Scattering microscope inspection

Abstract

To provide a scattering microscopy arrangement.SOLUTION: A scattering microscopy arrangement uses a microscope to image an object comprising a surface. A light source emits illuminating light, and a light detector detects light elastically scattered from the object. An electrical potential is applied to the surface that affects electrochemical properties of the object during the imaging. The electrical potential provides a contrast mechanism improving the imaging, and allows characterization of the object and / or a surrounding environment.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to scattering microscopy. [Background technology]

[0002] Several types of scattering microscopy are known in which an object is illuminated with an illumination light and light elastically scattered from the object is detected by a photodetector. Examples of scattering microscopy include (a) interference scattering microscopy (iSCAT) as disclosed in, for example, Non-Patent Documents 1 to 3, and (b) dark-field scattering microscopy as disclosed in, for example, Non-Patent Document 4. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Kukura et al., “High-speed nanoscopic tracking of the position and orientation of a single virus”, Nature Methods 2009 6:923-935 [Non-Patent Document 2] Ortega Arroyo et al. “Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy”, Physical Chemistry Chemical Physics 2012 14:15625-15636 [Non-Patent Document 3] Cole et al., “Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy”, ACS Photonics 2017, 4, 2, 211-216 [Non-Patent Document 4] Jing et al., “Nanoscale electrochemistry in the “dark-field”, Current Opinion in Electrochemistry, Vol. 6, Issue 1, December 2017, pp 10-16 Summary of the Invention [Means for solving the problem]

[0004] The present invention relates to improvements over known techniques. According to a first aspect of the invention, there is provided a method of performing scattering microscopy, the method comprising imaging an object comprising a surface with a scattering microscope comprising a light source arranged to emit illuminating light and a photodetector, the microscope arranged to illuminate the object with the illuminating light and to detect light elastically scattered from the object with the photodetector, the method further comprising applying an electric potential to the surface which affects electrochemical properties of the object when imaging the object, the object being selected to have no plasmon resonance frequency at the wavelength of the illuminating light and the applied electric potential.

[0005] By applying an electric potential to a surface, it is possible to affect the electrochemical properties of the object in a way that provides advantages when performing scattering microscopy of the object. The change in electrochemical properties affects the scattering contrast of the object. In this specification, this is referred to as the potential dynamic contrast. This provides a contrast mechanism that allows the features of the object to be imaged more clearly and / or provides characterization of the object. For example, the potential dynamic contrast is sufficient to visualize the electrochemical state of particles on a surface. Similarly, since the change in electrochemical properties depends on the local topography and material composition, the potential dynamic contrast is also useful to identify surface features and / or particles that are deep sub-wavelength scatterers that would otherwise not be discernible from the background speckle. In addition, the electrochemical state of the object and / or the environment surrounding the object may be characterized. Similarly, the binding rate of particles to a surface can be controlled and monitored.

[0006] The surface may be a surface on which an electric double layer (EDL) is formed, where a potential applied to the surface may affect the electrochemical properties of the electric double layer, for example by causing its restructuring. By changing the surface potential, the configuration (physical structure and / or composition) of the EDL is changed. That is, a change in the surface potential changes the chemical activity of the surface and therefore the interaction between the object and constituents of the surrounding environment, e.g., molecules or ions. This results in a change in the amount of elastically scattered light.

[0007] The object does not have a plasmon resonance frequency at the wavelength of the illumination light and the applied potential. The wavelength of the illumination light is not the plasmon frequency of the object. As a result, the method uses a significantly different operating principle than microscopy, which uses plasmons, e.g., plasmonic nanoparticles or surface plasmons, and uses the plasmon resonance created in the object to provide a signal for imaging the object. In contrast, the method provides imaging using changes in electrochemical properties to provide dynamic potential contrast. In effect, the object does not have a plasmon resonance, increasing the types and properties of objects that can be observed.

[0008] The methods of the present invention also provide advantages over other analytical and microscopy techniques, for example:

[0009] In comparison to single particle electrochemistry, the present method offers the opportunity to perform coherent scattering microscopy to identify biomolecules that are too small to be detected by other optical methods.

[0010] In comparison to cyclic voltammetry, the present method allows potentiodynamic measurements on single (nanoscale) entities (instead of ensembles or extended surfaces). This feature reduces any drawbacks of surface heterogeneity and allows simultaneous measurements at multiple adjacent locations on the surface.

[0011] Compared to conductive atomic force microscopy, the method allows parallel (imaging) measurements on objects on the surface and is less invasive.

[0012] In one example, the surface may be a surface of a conductive material. In this example, a potential may be applied to the surface by applying a potential to the conductive material. Typically, the conductive material is not a metal that may avoid a plasmon resonance frequency at the wavelength of the illumination light.

[0013] In another example, the surface is a surface of a dielectric material, in which case the potential may be applied to the surface by applying the potential capacitively through the dielectric material.

[0014] In one type of experiment, the surface itself may be imaged without the object comprising it, in which case variations in surface properties such as roughness and / or chemical composition may be observed.

