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
  • G01B9/02—Interferometers
  • G01B9/02001—Interferometers characterised by controlling or generating intrinsic radiation properties
  • G01B9/02002—Interferometers characterised by controlling or generating intrinsic radiation properties using two or more frequencies
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
  • G01B2290/00—Aspects of interferometers not specifically covered by any group under G01B9/02
  • G01B2290/70—Using polarization in the interferometer
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/0092—Polarisation microscopes
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens

Definitions

• the present invention relates to a high-resolution surface plasmon microscope comprising a fiber heterodyne interferometer, that is to say substantially consisting of optical fibers.
• the technical field of the invention is that of the design of imaging systems and methods allowing the detection of small variations of refractive index in an observation medium and / or dielectric objects of the order of a few nanometers do not necessarily have remarkable optical properties (fluorescence, luminescence, localized plasmon resonance or Raman resonance) and located near a surface and immersed in any dielectric medium and in particular in air or in an aqueous medium.
• a surface plasmon is a surface electromagnetic wave that propagates at an interface between a metal and an observation dielectric medium.
• the surface plasmon excitation requires a polarized incident light and a coupling medium at the metal / dielectric medium interface at a particular angle which is generally referred to as plasmon resonance angle ⁇ p .
• 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 the production of images of objects of very small sizes located at the interface metal / dielectric medium, said objects modifying the optical properties of the surface plasmon at this interface, this 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.
• OCT Optical Coherence Tomography
• US 2004/100636 can not fill the gaps or be combined with current surface plasmon technologies insofar as the nature of the light beams observed is different, including the distribution in amplitude and in phase, not homogeneous in the case of surface plasmon, and which can not then be observed by OCT microscopy which focuses on the observation of light beams whose variations are uniform in amplitude and in phase .
• 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 that is compact and simple to use, and also suitable for a biological or medical laboratory environment.
• a high-resolution surface plasmon microscope comprising essentially: a) a coherent light source for emitting an excitation light beam, and b) a medium of optical coupling and confinement of a surface plasmon having a large numerical aperture lens, an immersion oil and a glass slide coated on one side which is not in contact with the immersion oil of a metal layer, and c) a heterodyne interferometer dividing the excitation light beam emitted by the light source into at least one reference beam and at least one measurement beam directed to the optical coupling medium to generate a surface plasmon, the interferometer being positioned between the light source and the objective of the optical coupling medium to form an interferometric beam between the reference beam and the measuring beam after reflection of each of them respectively by reflective element and by the metal layer, and d) scanning means of the metal layer with the aid of the measuring light beam, and e) means for detecting the interferometric beam coming from the interferometer, and f
• the heterodyne interferometer of the microscope consists essentially of at least four optical guide fibers, respectively, of the excitation beam, the measuring beam, the reference beam, and the interferometric beam and optically connected at a first of their ends to an optical coupler and each also connected optically at their second end respectively to the light source, to the optical coupling medium, to the reflecting element of the reference beam, and to the interferometric beam detection means.
• the microscope of the invention allows the detection of dielectric and metal objects with a diameter of less than 10 nm, without marking said objects. It has the advantage, compared to known surface plasmon microscopes, to provide a significant reduction in the size of the microscope and the optical settings thereof, since it allows the removal of any mechanical support of the optical elements of the microscope. 'interferometer.
• the microscope of the invention provides a significant and significant improvement in the stability of the interferometer and the quality of the optical beams involved, both the measurement and reference beams and the interferometric beam, which allows a much better quality and sensitivity of the images obtained.
• the optical fibers of the heterodyne interferometer may be monomode or multimode fibers.
• the choice of fibers is made according to the criteria of stability and sensitivity defined by the user.
• the optical fibers are polarization-maintaining fibers at the wavelength of the excitation light beam emitted by the source.
• the optical fiber connection coupler of the interferometer is adapted to the properties of the optical fibers used.
• the optical fibers whose second end is respectively connected to the reflecting element of the reference beam and to the coupling and confinement medium of the surface plasmon each cooperate with at least one acousto-optic modulator.
