EP0928433A1 - Mikroskop für die erzeugung einer dreidimensionalen darstellung von einem objekt - Google Patents

Mikroskop für die erzeugung einer dreidimensionalen darstellung von einem objekt

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Publication number
EP0928433A1
EP0928433A1 EP97943004A EP97943004A EP0928433A1 EP 0928433 A1 EP0928433 A1 EP 0928433A1 EP 97943004 A EP97943004 A EP 97943004A EP 97943004 A EP97943004 A EP 97943004A EP 0928433 A1 EP0928433 A1 EP 0928433A1
Authority
EP
European Patent Office
Prior art keywords
wave
image
microscope according
point
microscope
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97943004A
Other languages
English (en)
French (fr)
Inventor
Vincent Lauer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR9611773A external-priority patent/FR2754069A1/fr
Priority claimed from FR9615255A external-priority patent/FR2754070A1/fr
Priority claimed from FR9707469A external-priority patent/FR2757278A1/fr
Application filed by Individual filed Critical Individual
Publication of EP0928433A1 publication Critical patent/EP0928433A1/de
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/0005—Adaptation of holography to specific applications
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/04—Processes or apparatus for producing holograms
    • G03H1/08—Synthesising holograms, i.e. holograms synthesized from objects or objects from holograms
    • G03H1/0866—Digital holographic imaging, i.e. synthesizing holobjects from holograms
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/0005—Adaptation of holography to specific applications
    • G03H2001/005—Adaptation of holography to specific applications in microscopy, e.g. digital holographic microscope [DHM]
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
    • G03H1/04—Processes or apparatus for producing holograms
    • G03H1/0443—Digital holography, i.e. recording holograms with digital recording means
    • G03H2001/0454—Arrangement for recovering hologram complex amplitude
    • G03H2001/0458—Temporal or spatial phase shifting, e.g. parallel phase shifting method
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H2226/00—Electro-optic or electronic components relating to digital holography
    • G03H2226/11—Electro-optic recording means, e.g. CCD, pyroelectric sensors

