Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
  • G01N21/553—Attenuated total reflection and using surface plasmons
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
  • G01B9/00—Measuring instruments characterised by the use of optical techniques
  • G01B9/04—Measuring microscopes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y35/00—Methods or apparatus for measurement or analysis of nanostructures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/06—Illumination; Optics
  • G01N2201/067—Electro-optic, magneto-optic, acousto-optic elements

Definitions

• the present invention relates to a high resolution surface plasmon microscope comprising a heterodyne interferometer and using a radial polarization of the surface plasmon generation beam.
• the technical field of the invention is that of the design of imaging systems allowing the detection of small variations of refractive index in an observation medium and / or dielectric objects of the order of a few nanometers with no not necessarily remarkable optical properties (fluorescence, luminescence, localized plasmon resonance or Raman resonance) and located near a surface and immersed in any medium of refractive index less than 1.5 and in particular in the air or in a aqueous medium.
• a surface plasmon is a surface electromagnetic wave that propagates at a metal / dielectric medium interface.
• the angle ⁇ p (in other words the coupling condition) is very sensitive to the slightest changes in the optical properties at the metal / dielectric medium interface. This sensitivity makes exploitable the surface plasmon for producing images of very small objects located at the metal / dielectric medium interface, said objects modifying the optical properties of the Plasmon surface at this interface which allows to obtain a contrast between the object and its environment.
• the surface plasmon being an evanescent wave, it makes it possible to overcome the effects of volume within the observation medium.
• the principle of plasmon excitation in surface plasmon microscopy is very often illustrated in the literature with reference to the so-called Kretschmann-Raether configuration. This provides for the deposition on one side of a glass prism, in contact with a dielectric medium such as air, a metal layer of the order of 50 nanometers thick. When a high intensity light beam passes through the prism and meets the metal layer at the angle of incidence ⁇ p, a surface plasmon then takes birth in the metal and the resulting evanescent wave is confined to a few hundred nanometers at the interface metal / dielectric medium.
• illumination and detection are based on spatial filtering, at the entrance of the objective, light rays that contribute to plasmon excitation and elimination. of those who do not contribute.
• 1B and 2A a microscopy device which allows a measurement of the phase but which does not filter the light rays not contributing to the plasmon excitation by eliminating its uninteresting part. Coupling these two technical aspects has the advantage of having a resolution and sensitivity unequaled compared to other techniques known to date
• FIG. 2B represents the distribution of the intensity of the laser beam at the rear focal plane of the objective after retro-diffusion and ironing by the laser.
• objective two crescent-shaped rings that correspond to the area of the starting beam that participated in the excitation of the surface plasmon.
• the zone of the rays concerned has a privileged orientation which is that of the direction of polarization of the light at the entrance of the objective.
• no ray contributes to the excitation of the surface plasmon in the orthogonal direction (vertical passing through 0 in FIG. 2B) and there is only a fraction of the incident light energy which participates in the excitation of surface plasmon.
• US 2004/0100636 discloses the possibility of obtaining better image resolutions with a radially polarized excitation beam.
• this document does not disclose any example of a microscope structure implementing this principle nor any measurement carried out with a radial polarization which makes it possible to validate this mentioned measuring principle. It is an object of the present invention to provide a high resolution surface plasmon microscope which has increased resolution and sensitivity over existing surface plasmon microscopes.
• Another object of the invention is to provide a surface plasmon microscope which allows the observation of molecules and particles in aqueous dielectric media, and in particular in biological fluids.
• the object of the invention is in particular to provide a high-resolution surface plasmon microscope that enables the detection and visualization of objects of very small size, of the order of a nanometer, such as biological molecules for example, without having recourse to chemical, optical or radioactive markers of these objects.
• Another object of the invention is finally to provide a surface plasmon microscope simple to achieve and use.