• the optical fiber for guiding the excitation light beam is connected in its second end to the light source through at least one collimating lens.
• the microscope of the invention also advantageously includes an optical isolator and a half-wave plate disposed between the light source and the collimating lens.
• the microscope of the invention also advantageously comprises a polarization converter positioned between the half wave plate and the collimating lens.
• This polarization converter makes it possible to vary at will, if necessary periodically, the polarization of the excitation light beam linearly, circular, radial, or azimuth for example.
• the polarization conversion provided by the polarization converter makes it particularly advantageous to perform a differential mode imaging, which makes it possible to further improve the contrast and the dynamics of the images obtained. It is indeed possible to polarize alternately with the aid of the polarization converter the excitation beam in pure mode p (radial polarization) and in pure mode s (azimuthal polarization) and to scan linearly alternatively and synchronously the alternating polarization of the excitation beam the metal layer by the measuring beam polarized alternately in pure mode p and in pure mode s.
• the guide fiber of the measuring beam is connected at its second end to the optical coupling medium by means of a collimating lens which collimates the measuring beam on the objective of the coupling medium.
• the reflecting element of the reference beam is a mirror.
• this mirror is advantageously constituted by a metal coating deposited on the end of the guide fiber of the reference beam.
• the reflecting element of the reference beam is constituted by the glass plate of an optical coupling medium identical to the coupling medium connected to the guide fiber of the measuring beam, said plate being coated. on one side not in contact with the oil immersing a metal layer of the same quality as that which covers the glass plate of the optical coupling medium connected to the guide fiber of the measuring beam.
• This particular embodiment of the microscope of the invention advantageously makes it possible to perform surface plasmon interference imaging between the reflected beam generated by the measurement beam and the reflected beam generated by the reference beam.
• the objective of the optical coupling medium to which the guide fiber of the measurement beam is connected is replaced by a solid immersion lens and the collimation lens of the measuring beam is integrated. on the second end of the guide fiber of the measuring beam, by assembly or in the form of a lenticular fiber.
• the collimating lens connecting the guide fiber of the measuring beam to the optical coupling medium and the objective of the optical coupling medium are both replaced by an axicon formed directly at the second end of said fiber. guidance of the reference beam.
• the microscope of the invention comprises a scanning system of the metal surface of the optical coupling medium using the measuring beam.
• the microscope of the invention may also, in one embodiment, include a polarizer between the interferometric beam detection means and the second end of the guide fiber of said interferometric beam, in particular to increase the contrast of the images in the interferometer beam. configuration in linear polarization.
• FIG. 1 represents a first preferred embodiment of a surface plasmon microscope according to the present invention
• FIG. 2 represents a response diagram V (z) of the microscope of the invention in the configuration of FIG. 1;
• FIG. 3 represents an alternative embodiment of the optical coupling medium of the microscope, comprising a doublet of lenses; fixed on an optical fiber,
• FIG. 4 is a schematic representation of a first embodiment of the microscope of the invention
• FIG. 5 represents a second variant of the microscope of the invention adapted to perform a plasmon interference microscopy.
• the present invention provides a novel high resolution surface plasmon microscope configuration for observing nanoparticles or molecules without fluorescent markers in air and in an aqueous medium.
• the microscope comprises a heterodyne interferometer composed essentially of optical fibers.
• the microscope of the present invention firstly comprises a coherent light source 1, chosen in the particular example presented as a single-mode, polarized and stabilized helium-neon laser source. amplitude. This light source is however not limiting and one can consider the use of other types of coherent light sources.
• the light source 1 emits a laser excitation laser of a surface plasmon which is injected and directed by a heterodyne interferometer 6 to an optical coupling medium 7 comprising, in the example of FIG. conventional for a surface plasmon microscope, a high numerical aperture objective 8, an immersion oil 9 and a glass plate 10 covered with a thin layer of metal 11, preferably a thin layer of gold, at which a surface plasmon is generated by the excitation laser beam.
