WO2018122814A1

WO2018122814A1 - Method and optical microscope for detecting particles having sub-diffractive size

Info

Publication number: WO2018122814A1
Application number: PCT/IB2017/058544
Authority: WO
Inventors: Giovanni DE LELLIS; Andrey ALEXANDROV; Valeri TIOUKOV; Nicola D'AMBROSIO
Original assignee: Istituto Nazionale Di Fisica Nucleare
Priority date: 2016-12-30
Filing date: 2017-12-30
Publication date: 2018-07-05
Also published as: IT201600132813A1

Abstract

Optical microscope (100) for detecting particles having sub-diffractive size within a sample, comprising: a display system (50), having a first objective (01); a polarising device (6); an analyser system (60), having a second objective (02) and a reflection element (7), wherein an optical path between the first objective (01) and the second objective (02) is divided into two identical parts symmetric with respect to a coupling plane; a sensor device (S2) configured to detect a plurality of beams corresponding to a plurality of polarisation configurations of the polarising device (6) that is reflected by the reflection element (7), thus acquiring a plurality of images; and one or more processing units configured to perform a two-dimensional method of analysis of the plurality di acquired images.

Description

METHOD AND OPTICAL MICROSCOPE FOR DETECTING PARTICLES HAVING

SUB-DIFFRACTIVE SIZE

* * *

The present invention relates to a method and an optical microscope for the detection of nanometric particles, in particular for the observation through an optical microscope of nanoparticles with a sub-diffractive resolution limit.

The observation of nanometric quantities, for example the observation of traces of nuclear recoil in the field of nuclear emulsions (metal nanoparticles immersed in a transparent dielectric material) through conventional optical microscopes has physical limits of resolution due to the phenomenon known as "diffraction limit", that limits the ability of optical instruments to distinguish between two objects separated from each other by a distance of less than about half the wavelength of the light used to illuminate the sample.

In general, the diffraction process becomes relevant when the wavelength is comparable to the size of the obstacle, hence for visible light (having a wavelength that is around 0,5 μιη) diffraction phenomena occur when it interacts with objects of sub-micrometric size.

In case of nanometric dimensions, due to the diffraction of light, the image of a sample obtained through prior art optical systems does never perfectly represent the real details in the sample because there is a lower limit below which the optical system of the microscope cannot resolve structural details.

In fact, when focusing a point object, the lens of a microscope generates a diffraction pattern composed of a central disk surrounded by a series of concentric diffraction rings (Airy disk).

According to the Airy model, the size of the central disk is correlated to the wavelength of the incident light and the angle of aperture of the objective by the formula:

λ

R_Airy - J ^

where λ is the average wavelength of transmitted light illumination and NA is the objective numerical aperture.

The limit of resolution of a microscope is defined as the distance of two point sources at which their images show a separation such that the peak of an Airy model of a first point source coincides with the first dark ring of the Airy model of the second point source.

This is referred to as the Rayleigh criterion for resolution. The numerical expression of the Rayleigh criterion is:

λ

^Rayleigh ~ °-⁶¹ ^

However, even considering the most powerful objectives under ideal conditions, the resolution obtainable through an optical microscope is limited to values between 200 nm and 250 nm due to the transmission characteristics of the glass in correspondence of wavelengths below 400 nm and physical constraints on numerical aperture. Therefore, even in cases where an optical microscope is provided with the best quality lenses available on the market, is perfectly aligned, and has the highest numerical aperture, the resolution remains limited in the best case at about half of the wavelength of the incident light.

Some prior art microscopes are disclosed in documents EP 2081070 A2, DE 10 2005

005757 Al, US 5521705 A and WO 2016/191004 Al.

However, prior art microscopes suffer from some drawbacks.

A disadvantage of conventional microscopes is that they have a resolution that is not sufficient to distinguish a plurality of silver grains close to each other, which form a short particle trace (e.g., of length of less than 200 nm), from a single impure grain produced by a thermal excitation.

Each of the two appears as a single point.

Even the microscope disclosed by document EP 2081070 A2, that requires a coherent illumination and is based on a phenomenon of phase change and of recombination of non- deflected light with a deflected light having a phase shift, has a spatial resolution that is limited by diffraction and does not ensure any super-resolution capable of detecting sub-diffractive particles (i.e. nanometric particles). The microscopy technique directed to the study of birefringence properties of transparent materials illustrated in document US 5521705 A (as well as the very similar one illustrated in document DE 10 2005 005757 Al) is based on measurement (by means of a transmission illumination mode) of the deviation from the circular or quasi- circular polarisation of light passing through a sample; it is still limited by diffraction and does not allow any three-dimensional imaging (imaging). The microscopy technique illustrated in document WO 2016/191004 Al is a fluorescence microscopy technique for imaging of samples labelled by polymeric dyes, and it is based on the phenomenon of spatial dependence of the fluorescence of such dyes; however, it only allows a high lateral resolution, but it does not have any axial super-resolution and, therefore, it does not allow any three-dimensional imaging. Beyond optical microscopy, there are methods for observing structures characterised by size smaller than the diffraction limit size, such as X-ray microscopy and transmission electron microscopy (TEM).