Definitions

  • Microscope generating a three-dimensional representation of an object
  • the object of the invention is a device for obtaining a three-dimensional representation of the object and a complete representation of the diffracted wave by the latter in various lighting conditions, all in digital form then allowing any mode representation and any type of analysis
  • This device includes an optical part allowing the generation of interference figures between a reference light wave and a light wave diffracted by the object observed, sensors making it possible to digitize these interference figures, actuators allowing to act on the optical svstemc, and a computer receiving the digitized interference figures controlling the actuators.
  • a solution to reconstruct the light wave in the observed object is to make it interfere with a reference wave, the following principle of holography After registering l_ ⁇ interference pattern, the computer can perform a calculation equivalent rebuild operation to the optical operation used in holography But digital recording of a hologram is usually impossible because the variations in brightness are made over distances of 1 order of the wavelength, much less than the pitch of existing optoelectronic sensors.
  • the reference wave used is an approximately sphe ⁇ quc wave and centered, actually or virtually, in one point close to the observed part of the object This allows to obtain figures of interference in when the intensity variations are done over higher characteristic distances, allowing digital recording and reconstruction by calculation equivalent to optical reconstruction
  • the direct calculation of the light wave in the object according to a method simulating holographic reconstruction leads to defects which are the general defects of holography, namely the presence of a parasitic image symmetrical to the real image by relative to the point of origin of the reference wave and gaps of the second order with respect to the exact value of wave 1 in an advantageous version of the invention, the complex value (magnitude and phase) of the broadcast wave by the object and arriving on the receiving surface is calculated This then makes it possible to calculate the light wave in the object by direct application of the principle of reverse return of light, without the approximations linked to the holography. According to an advantageous version of the invention, this value is calculated from several interference figures differing from each other by the phase difference between the reference wave and the wave illuminating the object.
  • the light wave from the ob j ct alone is then obtained by a simple, linear formula, from the three intensity records
  • the interference pattern produced can be directly recorded by sensors located near the object, or it is possible to interpose optical elements deforming the wave between the object and the receiving surface.
  • An advantageous version of the invention is forming an enlarged intermediate image of the object using a microscope objective This method makes it possible to spatially filter the intermediate image by interposing a diaphragm whose opening determines the size of the observed part of the object By decreasing the image size observed, the pixel size of the sensors required is decreased.
  • a system of lenses located behind the diaphragm and suitably sized then allows the interference pattern to be formed.
  • the reference wave can be superimposed on the wave coming from the object using a semi-transparent mirror interposed on the path of the light coming from the object
  • the optical system is such that when a plane wave coming from the object, the image formed on the sensor in the reference wave is punctual abscence This allows to obtain directly a frequency representation of the wave coming from the object, each point of the sensor corresponding to a single frequency, that is to say a single wave vector, of the light wave coming from the object
  • the three-dimensional representation obtained is presented as a complex quantity depending on the three spatial coordinates, corresponding to the light vibration at any point of the ob j and The result strongly depends on the lighting mode It is possible to use a parallel beam , spatially coherent.
  • the representation obtained is then very precise in a plane orthogonal to the direction of the light beam but has defects in the direction parallel to this beam.
  • use is made of lighting or the spatial coherence has been broken, c that is to say where the object is illuminated at any point in all directions This lighting is produced using a condenser forming in the plane of the object the image of a diffusing element illuminates by the laser beam. But the representation obtained is then grainy due to the temporal coherence of the lighting wave.
  • This phenomenon called "Speckle” poses a problem already known in holographic microscopy.
  • improves the performance of the j ob and using the wave diffracted by the object under several different lighting In particular, one can use multiple representations obtained by slightly moving the diffusing element between each picture. We can then average over all the representations obtained the intensity of Tonde at each point. This provides a satisfactory spatial representation of the object
  • FIG. 1 shows the preferred embodiment of the invention.
  • FIG. 2 shows an example of sizing relating to this embodiment.
  • FIG. 3 indicates a particular configuration used during the adjustment procedure for this embodiment.
  • Figure 4 relates to a variant of this embodiment differing by the user sample illumination.
  • 5 shows a mechanical support for holding the whole device to the vertical.
  • a laser (100) emits a ray which passes through a filter (101) and is then separated into light beams Fe and of reference Fr by a semi-reflecting plate (102).
  • the light beam then passes through a lens (103) then is reflected by a mirror (104) and will strike a diffusing element (105) itself fixed on a motorized positioner with two axes (106) which can move it in an orthogonal plane to the optical axis of the objective (1 10).
