIC SCATTERING IMAGING TO DETECT AN OBJECT

Assignee

Inventors

Cpc classification

Classification Explorer

G01N15/1434

PHYSICS

Classification Explorer

G01N2015/1493

PHYSICS

Classification Explorer

G01N2015/0038

PHYSICS

Classification Explorer

G01N21/45

PHYSICS

Classification Explorer

G01N21/4795

PHYSICS

Classification Explorer

G01N15/1429

PHYSICS

Classification Explorer

G01N2015/1454

PHYSICS

Classification Explorer

G01N2015/0238

PHYSICS

Classification Explorer

G02B21/14

PHYSICS

Classification Explorer

G01N2015/1006

PHYSICS

Classification Explorer

G01N15/0227

PHYSICS

International classification

Classification Explorer

G01N15/14

PHYSICS

Abstract

Claims

Description