A disadvantage of known techniques is that, in transmission electron microscopy, sample preparation is a rather complex procedure.

In fact, the thickness of the sample must be less than 100 nm. High quality samples must have a thickness comparable to the average free path of electrons traveling through the samples which may be only a few tens of nanometers.

An X-ray microscope can reach a resolution of 30 nm and does not require a particular preparation of the sample, but it is an analysis that requires a long time.

Therefore, the technical problem posed and solved by the present invention is to provide a solution allowing to overcome the aforementioned disadvantages with reference to the prior art.

This problem is solved by a detection method and a microscope as defined by the independent claims.

Further embodiments of the present invention are defined by the dependent claims.

The present invention is based on the plasmon resonance phenomenon to overcome the diffraction limit and achieve three-dimensional super-resolution in the case of nuclear emulsions, allowing a three-dimensional imaging.

In particular, with respect to the prior art solutions, the present invention allows to provide a super-resolution simultaneously both in the vertical planes and in the horizontal planes, with consequent nanometric resolution along all the axes, allowing an efficient and reliable 3D reconstruction. The method and the microscope according to the invention are capable to acquire a plurality of images at different polarisation angles both in horizontal and vertical planes. The orientation of the vertical plane is adjustable: therefore, the invention allows to analyse a sample in two orthogonal projections by applying the two-dimensional method of image analysis, thus achieving the super-resolution for all the axes.

Consequently, the present invention permits the detection of nanometric structures in reduced times.

A further advantage is that the method, and the related microscope, according to the present invention allows a three-dimensional reconstruction of the sample and not only a surface resolution. Other advantages, features and modes of use of the present invention will be evident from the following detailed description of some embodiments, presented by way of example and not by way of limitation.

The present invention will be now described, by way of illustration and not by way of limitation, according to its preferred embodiments, by particularly referring to the Figures of the annexed drawings, in which:

Figure 1 shows a schematic view of an embodiment of a microscope used for the method according to the present invention;

Figure 2 shows a graph related to the spatial resolution of an optical microscope;

- Figure 3a and Figure 3b show, respectively, the positioning of a nanoparticle on an x-y plane, and the variation of the positioning along the x and y axes upon a change of a polarisation angle.

An embodiment of the method according to the present invention is directed to the reconstruction of ultrashort recoil traces, for example of some tens of nanometers, in nuclear emulsions. In any case, the method according to the present invention is extended to the detection of metal nanoparticles, optionally non-spherical ones, immersed in a dielectric medium.

The method for detecting particles having sub-diffractive size according to the present invention, comprising the steps of directing a monochromatic light beam in correspondence of the sample to be analysed, deflecting a light beam reflected by the sample to direct it towards a polarising device and obtaining a polarised light beam, rotating a polarisation direction of the reflected light beam, that passes through the polarising device, to obtain a plurality of polarised reflected light beams, and detecting an intensity of a polarised reflected light beam, in correspondence of a plurality of configurations of the polarising device, as will be better described in the following.

The Localised Surface Plasmon Resonance (LSPR) is defined as a free oscillation of electrons in the conduction band of metals excited by an incident radiation. However, for nanometric scale metals, the oscillation distance is limited by the size of the nanoparticles.

For Au or Ag nanoparticles, the Localised Surface Plasmon Resonance (LSPR) corresponds to the photon energy in visible wavelength regime, so said nanoparticles have been selected for their optical properties.

In particular, said optical characteristics include a strong plasmonic, resonant scattering, absorption and electromagnetic fields localised on the surface of the nanoparticles.

The size, shape, and dielectric constant of the nanoparticles, in particular the dielectric constant difference with respect to the surrounding medium, strongly affect the plasmonic absorption.

For instance, Au or Ag nanoparticles of spherical shape have single plasmonic absorption bands of about 540 nm and about 400 nm, respectively.

In general, what is referred in the following to conductive nanoparticles of Au or Ag immersed in a transparent dielectric material can be similarly intended for nanoparticles of other nature immersed in a contrast material, provided that the nanoparticles and the contrast material have different dielectric constants.