• a high-resolution scanning surface plasmon microscope comprising essentially: a) a coherent light source, and b) a coupling medium and a confinement of a surface plasmon having a high numerical aperture objective, an immersion oil and a glass slide, and c) a metal layer covering a surface of the glass plate of the coupling medium which is not in contact with the immersion oil of the latter, the metal layer being able to be brought into contact with a medium of observation containing a sample to be observed and to emit a surface plasmon generated by excitation of at least one light beam coming from the light source, and d) a Twyman-Green interferometer in heterodyne mode capable of dividing a light beam emitted by the light source into at least one reference beam and at least one measuring beam directed to the coupling medium and the metal layer to generate a surface plasmon, the interferometer being positioned between the light source and the lens of
• the microscope of the invention is characterized in that it comprises, arranged between the light source and the interferometer, at least one linear polarization converter in radial polarization of the light beams emitted by the light source, and allowing the detection of dielectric and metal objects with a diameter of less than 10 nm, without marking said objects.
• the microscope of the invention differs from the state of the art in that it allows a radial polarization conversion of the surface plasmon generation beam.
• the invention also relates to a high resolution surface plasmon microscopy method by heterodyne interferometry developed for the implementation of the microscope of the invention. According to this method:
• a sample to be imaged placed on a metal layer coating a glass plate of a coupling medium and confining a surface plasmon also having a lens (with a large numerical aperture and an immersion oil, and
• the surface of the metal layer is scanned by means of the measuring beam guided by scanning means, and
• an interferometric light beam at the output of said Twyman-Green interferometer is detected by detection means, and
• said interferometric beam is processed and an image of the surface plasmon emitted by the metal surface in contact with the sample to be imaged is formed by means for processing and forming an image
• This method is characterized according to the invention by the fact that the polarization of the beam of coherent light is polarized by a polarization converter before it enters the interferometer so as to illuminate the sample to be imaged on the metal layer of the coupling medium. using a measuring beam having a radial symmetry with respect to its axis of propagation.
• the polarization converter is polarized alternately with the coherent light beam in pure mode p (radial polarization) and in pure mode s (azimuthal polarization) and is scanned linearly. alternatively and synchronously the alternative polarization of the coherent light beam the surface of the metal layer by the measuring beam polarized alternately in pure mode p and in pure mode s.
• FIG. 1A represents the principle of excitation and confinement of the surface plasmon in a microscope of the prior art with a light beam focused at the metal / dielectric medium interface;
• FIG. 1B shows the principle of excitation and confinement of the surface plasmon in a microscope of the prior art with a defocused light beam in the metal / dielectric medium interface
• FIG. 1C represents an experimental curve V (z) in water obtained with a surface plasmon microscope of the prior art in linear polarization
• FIG. 2A represents a high-resolution surface plasmon microscope as known from the prior art implementing an excitation and a confinement of the surface plasmon as represented in FIG. 1B;
• Figure 2B shows the light distribution profile reflected by the surface plasmon generation metal surface at the exit of the microscope objective of Figure 2A;
• FIG. 2C represents the impulse response of the microscope of FIG. 2A in linear polarization by numerical calculation of the distribution of the focused light at an interface between a gold metal layer and a dielectric medium formed by water, with a numerical aperture objective equal to 1.65;
• FIG. 3A schematically shows a high resolution surface plasmon microscope according to the present invention
• FIG. 3B shows the light distribution profile reflected by the surface plasmon generating metal surface at the exit of the objective of the microscope of the invention shown in FIG. 3A
• FIG. 3C represents the impulse response of the microscope of FIG. 3A in radial polarization by numerical calculation of the distribution of the focused light at the interface between a gold metal layer and a dielectric medium formed by water, with a numerical aperture objective equal to 1.65;
• FIGs. 4A and 4B respectively show the image of a 50 nanometer diameter latex particle obtained with the prior art microscope shown in Fig. 2A and with the microscope of the invention shown in Fig. 3A;
• FIG. 5 schematically represents the polarization conversion effect of the electric field of the surface plasmon generation light beam implemented in the microscope of the present invention;
• Figure 6 shows in detail a preferred embodiment of the microscope of the present invention, as schematically shown in Figure 3A.
• the present invention provides a high resolution scanning surface plasmon microscope operating according to the so-called V (z) effect principle which provides that the V response of the microscope varies as a function of the defocus distance z relative to the metal layer interface / dielectric observation medium of the microscope.