  • This condenser is itself fixed to a manual positioner an axis (108) which can move it in the direction of its optical axis.
  • the focal distance of the lens (103) must be chosen so that the area of (105) illuminated by the beam is large enough so that its image by the condenser covers at least the entire area to be observed.
  • This objective is a plane objective (which gives a plane image of a plane), with large aperture (for example 1.25), with immersion , and giving an enlarged image of the object at a finite distance.
  • This microscope objective is attached to a focusing device (111).
  • a diaphragm (112) is interposed allowing spatial filtering of the image. Behind this plane, an achromat (113) is positioned, the object focal plane of which must be merged with the image focal plane of the objective (1 10) A second achromat (115), the image focal plane of which is in the plane of a CCD sensor (116) forms in the plane of this CCD the image of the image focal plane of the objective (1 10).
  • the CCD (116) is integrated into a camera (117) outputting an analog video signal.
  • the reference beam first passes through a filter (1 18) then is reflected by a mirror (1 19) mounted on the mobile end of a piezoelectric translator (120) It then passes through a lens (121) which focuses the beam at a point
  • the divergent beam from this point is partially reflected by the semi-reflecting mirror (114), which superimposes it on the beam from the object cl makes it possible to record their interference on the CCD (1 16)
  • the point focusing the beam coming from the lens (121) must have its virtual image after reflection on the semi-transparent mirror (1 14) to the center of the image of the diaphragm (112) by one achromat (113) the piezoelectric translator (J20 ) modulates the phase of the reference beam
  • the plane of the object must be horizontal so that the optical oil necessary to use the objective and the immersion condenser does not flow optics of 1 set is therefore vertical
  • the camera (117) is mounted on a three-axis positioner in translation
  • the laser (100) is mounted on an angular positioner allowing its direction to be adjusted
  • the piezoelectric translator (120) is mounted on a positioner making it possible to adjust the direction of a axis orthogonal to the mirror plane (119) and to move the assembly (119) (120) in translation along this axis
  • the mirror (104) is molded on a positioner making it possible to adjust the direction of an axis orthogonal to the plane of the mirror Le adjustment of these positioners is in principle carried out once and for all and in a mass production version of the system they can be eliminated in favor of more careful manufacturing. However, in this version intended for small series.
  • the set is fixed on a support plate (500). the side of the plate opposite to the point of view of Fig 5 This plate is fixed to two triangular plates (501) and (502) themselves fixed to a square base (503)
  • the plates (500) (501) (502 ) (503) are made of rigid aluminum alloy AU4G, for example 20 mm thick.
  • the fixing of the plates can be done by screws and tapped holes, and must be made at a sufficient number of points to ensure perfect rigidity of the assembly This allows the system to be kept vertical while ensuring sufficient rigidity
  • the assembly is placed on an anti-vibration support constitutes, for example, a granite plate placed on truck air chambers which absorb vibrations
  • the assembly various elements on the plate (500), and in particular mirrors and semi-transparent mirrors, must be carried out in such a way as to ensure maximum rigidity of the assembly All the usual precautions must be taken in order to limit vibrations
  • the condenser is a bright field condenser with immersion opening 1.2 whose opening diaphragm has in the open position a diameter of 28 mm
  • the area of the diffusing element which is illuminated by the laser beam has a diameter D6 of about 1 cm.
  • the distance D5 between this diffusing element and the condenser is 100 mm
  • the microscope objective is a plane objective lOO with aperture 1.25, at a finite distance, forming the image at 160 mm from the neck of the objective, focal length approx.
  • La distance D4 between the lens neck and the diaphragm is 160 mm
  • Distance D3 between the diaphragm (1 12) and the achromat (1 13) is 20 mm
  • the achromat (1 13) has a focal length of 200 m and a diameter of 30 mm and its most curved face is oriented towards the scmi -transparent mirror (114)
  • the achromat (115) has the same characteristics and its most curved face is also oriented towards the mirror (1 14)
  • the distance D2 between the two achromats is 85 mm, allowing the insertion of a semi-transparent mirror (1 14) of sufficient dimensions
  • the distance between the achromat (1 15) and the CCD (1 16) is 200 mm
  • the lens (121) has a diameter of 4mn ⁇ and a focal length of 6 mm the distance D7 between this lens and the optical axis is about 70 mm the distance D8 between the achromat (1 12) and the center of the mirror semi- transparent (1 14).
  • the laser is a hey u -Neon wave length 633 nui has random polarization power of about 0.5 mW, 0.47 mm diameter beam from the sensor CCD is a square pixel sensor. the pixel surface being approximately 8.5 x 8.5 micrometers
  • the camera outputs a CCIR video signal and a pixel clock
  • the piezoelectric positioner (122) is a piezoelectric battery in the shape of a cylinder whose body is fixed and l he end moves 15 micrometers for an applied voltage of 100 Volts
  • the calculation system is for example a PC type computer.
  • the video signal acquisition card operating in real time, samples the signal on 8 bits and acquires images of 512x512 pixels sampled according to the clock pixel, therefore corresponding exactly to the CCD pixels
  • the piezoelectric positioner is controlled directly by a digital / analog conversion card outputting a signal for example between zero and I ' m ⁇ , with for example