The localised surface plasmon resonance (LSPR) is an optical phenomenon generated by a light wave trapped inside conductive nanoparticles of size smaller than the wavelength of light.

The phenomenon is the result of interactions between the incident light and the surface electrons in a conduction band.

This interaction produces localised plasmonic coherent oscillations having a resonance frequency that strongly depends on the composition, size, geometry, dielectric environment and distance between the conductive nanoparticles.

The interaction of the conductive nanoparticles with light allows some photons to be absorbed and others to be scattered.

The scattering section is given by the formula:

where λ is the wavelength of the incident light and a is the polarisability.

The polarisability represents a distortion of the electron cloud in response to an external electric field.

In fact, in metal nanoparticles the absorption of light (and therefore the color) is due to the collective oscillations of the valence electrons, i.e. the plasmons, as in the macroscopic case. Moreover, at the nanoscale level, these oscillations depend not only on the material, but also on the size of the objects.

For a nanoparticle of homogeneous spherical shape, the polarisability is: «i = 3Ve_m ^e _r ^ε?"^} , (2)

1^m l e+(i + l)e_m' ' where V is the volume of the particle, ε = ει + ϊε₂ a nd £_m are the permittivities of the particle and the surrounding medium, respectively, and I is the number of orbital moment (for instance, I = 1 for a dipole).

The conductive nanoparticles respond substantially as induced dipoles for diameters less than about 150 nm, thus for this type of analysis it will be always considered I = 1.

Even the polarisability of an ellipsoidal particle can be described through an analytical expression. The dipolar polarisability along one of the main axes j is equal to:

where, j = x, y, z indicates the main axis of the ellipsoid and Lj is the depolarisation factor given by:

ds

(S+Rpj(s+R (s+R0(s+Ri)

where L_x + L_y + L_z = 1, and Rj are the respective lengths of the semi-axis of the ellipsoid.

The case of a spherical particle is obtained considering:

R_x = R_y = R_z, and Lj = 1/3.

In the case of a revolution ellipsoid, wherein R_x = R_y, the depolarisation factors depend on the aspect ratio R = R_z/R_x: l-e

^■f I-2e ln f \—l-e )J (5)

Lx— Ly— (7)

Therefore, the scattering section along the j axis becomes: (ei-e_m)² +

A (8)

^ ¹ (e_{1 +Xj}e where A_j = 1/Lj are the weighting factors and X_j = (1— LJ)/LJ are the form factors.

A value equal to 2 for a spherical nanoparticle is assigned to the form factor and it can be even greater than 20 for particles with a high aspect ratio.

The LSPR peak that gives rise to the color of the nanoparticle is observed when e =—X_j€_m, wherein the denominator of equation 8 has a minimum.

For Ag and/or Au nanoparticles, this condition is reached in the visible region, making these materials adapted for many applications involving the visualisation of a color.

The factor y increases with the aspect ratio, moving the plasmonic peak towards the red.

The resulting dispersion spectrum has two peaks, one corresponding to the transverse plasmonic mode from the x and y contributions, and the other corresponding to the longitudinal plasmonic mode by the z contribution.

The relative heights of the peaks are defined by the weighting factors Aj , which become strongly disproportionate as the aspect ratio increases, with a rapidly growing longitudinal fraction, causing a partial polarisation of the scattered light that becomes parallel to the major axis of the nanoparticle.

Therefore, the ellipsoidal particles have spectra strongly dependent on the polarisation, wherein small changes in the aspect ratio entail significant changes in the observed response.

The level of polarisation can be written as P = A_Z/(A_Z + A_x).

This value can be measured, as will be better described in the following, through the analysis of the polarisation distribution of the scattered light, for instance through a rotating polarising device.

The brightness of the non-spherical nanoparticles visible through the microscope changes with the rotation of the polarising plate that goes from a minimum value B_min to a maximum value B_max.

From the measurement of the ratio a = B_max/B_min it is possible to estimate the polarisation level P = α/(α + 1) and, therefore, also to detect the aspect ratio.

The optical microscope according to the present invention is optionally completely automatic and is configured to perform the analysis with polarised light that advantageously allows to exploit the effect described above.

As known, in a dielectric medium with metal nanoparticles, the scattered light has a resonance if the particles have size around 50 nm.

Moreover, if the nanoparticle has ellipsoidal shape instead of a spherical one, there is a dependence of the scattered light on the polarisation direction of the light: when the light has a polarisation directed in parallel to the major axis of the ellipsoid, there is a very pronounced resonant effect, unlike when it is polarised in the orthogonal direction.