• the heterodyne interferometer 6 of the microscope of the invention is essentially composed, as represented in FIG. 1, of at least four optical fibers 12, 13, 14, 15 optically connected to each other at a first of their ends 12a. , 13a, 14a, 15a by an optical coupler 16.
• These optical fibers 12, 13, 14, 15 are preferably monomode polarization-maintaining fibers.
• polarization-maintaining optical fiber means that the optical fibers 12, 13, 14, 15 of the interferometer are single-mode at the wavelength of the excitation laser beam of the source 1, that is, the propagation of light in these optical fibers is effected in a single guided mode.
• a first optical fiber 12 forms a guide fiber of the excitation beam.
• This beam is injected into the guide fiber 12 by the second end 12b of the fiber by means of a collimating lens 5.
• the excitation beam emitted by said source through an optical isolator 2, a half-wave plate 3 and a polarization converter 4.
• the optical isolator 2 has the function of eliminating the beam return associated with the interferometer 6 which behaves like a mirror and which destabilizes the laser.
• the half-wave plate 3 for its part makes it possible to control the orientation of the polarization of the excitation beam, the polarization converter 4 being intended for generating a selected polarization of the excitation beam before it enters the guide fiber. 12, this polarization being linear, circular, radial, or azimuth for example.
• the guide fiber 12 is welded at its first end 12a to the optical coupler 16.
• This coupler 16 is of the polarization maintaining type. It divides the excitation beam guided by the fiber 12 from the light source 1 into two identical beams of measurement and reference respectively, the measurement beam being transmitted and guided in a second optical guide fiber 13 to the middle of the optical coupling of the microscope, the beam of reference being transmitted and guided by a third optical fiber 14 to a reflecting element 17, this element being in this embodiment a mirror 18.
• the optical fibers 13, 14 respectively form the measurement arms 19 and 20 of the heterodyne interferometer 6. These two arms, and therefore the two fibers 13, 14 are each connected to an acousto-optical modulator
• the acousto-optical modulator 22 of the reference arm 20 is connected to a polarization-holding optical fiber 23 whose end 24 is covered with a metallic deposit acting as a mirror 18.
• the light transmitted in this arm 20, whose frequency has been shifted by ⁇ ref is reflected by this mirror 18, and passes through the acousto-optical modulator 22, the optical fiber 14, the coupler 16 and is then coupled in a fourth arm 25 of the interferometer 6 formed by a fourth fiber optical 15. It undergoes a shift in total frequency of 2 ⁇ ref.
• the optical fiber 13 is connected to an acousto-optical modulator 21 which shifts the optical frequency of the transmitted light with a frequency ⁇ test.
• the acousto-optical modulator 21 is connected to a polarization-maintaining optical fiber 26, at the output of which the light, which is in fact the excitation beam, is collimated by a collimating lens 28 on the high-aperture lens.
• digital microscope optical coupling medium to illuminate the glass slide on which a sample E is placed to be observed.
• the light of the excitation beam transmitted by the objective 8 is reflected by the optical system 10, 11, E, passes through the objective 8 and returns to the lens 27 which focuses the light at the end of the fiber optical 26 to allow its reinjection.
• the light is again shifted in frequency at the output of the acoustic modulator 21 of ⁇ test.
• the reflected light After passing through the optical fiber 13 and the coupler 16, the reflected light is transmitted in the fourth arm 25 of the heterodyne interferometer 6 with the reflected beam from the reference arm. It undergoes an overall frequency shift of 2 ⁇ test. So the A bright field propagating in the fourth arm of the interferometer is the superposition of the reflected beams from the reference arm and the measuring arm.
• a polarizer 29 may also be added in the case of a linear polarization of the starting excitation beam.
• the microscope of the invention as shown in Figure 1 and described above, provides a number of advantages over known surface plasmon microscopes.
• the microscope of the invention provides a miniaturization of the microscope (partial or total) related to reducing the size of the interferometer 6 enabled by the use of optical fibers.