  • V m ⁇ X 10 volts
  • An RC filter with a time constant of approximately 0.1 ms is interposed between the output of the conversion card and the terminals of the piezoelectric actuator
  • the positioner (106) is powered by stepper motors also controlled from the computer
  • the computer will move it along the two axes in steps of 0.2 mm for example
  • supp ⁇ mant (103) (105) (106 ) (107) (108) by inserting a mirror (300) returning the beam towards the lens, and using as a filter (118) a completely opaque element
  • the miron (300) is mounted on an angular positioner which must be adjusted so as to target the input of the objective (1 10) and to maximize the intensity of the image received on (1 16)
  • the position of (116) is then set in the a direction of the optical axis so that the image received on the sensor (1 16) is a point,
  • the image received on the sensor (116) can be displayed in real time on the screen of the computer
  • the reference beam must then be restored and the beam from the object must be removed by placing a cover behind the lens (1 10)
  • the direction of the laser (100) must be adjusted so that viscr the center of the mirror (120)
  • the position of the assembly (1 19) (120) must then be adjusted so that the beam from (120) actually passes through the lens (121)
  • These settings can be controlled simply using a piece of light-diffusing paper
  • the position of the unit (1 1) (120) must be refined so as to obtain the most intense and homogeneous lighting possible on the sensor (116)
  • the reference beam must then be supp ⁇ me again and the beam from the object must be restored, the object used then being a simple transparent blade
  • the position of (104) must be ieglec so as to target the diffusing element ( 105), then refined so that the image received on the sensor (1 16) is correctly centered.
  • the positioner (108) must be governed so that the image received on the sensor, which represents the frequencies from the object or also a clear granular wide disc as possible During a change of observed object, it may be necessary to change the setting (108) but of (104) is normally fixed once and for all
  • the values of optical density of the filters (101) and (118) must be constantly modified to have the most easily observable beam when it is observed with the naked eye or to have an optimal illumination of (1 16) when 1 image received on (1 16) is observed on the screen
  • the automatic gain adjustment on the camera can advantageously be used
  • the optical density values of the filters (1 1) and (1 18) must be chosen so that the reference beam and the beam coming from the object have close intensities when arriving at the CCD sensor, and that the interference pattern produced is as intense as possible without, however, the maximum value of the pixel recommended by the camera and the video digitization card being exceeded
  • the lighting is changed by moving the diffusing element (105) using the positioner (106) At each position of the positioner, a different light is chosen
  • the computer calculates the value complex (that is to say the complex number representing the phase and the intensity) of the light wave at any point of the object
  • the final image of the object is obtained by averaging the intensities thus obtained for a number sufficient to separate illumination intensity of the wave at a point is the edge of the corresponding complex value Module to obtain the complex value of the light wave at any point on the objel
  • I computer first calculates the complex value of the light wave coming from the object at any point of the CCD sensor This value is obtained from the recording of three interference patterns received on the sensor, the phase of the reference wave being offset by 120 degrees between each of these figures
  • I ⁇ P, ⁇ the intensity at a point P of the sensor of the interference figure produced by the wave coming from the object and the reference wave for a phase shift ⁇ radians of the reference wave
  • I re f
  • Each pixel P of the sensor corresponds to a pure frequency of the wave in the object If we note (/,./) the coordinates (distance in pixels) of a point of the sensor relative to the central point of the area of interest, the pure frequency to which the point of coordinates (', /) corresponds, expressed in a coordinate system whose third axis is orthogonal to the plane of the sensor is then worth, has a constant factor near
  • the computer generates the table S [i j] of complex numbers, for i and J varying from 0 to 51 1 by assigning to the elements of the table the following values
  • the computer generates the table H of complex numbers representing the frequency of dimensions 512x512x512. in initializing to zero and then browsing the set of indices i and j varying from 0 51 1 by performing the operation
  • the virtual point of origin of the reference wave is in the center, i.e. at the point of coordinates (256,256,256)
  • Table F 1 constitutes the representation bidnnensionnelle of the wave in the plane of the object passing through the point of origin of the virtual orthogonal wave el reference to the optical axis (cutting) a section in a plane orthogonal to the above can be generated by calculating d 'first the table S2 [ij] of dimensions 512 ⁇ 512 in 1 adding to 0 then by browsing all the indices (i, j) by performing
  • S [i, j] has the complex value of S2 i, - K 2 - (i - 256) " - (j - 256) C initially present
  • the microscope is focused so as to have the sharpest possible picture bidnnensionnelle
  • the above cycle is the simplest, but it can be optimized to take account overlaps between sensor exposure time and image transfer time
  • the light wave calculation can be done on the whole object or on a part of the object, pa r example a cut, as a function of time and available calculation capacity In the simplest case.
  • the computer generates and displays a simple section of the object
  • the movement of the positioner (106) is done as indicated above, in steps of 0.2 mm in both directions