In fact, since Ag grains are ellipsoids with a randomly directed axis, in a trace made up of two grains, the light highlights one or the other grain as the polarisation direction changes, as will be better described in the following, allowing its detection even if they are far less than the diffraction limit (about 200nm).

Figure 1 shows an embodiment of the optical microscope 100 according to the present invention comprising a sample display system 50 and an optical analyser system 60.

In particular, as will be better described in the following, the display system 50 and the analyser system 60, which respectively comprise a first objective 01 and a second objective 02, are to be understood as two substantially identical microscopes coupled to each other, the term identical meaning that the optical path between the objectives 01 and 02 is divided into two identical parts symmetric with respect to a coupling plane.

The display system 50 comprises a light source 1, for example a LED source that advantageously involves a low thermal dissipation with respect to other light sources under the same lighting power. In particular, as shown in Figure 1, the microscope 100 has an illumination of the sample from above and operates with reflected light. In this way, the optical contrast of the image is optimised.

The display system 50 further comprises an objective 01, and directing optical components for directing a monochromatic light beam from the light source 1 towards the objective 01 to allow an illumination of the sample to be analysed positioned in correspondence of a sample holder 4.

In the preferred embodiment, the said directing optical components comprise a plurality of lenses, in particular four converging lenses positioned in series with respect to each other and in series with respect to the objective 01, spaced from each other and with respect to the objective 01 so as to create a sample image on the secondary focal plane of the objective 01. As shown in Figure 1, the display system 50 further comprises a beam splitter BSl positioned between said lenses and the objective 01. A reflection surface of the beam splitter BSl is positioned, downstream of the directing optical components, with an inclination of 45 degrees with respect to the path of the incident light. In particular, the beam splitter BSl is implemented, for instance, as a semitransparent mirror or as a glass sheet with an aluminum coating of such thickness that half of the incident light at 45 degrees is transmitted and the other half is reflected.

In particular, an incident light beam coming from the light source 1 is divided into two beams by the optical device, one of which is deflected with respect to the main direction of the beam, while the other one does not undergo any deflection and is used to illuminate the sample.

As far as the light beam reflected from the sample is concerned, it is divided into two by the beam splitter BSl, and only the portion of the reflected beam that is deflected, in particular orthogonally, with respect to a reflection direction is taken into consideration in the preferred embodiment of the method according to the present invention. The reflected light beam is deflected with a deflection angle of about 90 degrees with respect to a main direction of reflection.

The portion of the reflected beam that is orthogonally deflected is directed towards a first lens LI (tube lens), as shown in Figure 1, the function of which is to collect the parallel light beams deflected by the beam splitter BSl and to bring them towards the optical analyser system 60, in particular through a second lens L2.

The optical analyser system 60 comprises a further BS2 beam splitter configured to separate the polarised light beam coming from the lens L2 into two portions.

A first portion is not deflected with respect to the incident beam and is detected by a sensor SI. In particular, the first non-deflected portion is focused, through a passage through a lens L4, in correspondence of the sensor SI, for instance a camera, as shown in Figure 1.

The deflected beam portion is instead deflected with a deflection angle of about 90 degrees with respect to a main direction of polarisation of the light beam.

Instead, the deflected beam portion creates a three-dimensional image of the sample around a focal plane of the objective 02. In fact, this portion is reflected by a reflection element, for instance a mirror 7, positioned downstream of the objective 02 so that it passes through the focal plane of the objective 02. Advantageously, the mirror 7 is inclined by 45 degrees with respect to the direction of the incident light beam. In particular, the image reflected by the mirror 7 is detected by an acquisition sensor S2, for instance a camera.

The mirror 7 is advantageously fixed to the pupil of the objective 02. Optionally, the short working distance of the objective 02 in use (about 0,12 mm) makes only the surface near the edge usable. This requires the mirror 7 to have a high quality reflecting surface and a straight edge. A high numerical aperture NA of the objective 02, optionally equal to 1,49, and the proximity of the image reflected from the mirror 7 to the pupil of the objective 02 allow a sufficient fraction of the beams forming the image to be collected by the objective 02.

The rays reflected by a spot in the sample, for instance by a grain that is around the focal plane of the objective 01, after having passed through the entire optical path intersect around the focal plane of the objective 02 creating a virtual image of such grain. If the grain is at a certain distance from the focal plane of the objective 01, its image is at the same distance from the focal plane of the objective 02. Thanks to the symmetric structure of the display system 50 and analyser system 60, the distortions introduced by the display system 50 are deleted by the analyser system 60 that is symmetric thereto. Therefore, such symmetricity ensures the best quality of the image produced in the focal plane of the objective 02.