[0015] In another type of experiment, the object may further include at least one particle on the surface. The potentiodynamic contrast caused by the restructuring of the EDL by the applied potential improves the contrast of the particle relative to the surface and is sufficient to visualize the electrochemical state of the particle on the surface, allowing for long-term continuous measurement of the particle on the surface. Because the particle has a different potentiodynamic response than the surface, the position of the particle can be identified and continuously monitored with sub-wavelength precision. Similarly, the electrochemical state of the particle and / or the electrochemical state of the environment surrounding the particle can be characterized and monitored.

[0016] The potential may have an amplitude that is constant over the response period of the electrochemical property of the object affected thereby.

[0017] Alternatively, the potential may have an amplitude that varies over a period that is shorter than the response period of the electrochemical property of the object it affects, for example.

[0018] A number of different potentials may be applied to the surface, where detection of the light elastically scattered from the part of the object for each potential allows characterization of the part of the object and / or the surrounding environment, for example by determining the similarity of the profile of the light elastically scattered from the part of the object for each potential to a reference profile. Typically, the potentiodynamic response of the contrast (change in contrast as a function of the applied potential and its hysteresis) is non-linear, which can be used to characterize the object (e.g., the surface or an object on the surface) and / or the surrounding environment.

[0019] The method may be applied to any microscope arranged so that the signal detected by the photodetector is sensitive to the amplitude of light elastically scattered from an object, for example a total internal reflection scattering microscope, an interference scattering microscope, or a dark field scattering microscope.

[0020] The microscope may output a two-dimensional image of the object.

[0021] The light may be ultraviolet, visible, or infrared.

[0022] According to a second aspect of the present invention there is provided a scattering microscope apparatus comprising an object including a surface, a microscope including a light source and a photodetector arranged to emit illuminating light, the microscope being arranged to illuminate the object with the illuminating light and to detect light elastically scattered from the object with the photodetector, the object being selected such that it has no plasmon resonance frequency at the wavelength of the illuminating light, and a voltage source arranged to apply a potential to the surface which affects the electrochemical properties of the object.

[0023] The microscope apparatus operates according to that method and offers similar advantages.

[0024] To enable a better understanding, embodiments of the invention will now be described, by way of non-limiting example, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 is a diagram of a total internal reflection scattering microscope. [Diagram 2] FIG. 1 is a diagram of an interference scattering microscope. [Diagram 3] FIG. 2 is a schematic diagram of a first type of object. [Figure 4] FIG. 2 is a schematic diagram of a second type of object. [Diagram 5] This is a scattering image of an indium tin oxide (ITO) surface. [Figure 6]1 is a surface profile of ITO measured with an atomic force microscope (AFM). [Figure 7] FIG. 6 is an enlarged view of FIG. [Figure 8] 1 is a graph of the scattering intensity at a point as the applied potential varies with a triangular waveform. [Figure 9] 7 is a surface profile measured by AFM along the line in FIG. 6. [Figure 10] 1A and 1B are time diagrams and scatter plots of applied potential and detected light elastically scattered from the surface of ITO at a single spot exposed to a solution; the time diagram is a plot of applied voltage V (volts) against time (s) and the scatter plot is a plot of relative differential intensity dI / I against applied voltage (V). [Figure 11] 1A and 1B are time diagrams and scatter plots of applied potential and detected light elastically scattered from the surface of ITO at a single spot exposed to a solution; the time diagram is a plot of applied voltage V (volts) against time (s) and the scatter plot is a plot of relative differential intensity dI / I against applied voltage (V). [Figure 12] 1A and 1B are time diagrams and scatter plots of applied potential and detected light elastically scattered from the surface of ITO at a single spot exposed to a solution; the time diagram is a plot of applied voltage V (volts) against time (s) and the scatter plot is a plot of relative differential intensity dI / I against applied voltage (V). [Figure 13] 1A and 1B are time diagrams and scatter plots of applied potential and detected light elastically scattered from the surface of ITO at a single spot exposed to a solution; the time diagram is a plot of applied voltage V (volts) against time (s) and the scatter plot is a plot of relative differential intensity dI / I against applied voltage (V). [Figure 14] Scattering microscope image of TiO2 particles on an ITO surface plotting the logarithm of the average intensity. [Figure 15] FIG. 15 is the image of FIG. 14 processed to show potential contrast. [Figure 16]13A-13C are time and scatter diagrams of the applied potential and the detected light elastically scattered from the TiO2 particles. [Figure 17] 13A-13C are time and scatter diagrams of the applied potential and the detected light elastically scattered from the ITO surface. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] 1 shows a first microscope arrangement 10 including a total internal reflection (TIR) ​​scattering microscope 20, and FIG. 2 shows a second microscope arrangement 20 including an interference scattering (iSCAT) microscope 40, which are examples of microscope arrangements to which the present technique may be applied. The first microscope arrangement 10 and the second microscope arrangement 20 each further include an object 50, which is the same in both cases and will be further described below.

[0027] The iSCAT microscope may additionally comprise a spatial filter positioned to filter the output light, the spatial filter being arranged to pass the output light, but with a greater reduction in intensity within a given numerical aperture than at higher numerical apertures. Most of the light scattered from nanoscale objects is scattered at high numerical apertures, so that the intensity of low NA light can be reduced without reducing the intensity of the scattered light from nanoscale objects. By reducing the intensity of the reflected light but leaving the scattered light relatively unattenuated, contrast and sensitivity are improved, resulting in a better image.

