FR2821935A1 - Microscope for diffracting objects, comprises light source, condenser, light beam not focussed on observed object, mirrors, microscope objective and attenuating and phase modifying filters - Google Patents

Abstract

The microscope sends light from a source (2000) to an object (2040) via galvanometric mirrors (2003,2007), fixed mirrors (2004,2009,2043,2010) and a condenser (2011). Light from the object travels through an objective (2012), lenses (2044,2045), fixed mirrors (2014,2015) and the galvanometric lenses to CCD sensors (2023,2028,2031) and synchronized cameras (2025,2030,2033) via a filter (2019,2047)

Description

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Domaine de l'invention :
L'invention concerne un microscope pour l'observation d'objets diffractants. Microscope for diffracting objects

Field of the invention
The invention relates to a microscope for the observation of diffracting objects.

Prior art.

 Images of high resolution, of very small or very deep depth of field, can be obtained by means of the microscope described in the following publication: [Lauer 1]: "Observation ofbiological objects using an optical diffraction tomographic microscope", by Vincent Lauer, proceedings of SPIE vol. 4164 p. 122-133.

 However, the apparatus described in this publication makes it difficult to perform real-time imaging.

 In a microscope operating in transmission and in which the illumination wave is not focused at a particular point of the sample, it is useful to be able to independently modify the diffracted portion of the illumination wave and the non-illuminated portion. diffracted from this wave. The phase contrast microscope, for example, is based on this principle. However, in a phase contrast microscope, the phase shift applied by means of a phase ring affects the non-diffracted part of the wave, but also a non-negligible part of the diffracted wave. This results in significant disturbances of the image, for example the phenomenon of halo or the fact that the depth of field is poorly defined and higher than it is in brightfield, the image quality in general being very less than that obtained by means of the microscope described in [Lauer 1].

 In brightfield, the image formed by a conventional microscope also has an effective resolution which is less than half the theoretical maximum reached for example in [Lauer 1].

Description of the invention
The subject of the invention is a microscope whose performances in terms of resolution and depth of field are similar to those described in [Lauer l], but which makes it possible to obtain images in real time and in color, for an acceptable cost.

 The invention consists of a microscope operating in transmission, in which the illumination beam is not focused on an observed point of the object, characterized in that it comprises: a beam deflector A placed between the lighting source and the condenser, and making it possible to modify the direction of the illumination wave in the sample, - a beam deflector B placed between the objective and the detector, and making it possible to modify the direction of the wave coming from the sample and having crossed the objective.

 The detector can typically be a camera or the retina of the eye. The deflector A has the purpose of varying the lighting conditions. For example, the light source may be a laser beam. By illuminating the entire sample and varying the lighting conditions over time, we obtain on average time lighting roughly equivalent to incoherent lighting. The deflector B serves to deflect the beam collected by the lens, so that the part of this beam that

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 was not diffracted by the observed object finds characteristics approximately constant after passing the deflector B. This non-diffracted part of the beam, after passing the deflector B, can be focused in an intermediate plane or it will therefore, by means of appropriate use of the deflector B, at a point almost fixed, or mobile in a reduced area. In this intermediate plane it is possible to place different devices for example to increase the resolution or to change the mode of observation.

 For example, it is possible to place in this intermediate plane a differentiation device for applying an average modification of phase and / or of different attenuation and / or polarization to the non-diffracted portion of the illumination wave and to the wave diffracted by the observed object. This device therefore acts differently on the non-diffracted portion of the illumination wave and on the diffracted portion of the wave. This is made possible by the fact that the non-diffracted part of the beam reaches a point approximately fixed in the intermediate plane. If the differentiation device is, for example, a window carrying a thickness over a reduced area comprising all the points reached by the non-diffracted portion of the wave, then the phase shift produced by this extra thickness affects essentially the non-diffracted part. diffracted from the wave.

An improved phase contrast is thus achieved in this manner because the phase shift only affects the non-diffracted part of the wave. The image quality is also improved because the illumination wave can scan over time the entire focal plane of the condenser, whereas in a phase contrast microscope the illumination wave is zero in this plane outside a transparent ring.

If the differentiation device changes the amplitude and not the phase, we obtain a bright field image but whose contrast can be enhanced. If the differentiation device totally absorbs the non-diffracted part of the wave, a black background image is obtained. If the differentiation device modifies the polarization direction, this polarization modification can subsequently be transformed, by means of a polarizer or a wave plate for example, into phase modification or attenuation. Such a device then allows a continuous transition from light background to black background and phase contrast.