• the principle of surface plasmon excitation in high resolution surface plasmon microscopy is analogous to the so-called Kretschmann configuration. It implements in the first place a coupling medium that replaces the glass prism of the so-called Kretschmann configuration.
• This coupling medium consists of an O objective with a high numerical aperture, typically at least 1.2 NA, bathed by the surface of one of its diopters on an immersion oil Hi which itself is in contact with a first face of a glass slide Gs.
• the free face of the glass plate Gs (that is to say that not in contact with the oil) is covered with a metal layer M s of about 45 nm in thickness, for example gold or silver.
• This analysis medium D can be air, water or an aqueous solution or more generally any dielectric medium having a refractive index less than or equal to 1.5.
• a light beam L such as a laser beam, represented by the double arrows in FIGS. 1A and 1B, is sent towards the metal layer M 5 through the coupling medium.
• the rays reaching the metal layer M s and the interface thereof with the dielectric medium D only those which are reflected on the metal layer with an angle of incidence close to the plasmon resonance angle ⁇ p excite the surface plasmon, represented by the solid lines in FIG. 1A, at the interface between the metallic layer Ms and the dielectric observation medium D.
• the defocusing of the focal plane of the objective O behind the metal surface M s to be observed enables the surface plasmon, excited by the ray Ri to propagate, to reemit, throughout its propagation at the interface of the radii with an angle ⁇ p and only that which passes through O, ie Rp propagates towards the photodetector.
• This phase delay varies with the defocus distance z along the Z axis in the XOZ mark and the propagation speed of the surface plasmon.
• a measurement of this phase shift and a scanning on the surface point by point make it possible to probe the local variations of the plasmon resonance and thus make it possible to visualize, with a resolution of the size of the focused light spot, the local variations of the optical properties at the of the interface.
• the image is then formed point by point.
• the device comprises a laser source LG whose beam L is divided in two by a beam splitter BS, thus forming an interferometer.
• a beam expander BE passes through a coupling medium comprising an objective O which allows the excitation of the plasmon due to its very large numerical aperture and which confines the plasmon thanks to its high magnification, an oil to immersion Hi and a glass plate covered on its outer surface with a metal layer M s of gold or silver in contact with an observation dielectric medium D.
• the beam reflected by the metal layer M s passes through the Objective O and is recombined with the other beam which has been reflected by a mirror M.
• the two beams generate an interference signal having a temporal modulation generated by a shift of the different optical frequency in each arm of the interferometer.
• the interferometric signal is collected by an optical detector PD and an electronics demodulates the modulated signal.
• the signal thus obtained represents the intensity of a pixel of the image.
• the present invention provides a significant improvement to the prior art, in particular by allowing a uniform light distribution at the objective output of the microscope to be obtained, which provides a greatly improved resolution and sensitivity as will be presented hereinafter. after.
• a particular embodiment of the microscope of the invention is shown schematically in Figure 3A in which the elements common with the microscope of Figure 2A have the same references.
• the microscope of the invention similarly to the microscope of FIG. 2A, comprises a coherent LG light source, for example a L.A.S.E.R. source, and in particular a helium-neon gas (He-Ne) laser.
• a coherent LG light source for example a L.A.S.E.R. source, and in particular a helium-neon gas (He-Ne) laser.
• He-Ne helium-neon gas
• a polarizer P supplemented with a beam magnifier BE to expand the laser beam L before entering a Twymann-Green interferometer operating in heterodyne mode which comprises firstly a beam splitter BS to form two laser beams L Re f and L Mes propagating in two distinct arms of the interferometer.
• a first laser beam L Ref propagates in a first arm, referred to as reference, which comprises a reflection mirror M R e f of this first reference light beam.
• the second laser beam L M e / which we will call measurement propagates in a second arm, said measuring, towards a coupling and confinement medium of a surface plasmon comprising a lens O with a large numerical aperture, a filter oil. Immersion Hi and a glass slide G 5 .
• the measuring arm also comprises at least one beam expander BE placed between the beam splitter BS and the coupling medium.