[0028] The TIR scattering microscope 20 and the iSCAT microscope 40 are each used to image an object 50 and have in common a light source 30, an objective lens 31, and a photodetector 32. In each case, the light source 30 emits illuminating light and the photodetector 32 detects light elastically scattered from the object 50.

[0029] In general, in all of the techniques described herein, the illumination light may be either visible light (wavelength range from 400 nm to 700 nm), ultraviolet (wavelength range below 400 nm to a lower limit of 10 nm), or infrared (wavelength range above 700 nm to an upper limit of 1 mm).

[0030] The TIR scattering microscope 20 and the iSCAT microscope 40 may be operated in a wide-field mode, in which case the photodetector 32 may be an image sensor, for example a CMOS (complementary metal-oxide semiconductor) image sensor, that captures a two-dimensional image of the object 50. Conversely, the TIR scattering microscope 20 and the iSCAT microscope 40 may be adapted to operate in a confocal mode, in which case the photodetector 32 may be a simple photodiode. In this case, the two-dimensional image of the object 50 may be obtained by scanning the object 50.

[0031] The TIR scattering microscope 20 and the iSCAT microscope 40 are each positioned to illuminate an object 50 with illuminating light and direct elastically scattered light to a photodetector 32 .

[0032] The TIR scattering microscope 20 and the iSCAT microscope 40 are each configured such that the signal detected by the photodetector 32 is sensitive to the amplitude of light elastically scattered from the object 50, but with different configurations to achieve this, as follows.

[0033] The TIR scattering microscope 20 shown in FIG. 1 is a type of dark field scattering microscope arranged as follows.

[0034] In this case, the light source 30 emits illumination light perpendicular to the optical axis O of the objective lens 31. The first reflective surface 21 deflects the illumination light from the light source 30 through the objective lens 31 parallel to but offset from the optical axis O. As a result, the objective lens directs the illumination light towards the object 50 at an angle of incidence greater than the critical axis such that the light is reflected from the object 50 by TIR at a corresponding angle of reflection.

[0035] The reflected light passes back through the objective lens 50 and emerges parallel to but offset from the optical axis O. In this manner, the illumination light and the reflected light do not pass through the objective lens 31 along the optical axis O.

[0036] A second reflective surface 22 deflects the reflected light perpendicularly from the optical axis O to a quadrant photodiode (QPD) 23 that is used as a position sensing device (PSD) for focus stabilization.

[0037] In TIR scattering microscope 20, photodetector 32 is aligned along optical axis O. As a result, photodetector 32 receives light elastically scattered from object 50, but does not receive reflected light that does not pass through objective lens 31 along optical axis O. The configuration of TIR scattering microscope 20 thus allows photodetector 32 to detect light elastically scattered from object 50 in dark field mode with a signal sensitive to the amplitude of the elastically scattered light.

[0038] Although the TIR scattering microscope 20 is described as an example of a dark-field scattering microscope, the techniques described herein can be performed with any dark-field scattering microscope, for example, as disclosed in non-patent document 4.

[0039] The iSCAT microscope 40 shown in FIG. 2 is arranged as follows.

[0040] In this case, the light source 30 is a coherent light source such as a laser that emits illumination light perpendicular to the optical axis O of the objective lens 31 to a beam splitter 41. In this example, the beam splitter 41 is a plate 42 with a reflective film 43, which may be metallic or dielectric, arranged at 45° to the optical axis O, although the beam splitter 41 could have other forms such as a cube beam splitter formed by a matched pair of prisms with a partially reflective film at the interface between them.

[0041] A condenser lens 42 for condensing the illumination light is provided between the light source 30 and the beam splitter 41 .

[0042] The beam splitter 41 is configured to deflect the illumination light along an optical axis O to the objective lens 31. The objective lens 31 then focuses the illumination light onto the object 50.

[0043] Objective lens 31 also collects output light from the object and directs it to photodetector 32 through beam splitter 41, tube lens 43, and optionally a pair of telescope lenses (not shown) that focus the output light onto photodetector 32. The output light includes both (a) light reflected from object 50 and (b) light elastically scattered from object 50. As a result, the elastically scattered light constructively interferes with the reflected light, so that the signal detected by photodetector 32 is sensitive to the amplitude of the light elastically scattered from object 50.

[0044] Although an iSCAT microscope 40 is described as an example, the techniques described herein can be performed with any iSCAT microscope, for example as disclosed in non-patent documents 1-3.

[0045] Although the TIR scattering microscope 20 and the iSCAT microscope 40 are given as examples, the techniques described herein can be performed with other forms of scattering microscopes.

[0046] Next, the object 50 will be described. Two alternative forms of the object 50 are shown in Figures 3 and 4. In all cases the object 50 comprises a surface 51 to which a potential is applied. The potential is applied by a voltage source 53 which is also connected to a counter electrode 54.

[0047] A fluid 52 may be provided to the environment around the surface 51. The fluid 52 may be a solution or may be an aqueous solution. The fluid 52 may contain dissolved ions.