 In the intermediate plane placed after the deflector B, it is also possible to place a filter blade whose transmissivity depends on the distance to the optical axis. Preferably the transmissivity increases with the distance to the optical axis. This makes it possible to increase the resolution of the microscope.

 Laser lighting can be used, but it is also possible to use white light. For example, it is possible to use a limited incoherent wave, in the object focal plane of the condenser, with a small disk-shaped zone, which moves in the focal plane because of the deflector A. This wave can be reduced by means of the set B, to a roughly fixed zone of the intermediate plane, in which it can be applied for example a phase shift different from that applied on average to the diffracted wave. It is also possible to use a coherent or incoherent wave limited in a pupillary plane by the crossing of several holes, all of these holes being movable in a pupillary plane. By returning, by means of the set B, the wave coming directly from each of these holes towards as many fixed points, and by using an attenuation or delay device occupying a reduced zone around each fixed point, we obtain a similar effect, with more disturbances but also with more brightness.

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 The invention does not relate to scanning microscopes in which the illumination wave is focused at an observed point of the object. Indeed, in the case where the light wave is focused at an observed point of the object, this point affects the whole of the wave and it is therefore difficult to differentiate (at least at this point) a wave diffracted from an undiffracted wave. On the other hand, the illumination wave may not be flat. As soon as a significant part of the plane on which the objective is focused is illuminated, it is possible to differentiate later the light wave of the diffracted wave.

 However, according to a preferred version of the invention, the illumination wave is a plane wave (case of the coherent plane wave) or a sum of uncorrelated plane waves (case of the incoherent wave constituting a sum of planar waves inconsistent with each other). In this case, the intermediate plane may advantageously be in a plane conjugated to the image focal plane of the microscope objective. If for example the light wave is a plane wave, it is focused at a specific point of the intermediate plane, which makes it easier to differentiate it from the diffracted wave. If it is a sum of plane waves, it can be easily limited to a reduced portion of the object focal plane of the condenser and therefore to a reduced portion of the intermediate plane.

 For maximum system efficiency, it is preferable that said beam deflectors A and B be controlled or arranged so that the direction of the illumination wave in the sample is variable and so that that the non-diffracted portion of the illumination wave arrives at a fixed point of said intermediate plane. Indeed, if the non-diffracted part of the illumination wave arrives at a fixed point, the differentiation device placed at this point can differentiate a very small area around this point, which avoids that part of the diffracted wave is not differentiated from the undiffracted wave.

 Various types of deflectors can be used. For example it is possible to incorporate accousto-optical deflectors. However, the deflectors that create the least parasitic disturbances are movable mirrors, for example galvanic mirrors.

 Obtaining a fixed position from the point at which the non-diffracted part of the wave reaches the surface requires, if the deflectors A and B are independent, a very precise control of each of these deflectors. This problem can be solved if each moving mirror of said deflector B coincides with a corresponding mobile mirror of said deflector A, or is integral with a corresponding mobile mirror of said deflector A. In this case, the deflection by B is proportional to the deflection by A, and by means of an appropriate optical system it can compensate exactly the deflection by A. This makes it possible to avoid any complex control of the galvanometric mirrors, the compensation being done automatically and purely optically.

 The part not diffracted by the observed sample of the wave, having been deflected successively by the sets A and B, has a constant diection. This property is useful in that it allows the application of phase shift and / or attenuation to that part of the wave only. On the other hand, especially if the light wave is from an intense laser beam, direct observation of this wave is dangerous.

Indeed, this wave is likely to focus in a fixed point of the retina and cause lesions.

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 Moreover, if the mirrors of the set B are not placed exactly in image planes, the deflection on these mirrors is accompanied by a translation of the image which depends on the orientation of the mirrors and which therefore disturbs the final image obtained from a series of different orientations of the mirrors.

To avoid this disturbance, the mirrors of the set B can be placed in image planes.

However, this solution is not robust, an accidental translation of a mirror of the set B may result in a disturbing disturbance of the image.

 According to one version of the invention, these two problems are solved by means of a deflector C making it possible to modify the direction of the wave coming from the sample after it has been deflected by said deflector B. In fact, the deflector C makes it possible to vary over time the direction of the non-diffracted portion of the wave after deflection on sets A, B and C. This allows direct observation in better safety conditions, the laser beam can not be focused on a fixed point. Moreover, an appropriate optical configuration makes it possible to compensate exactly, by means of the deflector C, the translations of the image which accompany the deflection on the set B.

 If the set C is independent of the set B, the control of this deflector must be perfectly synchronized with the control of the deflector B. This difficulty is solved if: said deflector C is a moving mirror or a pair of moving mirrors, each movable mirror of said deflector C coincides with a corresponding mobile mirror of said deflector B, or is integral with a corresponding mobile mirror of said deflector B.