• each arm of the interferometer at least one AOM Ref acousto-optical modulator
• AOMM ⁇ S for example consisting of a Bragg cell, capable of introducing an offset of the optical frequency of the light beam L Ref and L Mes reference and measurement respectively.
• the objective O of the coupling medium has a numerical aperture greater than or equal to 1.2 in the air and at 1.55 in an aqueous solution and a magnification greater than sixty times. These characteristics of the objective O thus ensure good excitation and good confinement of the surface plasmon.
• An outer surface of the glass plate G s of the coupling medium is covered with a metal layer M s in contact with a dielectric observation medium D of refractive index less than 1.5, for example from air or water, containing a sample to be observed.
• the measuring beam L Mes is thus directed towards the coupling medium and the metal layer M 5 to generate a surface plasmon at the metal layer / dielectric medium interface D.
• the measurement beam L Mes reflected from the metal surface M s passes through the objective O and is then recombined with the beam L R ⁇ f reflected by the mirror M Ref in the reference arm.
• the two beams generate an interferometric signal collected by optical detection means PD such as a photomultiplier or a CCD camera for example or a photon counter, or an avalanche photodiode.
• This interferometric signal has a temporal modulation generated by an offset of the optical frequency of each light beam reflected in each interferometer arm.
• the microscope comprises an appropriate demodulation electronics as well as means for processing and forming an image from the interferometric beam essentially consisting of computer processing and visualization means.
• the microscope of the invention differs from microscopes of the prior art and in particular that of Someck et al. in that it comprises, arranged between the light source LG and the interferometer, at least one linear polarization CP converter in radial polarization of the laser beam L emitted by the light source LG.
• the passage in radial polarization of the laser beam L emitted by the light source LG makes it possible in fact to illuminate the input of the objective O of the coupling medium with a polarization which presents, as represented in FIG. 5, a radial symmetry by relative to the axis of the beam.
• the polarization converter CP advantageously makes it possible to modify the polarization and therefore the orientation of all the electric field vectors Eo with respect to the axis of propagation of the beam L so that these vectors are all oriented radially to the axis of propagation so that the contribution to the generation of surface plasmon by the incident wavefront on the metallic surface Ms is uniform and optimal.
• the distribution of light reflected at the exit of the objective is quite uniform and circular, which can be concluded, by a simple comparison of FIGS. 2B and 3B, that the zone of the beam which contributes to the the excitation of the plasmon is greater and its surface substantially multiplied by two compared with the microscopes of the prior art.
• the radial polarization operation of the surface plasmon microscope of the invention substantially improves the resolution and sensitivity of the microscope as distinctly shown in FIGS. 2C and 3C which represent the light intensity at the level of the microscope. the interface calculated respectively in linear polarization and in radial polarization.
• the spot in radial polarization, the spot has only one intense peak which makes it possible, on the one hand, to reconcentrate the light beam and, on the other hand, to improve the impulse response of the microscope. .
• the intensity of the focused spot obtained with the microscope of the invention is accordingly four times more intense.
• the microscope of the invention provides an improvement in the profile of the optical response since a single peak is obtained instead of two with the previous microscopes, but also a 3-fold increase in resolution, which passes from 600 nm to 200 nm.
• the microscope of the invention comprises means for scanning the metal layer using the measuring light beam, in particular piezoelectric means for translational movement of the lamella and or the objective of the coupling medium in two orthogonal directions X, Y in the same plane.
• the scanning means of the microscope of the invention comprise piezoelectric means for moving the objective in translation along a Z direction normal to the plane of each of the surfaces of the glass slide of the coupling medium. and the metal layer, thus ensuring a knowledge of the distance of the lens with respect to the lamella.
• Figure 6 shows in detail a surface plasmon microscope in a preferred embodiment according to the invention. It comprises in the first place a light source LG formed by a laser
• This LG light source emits a laser beam L towards two successive deflection mirrors Ml and M2 with an angle of incidence of approximately 45 °.
• These two mirrors M1, M2 allow precise adjustment of the height and parallelism of the beam L with respect to a horizontal plane and an axis which will define the centering of all the optical components and in particular the axis of symmetry of the focusing objective. and the direction of normal incidence of the surface of the coverslip.