[0048] In the embodiment shown in FIG. 3, the object 50 comprises a conductive layer made of a conductive material, and the surface 51 is the surface of the conductive layer 60 .

[0049] In this case, the potential is applied directly to the surface 51, since it is applied to the conductive layer 60 to which the voltage source 53 is connected. The conductive material of the conductive layer 60 may be any suitable material, for example indium tin oxide (ITO). The conductive layer 60 can be made of any material commonly used for optical imaging, in particular gold, graphene, ITO, silver, platinum, and graphene oxide. Ideally, the conductive material of the conductive layer 60 is not metallic to avoid the formation of plasmons, as discussed below. The conductive layer is configured such that its plasmon frequency is not the wavelength of the illumination light. This may be due to the choice of material or the particular geometry. Alternatively, the illumination frequency is selected such that it is not the plasmon frequency of either the object 50 or the conductive layer.

[0050] 4, the object 50 comprises a dielectric layer 70 of a dielectric material, and the surface 51 is the surface of the dielectric layer 70. The dielectric layer 70 is disposed on an electrode layer 71 made of a conductive material. The conductive material of the electrode layer 71 may be any suitable material, for example indium tin oxide (ITO). Ideally, the conductive material of the electrode layer 71 is not metallic to avoid the formation of plasmons, as discussed below.

[0051] In this case, the potential is applied capacitively to the surface 51 as it is applied to the electrode layer 71 to which the voltage source 53 is connected.

[0052] 3 and 4 show examples of objects 50, but more generally the techniques disclosed herein may be applied with objects 50 taking any form that includes a surface 51 to which an electrical potential may be applied. Some other examples are as follows:

[0053] Surface 51 may be the surface of a material that is a solid, liquid, or gas.

[0054] In one type of object 50, surface 51 may be the surface of a layer of material. The material may be a solid, liquid, or gas.

[0055] In another type of object 50, the surface 51 may be the surface of a three-dimensional shaped structure. In one example, the structure may be a liquid droplet, in which case the surface may be the surface of the liquid or the surface of a surfactant surrounding the liquid. In another example, the structure may be a gas bubble, in which case the surface may be the surface of a gas or the surface of a surfactant surrounding the gas.

[0056] The counter electrode 54 is shown diagrammatically in Figures 3 and 4, but can take any suitable form, for example being a separate element positioned adjacent to the object 50 or being integrated into the structure on which the surface 51 is formed.

[0057] The object 50 may further include at least one particle 55 on the surface 51, for example as illustrated diagrammatically in Figures 3 and 4. The presence of the particle 55 as part of the object 50 along with the surface 51 is not necessary, as the present technique may equally be applied to the surface 51 alone, for example to image or characterize the surface 50 or to characterize the surrounding environment.

[0058] Particles 55 typically have a different scattering effect than surface 51, thereby providing contrast between particles 55 and surface 51 and allowing particles 55 to be identified in a two-dimensional image. Furthermore, as discussed in more detail below, application of a potential provides a potentiodynamic contrast that increases the contrast between particles 55 and surface 51. As also discussed below, application of a potential also allows particles 55 to be characterized by observation of a signal from particles 55.

[0059] Particles 55 may generally be any type of particle that has a scattering cross section for the illuminating light. Some non-limiting examples are as follows:

[0060] The particles 55 may be dielectric particles.

[0061] Particles 55 may be proteins or protein aggregates.

[0062] The particles 55 may be metal particles.

[0063] The particles 55 may be nucleic acid molecules such as RNA (ribonucleic acid) or DNA (deoxyribonucleic acid), or artificial nucleic acid molecules, or any aggregates thereof.

[0064] Particle 55 may be a polypeptide, a glycopolypeptide or glycoprotein, a lipopolypeptide or lipoprotein, or any aggregate thereof.

[0065] Particle 55 may be a lipid, a proteoglycan, or a sugar polymer.

[0066] Particles 55 may be any biopolymer.

[0067] Particles 55 may be aggregates of the different particles enumerated herein, for example, aggregates of proteins and nucleic acids, aggregates of glycoproteins (eg, antibodies) and protein or polypeptides.

[0068] Particles 55 may be naturally occurring, man-made, or a hybrid of natural and man-made.

[0069] Particles 55 may be inorganic particles or aggregates, such particles being non-biological and non-derived.

[0070] Particles 55 may be any nanomaterial, especially engineered nanomaterials.

[0071] Particle 55 may be a charged particle, since many biopolymers are naturally charged, for example naturally occurring nucleic acids are highly charged due to their phosphate backbones.

[0072] Typically, the particles 55 are 10 -17 m 2 and / or have a scattering cross section for illumination light of 10 -26 m 2The scattering cross section may be a fundamental measurable property related to the effective size of an object for incident light of a particular wavelength, regardless of the technique used to measure it. The scattering cross section can be measured, for example, by inspection with a dark field microscope.

[0073] Particles 55 may have a mass of 10 kDa or more.