Figure 4 schematically shows the directions of propagation of beams within the microscope 100 when they interact with a sample ("Sample"), i.e. the vertical tracing of these beams, wherein Fig. 4a shows in detail a pattern within the sample, Fig. 4b shows the rotated pattern of Fig. 4 that is focused on the image acquisition sensor S2, Fig. 4c shows the beams incident on the mirror 7 inclined by 45 degrees, and Fig. 4d shows the beams reflected by the mirror 7.

The function of the mirror 7, advantageously inclined by 45 degrees with respect to the optical axis of the objective 02, is to rotate the image in the focal plane of the objective 02. As shown in Figures 4c and 4d, the image is rotated by 90 degrees, thus transforming an originally vertical pattern into a horizontal pattern that is formed on the acquisition sensor S2.

As shown in Figure 4, if the rays reflected by the grain intersect before reaching the mirror 7, the rays reflected by the mirror create virtual intersection points downstream of the mirror 7. Instead, if the rays are reflected by the mirror 7 before intersecting, a point of real intersection of the reflected rays is created upstream of the mirror 7.

Advantageously, the mirror 7 is inclined by 45 degrees with respect to the optical axis of the objective 02 and all the intersection points, both real and virtual ones, are rotated by 90 degrees with respect to the three-dimensional image created by the incident light beam. In this way, an image of the vertical plane is produced.

In particular, the (inclined) mirror 7 mounted in front of the lens of the objective 02 of the analyser system 60 partially reflects the imaging rays in the objective lens 02 by rotating the virtual image by 90 degrees, thus making the vertical patterns as well as the horizontal ones visible. The unique combination of the display system 50 and of the analyser system 60 allows the simultaneous application of the super-resolution method both in the vertical planes and in the horizontal planes with consequent nanometric resolution along all the axes. In this way, the microscope 100 is capable to acquire the plurality of images at different polarisation angles both in horizontal and vertical planes. The orientation of the vertical plane is adjustable, whereby it is possible to analyse the sample in two orthogonal projections by applying the two-dimensional method of analysis of the image, thus achieving the super-resolution for all the axes.

Therefore, the light beam is reflected by the mirror 7 such that a reflected image appears rotated by 90 degrees, in particular the vertical structural features appear as horizontally oriented.

In other words, the presence of the mirror 7 creates a virtual image that reverses the vertical plane with the horizontal plane when the mirror 7 is inclined by 45 degrees with respect to an optical axis. The reversed, and thus virtual, image is detected by the acquisition sensor S2. In particular, the image is focused in correspondence of the camera S2, through the passage through a lens L3.

Advantageously, the orientation of the vertical plane of the image can be adjusted through a change of the positioning of the mirror 7. In particular, the mirror 7 is rotated about the optical axis maintaining its inclination of 45 degrees with respect to the direction of the light beam.

By rotating the mirror 7 around the optical axis, it is possible to change the vertical plane of the sample that is brought into focus. In this way, it is thus possible to acquire a three- dimensional image of the sample in the two planes of best focus, both horizontal and vertical one.

Advantageously, the lenses LI and L2 are positioned so as to have focal planes coincident with each other, i.e. at a reciprocal distance that is twice the focal length. In particular, the lenses LI and L2 are identical to each other, and in particular identical to the lenses L3 and L4, as the objectives 01 and 02 are identical, so as to avoid optical aberrations and distortions in the image created by the objective 02. ln this configuration, the first microscope, i.e. the display system 50, creates an enlarged image of the sample in the coupling plane and the second microscope, i.e. the optical analyser system 60, reduces it to the original size in the focal plane of the objective 02. In this way, an identical image of the sample is obtained in the primary focal plane of the objective 02.

As shown in Figure 1, the preferred embodiment of the optical microscope 100 according to the present invention comprises a polarising device 6, in particular a rotatable polarising plate.

The polarising device 6 is positioned, or can be positioned, along the optical path included between the display system 50 and the optical analyser system 60, allowing a polarisation of the incident rays.

In particular, the polarising device 6 is rotatable, for instance it is mounted inside a bearing to allow a free rotation around a main optical axis, optionally coinciding with an optical axis of the lenses LI and L2.

The polarising device 6 allows the passage of light of a specific polarisation depending on its specific angular positioning.

The rotation of the polarising device 6 to select a change of the polarisation can be carried out both mechanically, for instance through a motor, and with an optoelectronic device, for instance by providing a liquid crystal polariser driven by the system, so as to allow an automated control of the microscope.