• the beam L then passes through a polarizer P which polarizes the beam vertically, then a diaphragm D 0 .
• the beam L is collimated and enlarged by means of a first telescope Ti composed of an objective Oi and a lens Li. Its magnification factor is 2.3.
• the beam L then passes through a polarization converter CP which converts the uniform distribution of the initial vertical linear polarization into a spatial distribution of radial symmetry of the polarization with respect to the center of the beam.
• a polarization converter CP which converts the uniform distribution of the initial vertical linear polarization into a spatial distribution of radial symmetry of the polarization with respect to the center of the beam.
• the laser beam L passes through a diaphragm Di and then enters a Twyman-Green interferometer in heterodyne mode, the description of which is given below.
• the positioning of the CP converter before the interferometer is an important criterion because it limits the optical defects of the wavefront, those being subsequently eliminated by the interferometric technique.
• the interferometer comprises in the first place a separator cube BS through which the beam L passes and is divided into two beams L Me s, L Re f of equal intensities.
• the first beam L Mes is not deflected and continues its trajectory in a first arm of the interferometer which is called the measuring arm (on the right of the cube in the diagram of Figure 6).
• the second beam L Ref is deflected 90 ° with respect to the initial beam L. It continues its propagation in a second arm that is called the reference arm (below the cube in the diagram of Figure 6).
• the beam enters an acousto-optical modulator AOM Ref consisting for example of a Bragg cell with an incidence angle of 6.95 milliradians.
• An acoustic wave of frequency ⁇ ref 75 MHz generated and sent into AOM R ⁇ f by a synthesizer S makes it possible to generate a diffraction grating of the beam L Ref .
• the light undergoes a shift of its optical frequency ⁇ op t of + ⁇ ref .
• the angle of incidence of the beam is then adjusted to the Bragg angle in order to transfer all the light intensity in the +1 diffraction order.
• the adjustment makes it possible to obtain 85% of the starting intensity, the rest being distributed in order of decreasing intensity in the orders 0, -1, 2, -2 etc.
• This adjustment is possible by a rotation plate (not shown) fixed under the modulator AOM Re f and requires a positioning accuracy greater than 0.1 milliradian.
• the beam passes through a diaphragm D R ⁇ f of 2mm diameter in order to eliminate all the diffracted beams except the order 1, which is shifted in frequency by ⁇ Ref .
• This beam then arrives at normal incidence on a reference mirror M Ref of maximum optical quality and a flatness of ⁇ / 20, ⁇ being of course the wavelength of the laser beam L.
• the reflected beam L Ref goes back through the diaphragm D Ref and arrives on the acousto-optical modulator A0M Ref with the same Bragg angle as on the outward direction.
• the beam is again diffracted and shifted by + ⁇ Ref at the output of the acousto-optical modulator AOM Re f.
• This beam is shifted in frequency by 2x ⁇ ref with respect to the initial beam, and propagates on the same optical axis as the latter at the output of the separator cube BS. It goes back through the separator cube without deviation and arrives on an optical photodetector PD which has a diaphragm D 2 at its input thus eliminating all diffracted orders different from the order 1.
• the beam L Me s enters an acousto-optical modulator AOM Mes , also of type Bragg cell.
• An acoustic wave of frequency ⁇ Me s 75.05 Mhz generated and sent to AOM Mes by the synthesizer S makes it possible to generate therein a diffraction grating of the beam L Mes -
• At the passage of the beam L Mes in the modulator A0M MeS / la light is shifted by its optical frequency ⁇ op t of + ⁇ M es-
• the angle of incidence of the beam is adjusted to the Bragg angle in order to transfer all the light intensity in the +1 diffraction order.
• the adjustment of AOM Mes makes it possible to obtain 85% of the starting intensity, the rest being distributed in order of decreasing intensity in the order 0, -1, 2, -2 etc.
• the light beams diffracted at the different orders of L Me s pass through a lens O 2 with a magnification of 10 times and a spatial filter F s consisting of a hole of 50 ⁇ m in diameter, placed in the image focal plane of the objective O 2 .