[0074] In some applications, the particles 55 may have a mass of 5000 kDa or less. Typically, the present invention may be applied to particles 55 having a mass of 10 kDa or more, for example, objects having a mass in the range of 10 kDa to 5000 kDa. However, the particles 55 may be larger and may generally be any particle (or part of a particle) that fits within the field of view. These larger particles may be composed of linear biopolymers of the type discussed above. Examples of relatively large particles that may have a mass of more than 5000 kDa include fibrils, fibers, or cylinders. The larger particles may be biologically derived, for example, fibrils are structural biological materials found in nearly all living organisms. Exemplary larger particles that may include fibrils include collagen, actin, myosin, elastin, keratin, resilin, spider or insect silk, cellulose, amylose, or wood. Fibers may be described as plant tissue, mineral material, or threads or filaments from which textiles are formed. Fibers are also structures that form part of muscle tissue, nervous tissue, connective tissue, or other tissues of the human or animal body. Other types of exemplary biological or artificial larger particles include protein filaments, protein cages, protein aggregates, and protein channels such as membrane channels. Large nucleic acid origami structures may also be considered larger particles. Alternatively or additionally, the larger particles may be inorganic, such as carbon-based nanoparticles (e.g., rods, tubes, spheres, fullerenes). Similarly, object 50 may include chains of particles. As a result, the term "particle" does not imply a particular shape, such as a sphere, and may refer to any entity on surface 51.

[0075] Next, the potentiodynamic contrast provided by the change in the electrochemical properties of object 50 under an applied potential is described.

[0076] An electrical potential applied to surface 51 affects the electrochemical properties of the object. In the primary example, surface 51 is a surface on which an electrical double layer (EDL) is formed. In that case, an electrical potential applied to surface 51 affects the electrochemical properties of the EDL.

[0077] The EDL formed around the charged surfaces of nanostructures in liquids affects their light scattering intensity. The formation of EDL is a central component of virtually all electrokinetic processes. For example, the description of energy storage in batteries, molecular salvage, filtration processes in membranes, and most transport in liquid environments requires the consideration of EDL.

[0078] The EDL comprises first and second layers 56 and 57 of charges formed on the surface 51, as shown, for example, in schematic form in Figures 3 and 4. The first layer 56 is formed directly on the surface and contains ions that are adsorbed to the surface 51 due to chemical interactions. The first layer 56 provides a surface charge that may be positive or negative. The second layer 57 is formed on the first layer 56 and contains ions that are attracted to the surface charge of the first layer 56 via Coulomb forces. As a result, the second layer 57 electrically shields the first layer 55. However, since the Coulomb forces are weaker than the chemical interactions that hold the first layer 56 together, the second layer 57 is less strongly associated with the surface 51 than the first layer 56. The ions of the second layer 57 are free ions that move in the fluid 52 adjacent to the surface 51 under the influence of the electrical attraction and thermal excitation of the first layer 56, and are sometimes referred to as a "diffuse layer".

[0079] The thickness of the EDL typically varies between one nanometer and tens of nanometers, depending on the ionic strength of the adjacent fluid 52.

[0080] The formation of the EDL involves several time scales, the fastest of which is the molecular diffusion time scale D / λ. D 2 (D is the diffusion constant, λ D (where θ is the Debye length in the dimension of the EDL thickness). The small amounts and fast time scales involved in the formation of the EDL make direct access to its local dynamics an experimental challenge. Many observations in this area have been obtained by measuring local currents at high frequencies using scanning probe techniques or ultramicroelectrodes.

[0081] The application of the potential affects the electrochemical properties of the object 50 by causing a change in the configuration of the EDL. In particular, the physical structure and / or composition of the EDL may change. Such a change in the configuration of the EDL results in a change in the detected scattering signal. That is, the change in the surface potential changes the chemical activity of the surface and therefore the interaction between the object and constituents of the surrounding environment, e.g., molecules or ions. This results in a change in the amount of elastically scattered light, and thus affects the scattering contrast of the object, which is referred to herein as potentiodynamic contrast. This provides a contrast mechanism for microscopic examination that may be exploited in many ways.

[0082] Indeed, visualizing the optical contrast of the EDL provides direct access to wide-field measurements to observe spatial transport and combine the capabilities of optical microscopy with electrochemical analysis. Although the changes in light reflection from a surface due to the formation of an EDL are decades-long, its effect on elastic light scattering from nanoparticles is very small.

[0083] In some applications, potential dynamic contrast is used as a contrast mechanism to allow features of an object to be imaged more clearly, for example, to image surface features, e.g., structural and / or chemical composition features, of the surface 51 itself, and / or any particles 55 that may be present. Similarly, because changes in electrochemical properties depend on the local topography and material composition, potential dynamic contrast is also useful for identifying surface features and / or particles that are deep sub-wavelength scatterers that would otherwise not be discernible from the background speckle.

[0084] The principle of potential contrast is considered as follows.

[0085] First, an estimate of the expected electrokinetic contrast is derived as a function of the surface potential of the conducting nanosphere. Since we are primarily interested in the EDL, here the changes due to the reorganization of ions outside the particle are considered. Polarizability changes due to charge injection within the particle may also occur, as previously observed in plasmonic nanoparticles, but are expected to have much less effect on dielectric particles and semiconductors such as ITO. The optical contrast of the EDL can be used to observe particles that are dielectrics or metallic scatterers with plasmon resonance frequencies far away from the frequency of the illuminating light.