In particular, a damping element, for instance a soft O-ring, is present to absorb vibrations during the transfer of motion from the motor to the device.

Advantageously, the period of revolution of the polarising device 6 is synchronised with a shutter of the sensors SI and S2, optionally cameras, so as to ensure that during a complete rotation of the polariser 6, a sufficient number of images of the sample is collected to guarantee an analysis thereof.

The optical microscope 100 according to the present invention is also provided with moving devices. The moving devices comprise, in addition to the device for rotating the polarising device 6, a system MT for moving the sample holder 4 and a further system MS for moving the reflecting element 7.

In particular, the motorised arrangement allows a horizontal and vertical movement of the sample with respect to the microscope, so as to bring any point inside the sample into focus by vertically positioning the lenses and horizontally moving the same sample.

Moreover, as mentioned above, also the rotation of the polarising device 6 is automated, in particular to synchronise the positioning of the polariser 6 with the shutter of the sensors SI and S2, optionally cameras, so as to ensure that during a complete rotation of the polariser 6, a sufficient number of sample images is collected to allow an analysis thereof.

Likewise, the rotation of the mirror 7 around the optical axis of the objective 02 is motorised, for instance through the device MS, in particular to synchronise the positioning of the mirror 7 with the shutter of the camera S2, so as to ensure that during a complete rotation of the mirror 7, a sufficient number of sample images is collected to allow an analysis thereof.

Advantageously, both the movement of the various components and the acquisition of the images are controlled by one or more processing units, e.g. a dedicated computer, which also perform the image processing.

In particular, an analysis of the polarised component of the reflected light is indeed carried out. A rotation of the polariser 6 is provided and images are detected in correspondence of a plurality of polarisation angles different from each other (whereby a plurality of images at different polarisation angles are acquired).

A preferred embodiment of the method according to the present invention provides for a sample analysis through a step of bringing a grain into focus and a step of simultaneously detecting two images coming from the analysed grain, one in the horizontal plane and the other in the vertical plane.

The brightness of the images of a single grain, or of a cluster of grains, changes with the rotation of the polarising element in a rather random and independent way also for grains belonging to the same trace.

This characteristic allows to distinguish traces composed of a plurality of grains close to each other, from single grains, and at the same time it allows to isolate individual grains within the trace.

For example, the image of a single (not spherical) grain appears as a static cluster, with a variable brightness depending on the rotation of the polarising element.

The image of a trace comprising two grains close to each other appears as a cluster the centroid of which moves along the direction of the trace.

In particular, in Figure 3a the movements on the X-Y plane are shown. Each cross represents the coordinates of a centroid for a specific polarisation angle. The transverse dimension corresponds to a position accuracy of about 10 nm. The length of the trace measured in the example shown is 99nm. Figure 3b shows the curve of the movement along the X and Y axes upon the change of the polarisation angle.

In particular, the graph of Figure 3a refers to images which have been detected by rotating the polarising element by 180 degrees, with a fixed pitch of 22,5 degrees.

For each image, the movement (dx, dy) of the centroid of the cluster has been measured in x and y coordinates.

A movement exceeding the precision of the position of a single grain is an evidence that it is a cluster formed by two close grains and therefore it is produced by a trace signal.

A volumetric scan of the sample, for instance of a nuclear emulsion, is carried out by analysing a set of tomographic images equidistant from each other. Then, a vertical movement of the sample with respect to the microscope 100 is first made, in particular through the device MT, to focus the area to be analysed. Then, the field of view is horizontally moved, in particular by horizontally translating the sample with respect to the microscope 100.

In particular, in correspondence of each height, a set of images is detected, wherein each image corresponds to a specific angle of rotation of the polarising element.

The steps are then repeated until the entire volume of the emulsion is scanned.

The preferred embodiment of the method according to the invention acquires the images with the field of view containing a portion of the sample that is near the centre of the image (if the mutual arrangement of the microscope 100 and sample does not meet this condition, it is always possible to move the sample holder 4 to bring the sample to a proper position or to extract a sufficiently large sub-image). In particular, the sample is divided into one or more scanning portions which are subsequently acquired until the entire sample of interest to be analysed is scanned. In the following, an image processing implementing the two-dimensional method of image analysis illustrated above in general terms is described in greater detail. This images processing includes, for each one of the (one or more) portions of the sample, the following steps:

a) acquiring the plurality of horizontal images (similarly to what shown in Fig. 4b) at different polarisation angles with the sensor S2;

b) calculating the centroid of the spot of the sample portion (e.g. by means of a Gaussian fitting or other conventional appropriate method) for all the images corresponding to several different polarisation angles to obtain a respective plurality of centroids;