• the integration of a fiber interferometer 6 greatly reduces the volume of the system since the replacement of the optical components by optical fibers eliminates the need to use mechanical supports and thus decreases the volume occupied by the arms of the interferometer.
• the use of optical fibers also allows as will be described later and is shown in FIGS. 3 and 4 to replace the optical stage allowing the magnification of the beam and the microscope objective by an all-fiber system or a system. fiber / lens that excites the surface plasmon and confine it. This part of the assembly is therefore less bulky and can be fixed at the end of the optical fiber. It is thus possible to produce a version of this totally fiberized microscope.
• optical fibers to make the interferometer 6 of the microscope also provides a drastic reduction in the number of optical adjustments, an improvement in the stability of the interferometer, and an improvement in the quality of the optical beams of excitation, measurement, reference and the interferometric beam.
• a free-field interferometer as used in the microscopes known in the state of the art, it is necessary to superimpose the beams from each arm of the interferometer.
• Ir e f and I t are t the luminous intensities coming from the reference and test arms respectively, ⁇ is the phase of the interference signal,
• M (t) is a factor varying between 0 and 1, reflecting the superposition of the two beams at the detector.
• M (t) depends in this case on the quality of the overlap of the two beams and is therefore a functional of time t.
• This retrofit can be achieved for example by a motorization of both the mirror of the reference arm and a reflection mirror upstream of the beam separation. The positions of these mirrors are readjusted by a retrofit loop positioning the beam based either on the beam position detection or on the control of the optical signal itself (optimization).
• This time M does not depend on time.
• the interference process is obtained directly by coupling within the optical fibers and therefore does not require any post-adjustment or catch-up of beam superimposition.
• V (z) M ⁇ /c / /, ft , cos ⁇ 2 r £, / - ->)
• optical signal S (x, y) of each pixel of an image produced by the surface plasmon microscope being directly proportional to the modulus of the third term of equation (2) above, ie:
• Figure 2 shows the relevance and performance of a heterodyne fiber 6 interferometer surface plasmon microscope as proposed by the invention.
• This Figure 2 shows the module of the optical response of a surface plasmon microscope as a function of the defocus z of the microscope objective with respect to the interface of the gold metal layer 11 covering the glass slide 10 the optical coupling medium 7 with the viewing medium (here air), the metal layer 11 having in this example a thickness of 45 nm.
• the propagation of the electromagnetic fields in the core of the optical fibers used ensures a total superposition of the fields, eliminating the fluctuations of the interferometric signal related to the fluctuations of the respective positions of the measurement and reference beams. as it can be with free-field microscopes.
• This stability greatly improves the signal-to-noise ratio of the phase signal ⁇ of the interferometer and makes easier fine measurements from the phase signal (by shortening the integration time of the measurement).
• free field surface plasmon microscopes it is therefore possible to use the microscope of the invention, which uses an optical fiber interferometer 6, the phase signal and to obtain images of the phase the response signal V (z) of the microscope.
• phase of the signal V (z) makes it particularly advantageous to distinguish the nature of a sample observed with the aid of the microscope and thus, for example, when an aqueous medium containing different particles or molecules is studied. to know which type of particle one observes at a moment t.
• a last singular advantage of the fiber interferometer microscope of the present invention is to allow an improvement in the quality of the optical beams involved, whether it be the excitation beam, the measurement and reference beams or the beam interferometer.
• the light field that propagates in the heart of the fiber possesses a profile based on the radial distance r to the center of the fiber which can be approximated by a Gaussian ⁇ r) TM ⁇ ffi ⁇ (r ⁇ i w J.
• the beam incident on the microscope objective has a Gaussian profile much more regular than that which can be obtained in free field.
• the light reflected by the sample E in the measuring arm 19 of the interferometer returns to the optical fiber 26 being spatially filtered during its reinjection into the fiber due to its passage through a heart of a few micrometers in diameter, thereby performing a cleaning of the beam.
• a first variant of the microscope of the invention is entirely fibered, from the light source to the optical coupling medium of the microscope.
• the collimation lens 27 and the high numerical aperture objective 8 of the microscope of FIG. 1 are replaced by a lens doublet (block B2 in FIG. 4).