[0086] A very simplified model of the EDL is that it consists of shielding ions of opposite charge with respect to the particle surface, with a thickness of λ s It is assumed that the nanosphere is uniformly distributed in the layer of ≪a, where a is the radius of the nanosphere. This model is based on the characteristic potential k B T / e ≒ 25 mV (k B This agrees well with the physical condition of a surface potential much larger than θ (θ = 1, ...

[0087] The total number of excess counterions N required to shield the nanosphere at potential V is V ea / k B T B (λ B In the Rayleigh scattering regime, the polarizability of the combined nanosphere and EDL system is the volume sum of its constituent materials. The Rayleigh polarizability and refractive index versus salt concentration n mix =n solvent +Kx s (x s Using the phenomenological relationship between the scattering of the EDL, α EDL Scattering of particles α P The final scaling result for the ratio to is given by:

number

[0088] In the case of water, ρ w =55nm ―3 and λ B = 0.7 nm, for a 10 nm (diameter) titanium dioxide particle in a NaCl salt solution with a surface potential of V = 1 volt, α EDL / α P = 0.008 is obtained. This small ratio suggests that such changes can only be measured interferometrically.

[0089] The results indicate the presence of a contrast mechanism that increases with the applied potential V and with decreasing particle size (ie, inversely proportional to the square of the nanosphere radius).

[0090] Repeating the same estimation for a cylinder of radius r gives:

number

[0091] The results also indicate the presence of a contrast mechanism that increases with the applied potential V and with decreasing size of the cylinder (i.e., inversely proportional to the square of the nanosphere's radius). It also indicates that it may be easier to observe potential contrast in the case of protuberances than in the case of nanoparticles on a surface.

[0092] Although these results relate specifically to spheres and cylinders, potential dynamic contrast is similarly provided by curved features of surface 51, or by particles 55 of any shape on surface 51. This provides direct visualization of such curved features or particles 55. As a result, the technique may be applied to provide wide-field imaging of objects that include surface 51 and optionally also particles 55. This allows the electrochemical properties of a variety of systems to be observed.

[0093] While the above analysis illustrates contrast mechanisms of structural features of surface 51 and particles 55 on surface 51, contrast mechanisms are likewise provided for variations in the chemical composition of surface 51 as affected by the applied potential.

[0094] Previously applied potentials have been used in plasmon resonance microscopy involving surface plasmons or plasmonic particles, and have been used to observe particles, although this is limited to surfaces and particles that have plasmonic resonances accessible by the illumination light. However, the present technique uses a significantly different principle of operation in which plasmonic resonances are not created. Instead, the present technique uses changes in electrochemical properties to provide potential dynamic contrast, as explained above.

[0095] As a result, in all examples, object 50 (including surface 51 and, if present, particles 55) is selected to have no plasmon resonance frequency at the wavelength of the illuminating light and the applied potential, to prevent plasmon resonance from causing a signal that masks the desired signal from the elastically scattered light affected by the change in electrochemical properties.

[0096] In other applications, the potentiodynamic contrast is used to provide characterization of the object 50 and / or the environment surrounding the object 50. The potentiodynamic contrast caused by the reconstruction of the EDL is sufficient to characterize the electrochemical state of the surface 51 as affected by the surface 51 and its environment, as well as the electrochemical state of particles 55 on the surface 51. The technique provides direct access to the dynamics of the EDL on time scales accessible for ultrafast and long-term optical measurements. Wide-field imaging of ion transport and other electrochemical activity across the surface is monitored and used to create feedback control of the electrochemical microscape.

[0097] By varying the electrochemical state of the surface 51, the binding rate of the particles 55 to the surface 51 can be controlled and monitored. By controlling the surface potential, the rate can be slowed down to avoid early crowding of the measurement surface, or increased to speed up the measurement process. The potential of the adsorption rate as a function of the surface potential is also an important indicator of the properties of the adsorbent.

[0098] Depending on the nature of the applied potential, various electrochemical properties may be observed.

[0099] In some applications, the applied potential has an amplitude that is constant over the response period of the electrochemical property of the object 50 that it affects. This provides a signal that depends on the static electrochemical property of the object 50.

[0100] In other applications, the applied potential has an amplitude that changes over a period shorter than the response period of the electrochemical property of the object 50 being affected thereby. For example, the amplitude of the applied potential may have an amplitude that alternates over a period shorter than the response period of the electrochemical property of the object being affected thereby, or may have an amplitude that undergoes a step change. This provides a signal that depends on the dynamic electrochemical property of the object 50.

[0101] The electrochemical properties at different potentials may be observed.

[0102] For example, multiple different potentials may be applied to a surface, and light elastically scattered from a portion of the object may be detected for each potential for characterization of that portion of the object and / or the surrounding environment, and the similarity of the profile of light elastically scattered from the portion of the object for each potential may be compared to a reference profile.

[0103] Experimental results showing the potential contrast were obtained as follows.

[0104] The object 50 was of the type shown in FIG. 3 and consisted of a surface 51 of a conductive layer 60 of indium tin oxide (ITO) provided as a 50 nm thick coating on a glass slide and to which an electrical potential was applied.