c) converting the plurality of centroids into the same coordinate system (e.g. the coordinate system of the field of view);

d) plotting the trace of the centroid movement upon the change of the polarisation angle (e.g. by plotting a graph similar to those shown in Figures 3a and 3b);

e) calculating the horizontal length of the movement plotted in step d) (e.g. as the length of the longest line interconnecting the centroids);

f) calculating the horizontal direction of the movement plotted in step d) (e.g. as the direction of the longest line interconnecting the centroids);

g) rotating the mirror 7 so that the vertical focal plane (similarly to what is shown in Fig. 4a) coincides with the direction calculated in step f);

h) acquiring the plurality of vertical images (similarly to what is shown in Fig. 4a) at different polarisation angles with the sensor S2;

i) repeating steps a) to f) for the plurality of vertical images to calculate the vertical length and the related vertical direction;

j) calculating the three-dimensional length of the trace (e.g. as the square root of the sum of the horizontal and vertical squared lengths);

k) calculating the three-dimensional direction of the trace (e.g. as the direction for which the horizontal direction is the azimuthal angle and the vertical direction is the inclination angle); and

I) if the three-dimensional length calculated in step j) exceeds a predetermined threshold (e.g. 3 times the spatial accuracy of the system, shown in Figure 2), marking this event as a trace.

As stated, the sequence of steps a) to I) is performed for each one of the (one or more) portions into which the sample is subdivided and which are subsequently acquired until the entire sample of interest to be analysed is scanned. Advantageously, the method according to the invention allows to reach a three-dimensional resolution of 10 nanometers, a value that is obtainable because this system is not limited by the Rayleigh criterion.

Thus, the entire volume can be scanned with a sub-diffractive resolution limit using a three-dimensional automated analysis method having a scanning speed that is far higher with respect to other systems with similar resolution.

The scanning speed depends on the specific configuration and it is of the order of few mm³ per hour.

The thickness of the analysed sample can range from 1 micron to 1 mm, depending on the optical components which are used.

A further embodiment provides for the execution of a scan of only of a series of selected points during a volume scan, instead of an entire volume, for instance through a traditional microscope.

This approach allows to further increase the scanning speed of an embodiment of the sub-diffractive resolution method according to the present invention.

The embodiment of the method according to the present invention that provides for the possibility of scanning on a sole surface of the sample allows a much higher scanning speed with respect to the embodiments described above.

In particular, the scanning speed is equal to about 20 mm²/h in case of a three- dimensional scanning, for instance for a thickness of 50 μιη, while in the case of surface analysis, the scanning speed can be about ten times higher than what stated above.

Therefore, the effects of the Localised Surface Plasmon Resonance (LSPR) advantageously give more accurate structural information than the optical resolution.

Although the optical resolution as the capability of a display system to discriminate two high-contrast objects adjacent to each other is limited by diffraction, the method according to the present invention allows a much higher spatial resolution, obtained by measuring the movement of the centroid of a particle or cluster of particles.

In particular, the spatial resolution obtained with the method according to the present invention depends on the shape of the object. In fact, a nanometric resolution is reached if the particles are substantially non-spherical in shape. The resolution improves as the pixel size decreases on the acquired image. In any case, such size must be less than about 50 nm.

As shown in Figure 2, through a repeated analysis carried out on the same object, the method according to the present invention allows to obtain a spatial resolution equal to about 10 nm. This result is made possible by the analysis of the movements of the position of the object centroid as the polarisation changes.

The preferred embodiments of this invention have been described and a number of variations have been suggested hereinbefore, but it should be understood that those skilled in the art can make other variations and changes without so departing from the scope of protection thereof, as defined by the attached claims.

Claims

1. Method for detecting particles having sub-diffractive size within a sample, comprising the steps of:

A. directing a monochromatic light beam towards the sample to be analysed through a first objective (01) having a first focal plane;

B. deflecting a light beam reflected by the sample, to direct it towards a polarising device (6);

C. changing a polarisation configuration of the polarising device (6) through which the light beam reflected by the sample passes, to obtain a plurality of polarised reflected light beams corresponding to a plurality of polarisation configurations of the polarising device (6);

D. deflecting the plurality of polarised reflected light beams, corresponding to the plurality of polarisation configurations of the polarising device (6), to direct them towards a reflection element (7) through a second objective (02) having a second focal plane, wherein an optical path between the first objective (01) and the second objective (02) is divided into two identical parts symmetric with respect to a coupling plane, whereby rays reflected by a point in the sample that is at a first distance from the first focal plane form an image at a second distance from the second focal plane that is identical to the first distance;

E. detecting a plurality of polarised reflected light beams that is reflected by the reflection element (7) according to a reflection direction, corresponding to the plurality of polarisation configurations of the polarising device (6), thus acquiring a plurality of images; and

F. performing a two-dimensional method of analysis of the plurality di acquired images.

2. Method according to claim 1, wherein the reflection element is a mirror (7) inclined with respect to an optical axis of the second objective (02).