• the F filter 5 makes it possible firstly to keep only one order diffraction and secondly to clean up the beam L My by spatial filtering.
• the diverging beam is collimated by a lens L 2 with a focal length of 100 mm.
• the telescope T 2 thus formed by the objective O 2 and the lens L 2 makes it possible to enlarge the diameter of the beam L Mes by a factor of 6.06.
• the diameter of the measurement beam L Mes is thus about 19 mm.
• This beam width makes it possible to cover the entrance pupil of the objective with a distribution of the luminous intensity optimized in the example presented for the operation of the microscope in a liquid medium, in order to observe in particular organic molecules in solution.
• a 45 nanometer gold metal layer M 5 is deposited on an outer face of the lamella G s used to allow the generation of a surface plasmon at the interface of this metal layer with a dielectric observation medium D .
• the beam L My penetrating into the objective O M is focused exactly at the interface between the metal layer Ms covering the glass plate Gs and the observation dielectric medium D, chosen in this case for to be a liquid.
• the light L Mes after passing through the coupling medium is reflected by the metal surface M 5 and back in the opposite direction by the objective OM-
• the position of the focusing point of the laser beam measurement L Mes relative to the layer of or M s being a fundamental parameter for the contrast of the images obtained by the microscope and that is why this position is controlled by means of a piezoelectric positioning device with a resolution of a few tens of nanometers over a range of 100 microns.
• the alignment of the axis of the lens O M with the normal of the surface of the strip Gs is for this reason carried out by a positioning system (not shown in FIG. 6) four manual axes on the lens O M and two manual axes controlling the support of the lamella G s .
• the objective as the plate are further carried on two platens PLi, PL 2 piezoelectric displacement for accuracy in moving in two directions X, Y orthogonal in the same plane and positioning greater than 10 nm.
• These plates PLi, PL 2 are advantageously controlled by EC electronic control means connected to a computer control and control COMP.
• the microscope of the invention also comprises piezoelectric means for moving the objective OM in translation in a direction Z normal to the plane of each of the surfaces of the glass plate G s and the metal layer M s covering one of the 'she
• the beam reflected by the metal layer M s then returns through the spatial filter F 5 , the objective O 2 and the acousto-optic AOM Mes , to be mixed with the reference beam L ref in the separator cube BS.
• This beam is shifted in frequency of 2x ⁇ Mes relative to the initial beam.
• a single light beam L v formed of the sum of the reference and measurement beams L R ⁇ f , L M ⁇ S and passes through a diaphragm D 2 is obtained to arrive at an optical detector such as, for example, a PD photodetector.
• the optical signal V resulting from the detection of the beam L v has a temporal modulation at the sum and at the difference of the optical frequencies of the two beams, ie 2 ⁇ mes + 2 ⁇ re f and 2 ⁇ mes -2 ⁇ r ef.
• the contrast of these images is based on the technique of defining profiles V (z), obtained by scanning in the direction Oz (normal to the lamella Gs) and whose variations are strongly correlated to the surface plasmon.
• the microscope 1 of the invention also has the advantage of great versatility of use and configuration.
• the microscope of the invention makes it possible to perform high-resolution plasmon microscopy imaging in differential mode.
• the polarization converter CP is used to linearly scan alternately and synchronously with the platens PLi, PL 2 by polarized beams in pure mode p (radial polarization) and in pure mode s (azimuthal polarization). sample that one wishes to observe. We thus gain in contrast images in the measure as well as in dynamics.
• optical signal obtained from the pure mode polarized beams can also be made for the purpose of servocontrolling the vertical position of the lens relative to the sample to be observed.
• the analysis of the electrical signals established from the reflected light beams polarized in mode s makes it possible to determine the absolute value of the position of the objective O M , and from this position, it is then possible to correct all mechanical and thermal drifts inherent in high resolution microscopy.
• Another advantage of the microscope of the present invention is to allow the construction of three-dimensional images of the measured function V (z).
• the construction of such three-dimensional "maps" of the function V (z) makes it possible to find the optical plane of section where the contrast of the image will be the best. To do this, we perform a post-processing of these 3D images and then by interpolation we determine the Z plane where the contrast is optimum.

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