[0105] Microscopic examination of the object 50 was carried out using a TIR scattering microscope 20 of the type shown in Figure 1. A 50 μm square sized area of ​​the object 50 was illuminated through an oil immersion type objective lens 31 having a numerical aperture of 1.45.

[0106] The mutual interference term of the scattered light from the substrate surface and the nanoparticles is proportional to the change in the particle polarizability and is used here to visualize the reconstruction of the EDL. The background scattering signal is kept stable with fluctuations (on the order of 1%) much smaller than the potential contrast. This constant level is achieved by paying special attention to the mechanical stability of the microscope and to the kHz bandwidth stabilization of the sample stage.

[0107] The counter electrode 54 was provided as another ITO slide or a floating Pt electrode kept at a distance of 100 μm. The use of a separate reference electrode such as Ag / AgCl was avoided to prevent contamination of the ITO surface by the deposition of small nanoparticles.

[0108] Unless otherwise stated, the potential was applied as a non-biased triangular waveform when recording the scattering images.

[0109] Figure 5 is a scattering image of ITO showing that the ITO surface has areas of high scattering in the form of parallelograms with sharp edges and angles, separated from relatively smooth areas where scattering is 10 to 100 times lower. Inspection by atomic force microscope of the ITO surface as shown in Figures 6 and 9 allows the detection of the presence of sparse particles of about 20 nm in these areas. The sharp boundaries that confine these particles indicate the crystallographic origin of their formation, probably related to a stoichiometric excess of tin or indium during the annealing treatment of the ITO.

[0110] Scattering images were recorded while varying the potential of the ITO substrate relative to the counter electrode. By subtracting the average scattering signal over the cycle from each, a potentiodynamic contrast image is obtained, as shown in Figure 7. First, the bare ITO substrate is measured in an area where both the rough and smooth surfaces are visible. Due to the relatively uniform size distribution of the particles on the ITO rough areas, they can be used as a reference for the potentiodynamic contrast of other types of nanoparticles.

[0111] To show that the reorganization of the EDL is the main contribution to the measured potentiodynamic contrast, the same area of ​​ITO for three different monovalent salts of the same concentration, and deionized water, were investigated. The applied potential was changed slowly compared to the response period of the electrochemical properties of the surface 54. As a result, the applied potential had, in effect, a constant amplitude over the response period, but the elastically scattered light at different potentials was detected at the sampling rate of the photodetector 32.

[0112] FIG. 8 shows how the scattering intensity of a single point changes in response to changes in the applied potential in a triangular waveform.

[0113] 10-13 show the applied potential and the detected light elastically scattered from the surface 51. Shown is the intensity variation of a single speckle versus applied potential, averaged over several tens of cycles. This uses potentiodynamic contrast to perform cyclic voltammetry at local current levels of 10-18 amperes, corresponding to the exchange of a few elementary charges.

[0114] The results characterize the solution in the environment of surface 51. For example, in our experimental conditions, the potential dynamic contrast of 2 mM NaI and NaCl was four times that of NaBr and DI water. Similarly, the elastically scattered light profile for each applied potential differs from solution to solution.

[0115] The change in optical contrast of a single titania (TiO2) particle with a diameter of 19 nm was then observed. To observe titania particles landing on the ITO surface, a suspension of the particles was inserted into the measurement cell. Excess non-deposited particles were replaced by a solution of 2 mM NaI.

[0116] Figure 14 shows a scattering microscope image of a TiO2 particle. In comparison, Figure 15 shows the amplitude of the intensity oscillations when applying the potential contrast to the average of each pixel, i.e. the amplitude to the average intensity of the signal measured on a curve similar to that of Figure 8.

[0117] The applied potential was changed slowly compared to the response period of the electrochemical properties of surface 54. As a result, in effect, the applied potential had a constant amplitude over its response period, but the elastically scattered light at different potentials was detected at the sampling rate of photodetector 32.

[0118] 16 and 17 respectively show the applied potential and detected light elastically scattered from TiO2 particles and rough spots on surface 51. This uses potentiodynamic contrast to perform cyclic voltammetry of a single 19 nm titanium dioxide at local current levels of 10-18 amperes, corresponding to the exchange of a small elementary charge. This comparison clearly shows the material specificity of potentiodynamic microscopy examination.

[0119] In summary, these results demonstrate experimental measurement of EDL reorganization directly from changes in optical contrast. Given that coherent scattering has the sensitivity to probe single biomolecules down to tens of kD, measurements of potential contrast facilitate long-term monitoring of oxidation and reduction processes of single proteins as well as other reactions.

[0120] At relatively low potentials compared to the electrochemical reaction potential, using fully polarizable electrodes, the potentiodynamic scattering contrast is mainly due to the reorganization of the EDL. That is, elastic light scattering is mainly affected by the physisorption of counterions, which have a different optical polarizability (refractive index) than the solvent. The dependence of the potentiodynamic contrast on the type of salt is shown, allowing for the characterization of the salt.