3. Method according to claim 2, wherein the mirror (7) is inclined by 45 degrees with respect to the optical axis of the second objective (02).

4. Method according to any one of claim 1 to 3, wherein the step of changing the polarisation configuration of the polarising device (6) comprises rotating a polarisation direction of the polarising device (6).

5. Method according to any one of claim 1 to 4, comprising a step of rotating the reflection direction of a polarised reflected light beam that is reflected by the reflection element (7), to obtain a respective plurality of polarised reflected light beams that is reflected by the reflection element (7) and that is detected in step E.

6. Method according to any one of claim 1 to 5, wherein in step B the light beam reflected by the sample is deflected by a deflection angle of about 90° with respect to a main direction of propagation of the light beam reflected by the sample.

7. Method according to any one of claim 1 to 6, wherein in step D the plurality of polarised reflected light beams, corresponding to the plurality of polarisation configurations of the polarising device (6), is deflected by a deflection angle of about 90° with respect to a main direction of propagation of the plurality of polarised reflected light beams, corresponding to the plurality of polarisation configurations of the polarising device (6).

8. Optical microscope (100) for detecting particles having sub-diffractive size within a sample, comprising:

a display system (50) including one or more directing optical components, configured to direct a monochromatic light beam emitted by a light source (1) towards the sample to be analysed through a first objective (01) having a first focal plane, and a first beam splitter (BS1) configured to deflect a light beam reflected by the sample;

a polarising device (6) configured to polarise the light beam reflected by the sample and deflected through the first beam splitter (BS1) according to a plurality of polarisation configurations;

an analyser system (60), including a second beam splitter (BS2) configured to deflect a polarised reflected light beam coming from the polarising device (6) to direct it towards a reflection element (7) through a second objective (02) having a second focal plane, wherein an optical path between the first objective (01) and the second objective (02) is divided into two identical parts symmetric with respect to a coupling plane, whereby rays reflected by a point in the sample that is at a first distance from the first focal plane form an image at a second distance from the second focal plane that is identical to the first distance;

one or more moving devices configured to change a polarisation configuration of the polarising device (6) through which the light beam reflected by the sample passes, to obtain a plurality of polarised reflected light beams corresponding to a plurality of polarisation configurations of the polarising device (6); a sensor device (S2) configured to detect a plurality of polarised reflected light beams that is reflected by the reflection element (7) according to a reflection direction, corresponding to the plurality of polarisation configurations of the polarising device (6), thus acquiring a plurality of images; and

- one or more processing units configured to control said one or more moving devices and to perform a two-dimensional method of analysis of the plurality di acquired images.

9. Optical microscope (100) according to claim 8, wherein the reflection element is a mirror (7) inclined with respect to an optical axis of the second objective (02).

10. Optical microscope (100) according to claim 9, wherein the mirror (7) is inclined by 45 degrees with respect to the optical axis of the second objective (02).

11. Optical microscope (100) according to any one of claims 8 to 10, wherein said one or more moving devices are configured to change the polarisation configuration of the polarising device (6) through a rotation of a polarisation direction of the polarising device (6).

12. Optical microscope (100) according to any one of claims 8 to 11, wherein said one or more moving devices also comprise a moving device (MS) configured to rotate the reflection direction of a polarised reflected light beam that is reflected by the reflection element (7), to obtain a respective plurality of polarised reflected light beams that is reflected by the reflection element (7) and that is detectable by the sensor device (S2).

13. Optical microscope (100) according to any one of claims 8 to 12, wherein said one or more moving devices further comprise a moving device (MT) of a sample holder (4) configured to receive the sample to be analysed.

14. Optical microscope (100) according to any one of claims 8 to 13, wherein the first beam splitter (BS1) is configured to deflect the light beam reflected by the sample by a first deflection angle of about 90° with respect to a main direction of propagation of the light beam reflected by the sample.

15. Optical microscope (100) according to any one of claims 8 to 14, wherein the second beam splitter (BS2) is configured to deflect the polarised reflected light beam coming from the polarising device (6) by a second deflection angle of about 90° with respect to a main direction of propagation of the polarised reflected light beam coming from the polarising device (6).