  • the order in which these movements are carried out is indifferent provided that the N mm lights used to calculate the three-dimensional representation of the object are obtained from all distinct positions of the actuator (106), so as not to have the same lighting several times
  • the passage at each cycle by the values 0 and V m ⁇ of the voltage limits the hysteresis effect of the piezoelectric actuator by always having the same cycle
  • the calculation methods indicated above are not limiting
  • the computer can generate three-dimensional representations showing portions of object more or lefs ranges (sections, projections, together sections to a limited thickness) It may filter these represenialioiis limiting 'grainy image at the cost of less definition treatment effeclue esl needed (speed of the image taken and definition calculation time RECL or different) and ii computing capacity of the computer
  • a variant of this embodiment is to use a beam of light unidirectional This eliminates the need to perform an intensity niovcnnagc on plusieuis images because each point of the object is then illuminates with the same intensity movennage
  • the intensity n elanl not necessary, the three-dimensional representation also preserves usable phase information.
  • the images obtained are precise in the three dimensions only for certain particular types of objects, for example sub-microscopic particles included in glass. very precise in the plane orthogonal to the direction of the beam and has defects in the direction of the beam This lighting mode little! however be used in cases where one is satisfied with a two-dimensional image.
  • This lighting can be made as shown in Figure 4 similar to Figure 3 to this close that the reference beam is no longer blocked and that an additional lens (400) must be inserted between the mirror (300) and the sample (109) This lens must be positioned so that its focal point coincides with the sample and its focal distance must be such that the diffraction spot formed on crossing the sample has approximately the diameter of the area observed.
  • Another variant of this embodiment comprises an 'use only one interference pattern to generate a three dimensional representation
  • This variant is the digital equivalent of the optical holographic reconstruction
  • the piezoelectric actuator (120) n' is not used
  • l (P) the intensity of the interference pattern produced at a point P of the sensor by the wave coming from the object and the reference wave
  • the focusing point of the beam from the lens (121) must have its virtual image after reflection on the semi-transparent mirror (1 14) on the side of the image of one aperture (1 12) by one achromat ( 1 1). and not cenlrc as before the operation cvclc the microscope is modified in the sense that one image per esl used instead of three cycle or so nx laughed more action on the phase control svstemc
  • the image is affected by second order approximations with respect to the ratio of the diffracted wave by the object to 1 reference wave
  • the filters (1 1) and (1 18) must therefore be chosen so that the reference wave has a substantially greater intensity at the wave coming from the object entering the CCD. and not near intensities as before This choice must be refined so as to have in the three-dimensional representation the best compromise between bnnt and second order distortions
  • the operation and the calculation mode are the same as before
  • the generated three-dimensional representation includes the observed part of the object bounded by the diaphragm, and symmetrically with respect to virtual point of focusing of the beam from the lens (121)
  • the aperture must be small enough to be visible on the representation obtained and its symmetric the area of the object that can be observed thus is smaller than in the preferred embodiment of this three-dimensional representation is also affected by second order approximations with respect to the ratio of the wave diffracted by the object at the reference wave, and the wave diffracted by the object must be much lower than the reference wave to obtain a convincing result II follows that the result obtained is much noisier than in the preferred embodiment
  • the system is less sensitive to vibrations and allows images to be obtained more quickly
  • This microscope can be used instead of conventional transmission microscopes in the field of biology or micromeasurement.
  • the recording of the three-dimensional image is faster than with other methods, which facilitates the observation of samples.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
EP97943004A 1996-09-27 1997-09-26 Mikroskop für die erzeugung einer dreidimensionalen darstellung von einem objekt Withdrawn EP0928433A1 (de)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
FR9611773A FR2754069A1 (fr) 1996-09-27 1996-09-27 Microscope enregistrant l'onde diffractee par l'objet observe et l'utilisant pour calculer une representation en trois dimensions de cet objet
FR9611773 1996-09-27
FR9615255 1996-12-12
FR9615255A FR2754070A1 (fr) 1996-09-27 1996-12-12 Microscope enregistrant l'onde diffractee par l'objet observe et l'utilisant pour carculer une representation en trois dimensions de cet objet
FR9707469 1997-06-17
FR9707469A FR2757278A1 (fr) 1996-12-12 1997-06-17 Microscope enregistrant l'onde diffractee par l'objet observe et l'utilisant pour calculer une representation en trois dimensions de cet objet
PCT/FR1997/001695 WO1998013715A1 (fr) 1996-09-27 1997-09-26 Microscope generant une representation tridimensionnelle d'un objet

Publications (1)

Publication Number Publication Date
EP0928433A1 true EP0928433A1 (de) 1999-07-14

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EP97943004A Withdrawn EP0928433A1 (de) 1996-09-27 1997-09-26 Mikroskop für die erzeugung einer dreidimensionalen darstellung von einem objekt

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US (1) US6249349B1 (de)
EP (1) EP0928433A1 (de)
WO (1) WO1998013715A1 (de)

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