[0121] At higher potentials, the potentiodynamic contrast from the adsorbed nanoparticles shows a different pattern than the underlying ITO substrate due to their electrochemical activation. This difference is used to perform the optical equivalent of cyclic voltammetry of single 19 nm titanium dioxide nanoparticles. The material and surface topography dependence of the potentiodynamic contrast creates a previously unexploited contrast mechanism for chemically specific optical microscopic examination of single macromolecules. Similarly, in the case of higher potentials where reactions such as water splitting begin, this contrast can be used to measure product formation.

[0122] The onset of these processes can best be identified by the nonlinearity of the contrast signal versus applied potential. In this regime, potential-dynamic contrast microscopy can provide similar information to cyclic voltammetry, with optical contrast indicating displaced charge instead of current measurements.

[0123] This technique has many applications, some non-limiting examples are as follows: Selectively repel or attract particles with a surface charge by applying an electric potential. This allows a variety of particles to be attracted to the surface and measured, for example, using mass photometry. Such a capability may be particularly useful in objects containing mixed particles, such as biological samples with proteins and nucleic acids. By measuring at neutral, negative and / or positive potentials, one checks for interaction bias with the measurement surface, i.e., to ensure that no additional species are attracted at the potential. · Compare measurements at different potentials, e.g. neutral and specific potentials. Alternatively, compare measurements at different potentials. Interactions with surfaces at potential compete with particle binding based on charge interactions. · To determine whether potential can selectively dissociate interactions of bound complexes. Monitoring the properties of single nanoscale objects and their (electro)chemical state, e.g. the oxidation / reduction of a single protein, the progression of catalytic activity of a single particle, changes in the abundance of "invisible" ions around a "detected" biomolecule, or changes in the chemical state of a biomolecule. · Monitoring the interactions of these objects with other molecules, e.g. binding / unbinding of other molecules, ion exchange with nearby objects or surfaces, affinity for specific molecules in a solvent. Identify and monitor surface unevenness and specific defects or surface structures. Observe the nucleation, formation, and dissociation of compounds on surfaces. Tuning the binding / unbinding rates of molecules in solution to surfaces for coherent scattering mass spectrometry. · Performing coherent scattering on one or more conductive surfaces with (continuously) adjustable surface potentials. Heterodyne measurements of reflection contrast are performed. Measuring the nonlinear response of reflectance contrast to changes in surface potential at the single particle level to infer material properties. · Performing optical contrast electrochemical measurements on dielectric objects (in contrast to previous work on metal particles). Detects solvent characteristics and the presence of reactive chemicals. · Investigating the dynamics of mixed phases (bubbles, polymers) on surfaces. The light scattering signal monitors the dynamics of objects passing through the nanopore. Many experiments which are essentially performed by cyclic voltammetry on surfaces can now be performed on single particles. Measuring specific variations in ion current and ion concentration on top of a surface, at an edge, or along a ridge. ·Measure the time dynamics of all of the above.

Claims

1. 1. A method for monitoring and controlling particle binding kinetics, comprising: imaging an object having a surface and at least one particle with a scattering microscope including a light source and a photodetector arranged to emit illuminating light, the microscope further arranged to illuminate the object with the illuminating light and to detect light elastically scattered from the object with the photodetector; applying an electrical potential to the surface of the object to change an electrochemical state of the surface, the change in electrochemical state allowing monitoring and control of the binding rate of the particles to the surface; Including, The method of claim 1, wherein the object is selected to have no plasmon resonance frequency at the wavelength of the illumination light and the applied potential.

2. The method of claim 1 , wherein an electric double layer is formed at the surface, and the electrical potential applied to the surface affects an electrochemical property of the electric double layer.

3. The method of claim 1 or 2, wherein the surface is a surface of a conductive material, and applying an electric potential to the surface comprises applying the electric potential to the conductive material.

4. The method of any one of claims 1 to 3, further comprising the step of selectively repelling or attracting particles to the surface by applying an electric potential to the surface.

5. The method according to any one of claims 1 to 4, further comprising the step of monitoring interactions between said object and constituents of the surrounding environment, said constituents being molecules or ions.

6. The method of claim 5 , further comprising adjusting the binding / unbinding rate of molecules in solution to the surface of the object.

7. A method according to any one of the preceding claims, comprising the step of applying a plurality of different electrical potentials to the surface.

8. The method of claim 7 further comprising the step of comparing measurements taken at different potentials.

9. The method of any one of claims 1 to 8, wherein the object is a single nanoscale object.

10. The method of claim 9 , comprising monitoring the properties and electrochemical state of the single nanoscale object.

11. The method according to any one of the preceding claims, wherein the particles are dielectric particles.

12. 12. The method of any one of claims 1 to 11, wherein the particle is a protein or protein aggregate, a polypeptide, a glycopolypeptide or glycoprotein, a lipopolypeptide or lipoprotein, or any aggregate thereof, a lipid, a proteoglycan, or a glycopolymer.

13. The method according to any one of claims 1 to 11, wherein said particles may be nucleic acid molecules such as RNA (ribonucleic acid) or DNA (deoxyribonucleic acid), or artificial nucleic acid molecules, or any aggregates thereof.

14. The method of any one of claims 1 to 13, wherein the particles have a mass of 10 kDa or more.

15. The method of any one of claims 1 to 14, wherein the particles have a mass of less than or equal to 5000 kDa.

Images