• the first lens of this doublet not shown in FIG.
• the optical fiber 26 is integrated on the end of the optical fiber 26, either in the form of a lenticular fiber, that is to say that the end of the fiber 26 is itself machined and constitutes a diopter, either as shown in Figure 3 in the form of an optical block 30 fixed to the end of the fiber 26 and having a first lens 31 and a second lens 32 which is a lens SIL (for "solid immersion lens” in English), a flat face 33 acts as a glass slide and is covered with a thin layer of gold 11 which is in contact with the observation medium in which is located for example a sample E to observe.
• This second lens 32 makes it possible to focus the measuring beam coming from the first lens 31 at the level of the gold layer / observation medium interface in order to generate the surface plasmon.
• This arrangement has several advantages, in particular to be much more compact than a commercial immersion objective as used in the microscope of Figure 1.
• the use of a lens doublet allows a direct attachment on the end of the optical fiber 26 transmitting the measuring beam, while providing numerical openings greater than the commercial objectives and consequently to allow the observation of higher-index media (dense polymers, liquid crystals, non-aqueous solvents ...) -
• the numerical aperture is increased.
• An alternative embodiment not shown is also to replace the objective 8 of the microscope of Figure 1 by a fiber whose end has an axicon.
• the axicon is machined directly from the end of the optical fiber to give a conical shape to the heart of the optical fiber.
• Such an axicon makes it possible to generate a ring of convergent light whose high angle of incidence makes it possible to excite the surface plasmon.
• the portions of the microscope of the invention that may or may not be fibered are represented in the form of Bl, B2 blocks.
• a block B2 such as, for example, the block 30 of FIG. 3, to replace the light source and the conditioning of the excitation light beam.
• an all-fiber system B1 such as for example a polarized fiber laser.
• the microscope of the invention comprises not one but two optical coupling mediums 7, T 1 each placed at the end of the measuring arm 19 and the reference arm 20 of the microscope.
• the sensitivity of the microscope is considerably increased because it is possible to discriminate, in the interferometric signal and the response V (z) of the microscope, the noise related to the gold layer and to the surface plasmon of that bound to the microscope. the observed sample.
• V (z) the response of the microscope
• the polarization converter is advantageous to use to perform, using the measuring beam, sweeps of the gold layer of the optical coupling medium in alternating lines. polarized in pure mode p (radial polarization) and in pure mode s (azimuthal polarization).
• the polarization converter is electronically piloted so as to switch at a selected frequency from an azimuth polarization to a radial polarization of the excitation light beam emitted by the laser source 1 synchronously with mechanical components. displacement along the three axes Z of the lens and X, Y; optical coupling medium.
• the displacement relates to the end of the optical fiber 26 supporting the doublet.
• This mode of alternating illumination of the gold layer of the optical coupling medium advantageously makes it possible to perform a differential imaging of surface plasmon. This gives better contrasting images, a gain in dynamic images, and a catch-up of the decrease of the response V (z) of the microscope when the defocus z increases relative to the sample.
• optical signal obtained from the pure mode polarized beams can also be made for the purpose of servocontrolling the vertical position of the objective 8 at the end of the measuring arm with respect to the sample E to be observed.
• the analysis of the signals established from the mode-polarized measuring beam and reflected by the gold layer of the optical coupling medium makes it possible to determine the absolute value of the position of the objective 8, and from from this position, it is then possible to correct all the mechanical and thermal drifts inherent to a high resolution microscopy.
• Such a technique of correcting the position of the objective of the microscope is not in itself totally new in microscopy, however, with the microscope of the invention, the peculiarity lies in the fact that it is the imaging system itself that allows the correction and not a system reported in parallel to the imaging system.
• the microscope is neither complicated nor significantly increased its adjustment cost, all without disturbing the optical measurement of plasmon.
• this ability to control the position of the objective 8 with respect to the observed sample makes it possible to have a greater accuracy in the measurements of the function V (z), both in amplitude and in phase.
• 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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