 Indeed, in this case, the deflection by the deflector C is proportional to the deflection by the deflector B, and by means of an appropriate configuration of the optical system it can compensate for exactly the deflection by the deflector B.

 Preferably, each movable mirror of said deflector C is formed on the opposite face of a corresponding movable mirror of said deflector B. This configuration is the most robust. Indeed, it avoids superimposing on the same optical path two beams of opposite direction, which would be likely to create disturbances due to parasitic reflections.

 The system can be modified to generate deep images of extended field. The "extended depth of field" mode corresponds to the generation of images whose depth of field is increased at constant resolution. Extended depth of field operation can be achieved by means of the present invention by using a mask placed in the image focal plane of the objective, or in an image plane of this focal plane which is placed on the beam path. before the deflection by said deflector B or after the deflection by said deflector C, which is absorbing everywhere except on an elliptical shaped strip. The deflector A must then be controlled to obtain a wave passing through this focal plane at a point of the elliptical band that moves over the entire band. By appropriately choosing the dimensions of this elliptical strip, an extended depth of field image is obtained. For example, if this band is a ring centered on the optical axis, we obtain a projection of the sample along the optical axis. If this band is a slightly off-center ring, the projection direction is slightly inclined.

If this band is strongly off-center, its ellipticity should be calculated appropriately

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 which results in a projection of the sample in a steeply inclined direction. The thinner this band is, the greater the depth of field.

 However, on such a projection, the diffracted wave tends to be strongly attenuated because the greater part of this wave is stopped by said mask, while the non-diffracted portion of the wave is not stopped by said mask . It is therefore necessary to attenuate the undiffracted part of the wave.

This can be done by means of an attenuator placed at the fixed point reached by the non-diffracted part of the lighting wave after redirection by the deflector B.

 It is possible to perform the acquisition, with a single camera, of a brightfield type image or phase contrast. However, it is also possible to use at least three cameras, each recording an image corresponding to a different phase shift and / or attenuation ratio between the diffracted portion and the non-diffracted portion of the illumination wave. Indeed, under these conditions, it is possible to recalculate from these images a complex image characterizing both the variations of index and absorptivity of the object, and linearly dependent on the diffracted wave without disturbances of the second. order. This image is more precise and more directly interpretable than the image obtained with a single camera. The simplest solution is to use three cameras, the phase shift between the diffracted wave and the non-diffracted portion of the illumination wave being respectively close to 0.120 and -120 degrees for the image formed on a snap camera.

 In addition, a three-dimensional image of the observed object can then be obtained without moving the object observed. For this, we perform the acquisition of a series of images each corresponding to a given direction of the light wave. From this series of images, and with appropriate calculations, we can reconstruct a three-dimensional representation of the object.

 The phase offsets and / or attenuations of the undiffracted portion of the wave can be obtained in various ways, however a particularly effective method is to use as a differentiating device a blade rotating 90 degrees the polarization of the incoming beam, pierced a hole coinciding with the fixed point of the intermediate plane or is focused the non-diffracted portion of the illumination wave, this hole being filled with an isotropic material index close to that of the blade. This birefringent element makes it possible to polarize differently the diffracted wave and the non-diffracted portion of the illumination wave. These two waves can then be shifted in phase relative to one another by means of a delay plate, and their relative intensity can be modified by means of a rotating polarizer. The delay blade may optionally be replaced by a variable phase shift assembly. Preferably, the hole drilled in the birefringent element must be filled with a material of optical index close to the average index of the birefringent element. In fact, in the opposite case, the system would have significant dysfunctions in polychromatic light, due to the fact that the part of the wave passing through the hole would be shifted in phase of several wavelengths with respect to the part of the wave. not crossing the hole.

 Quick description of the figures.

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 Fig. 1 is an overall diagram of a first embodiment. Fig. 2 is a diagram of a half-wave breakthrough plate used in this embodiment. Fig. 3 represents the trajectory of the point of impact of the illumination wave in a pupillary plane, during the integration time of the sensors, to obtain a section of the object using this microscope. Fig. 4 shows in section the two-dimensional frequency representation obtained by Fourier transform of the image obtained for a given plane lighting wave, as well as the part of the three-dimensional frequency representation of the object of which it is the projection. Fig. 5 represents a lighting system that can replace the laser. Fig. 6 shows a camera detection system which can replace the three-camera detection system described in FIG. 1. FIG. 7 represents an elliptical mask used to obtain a projection of the observed object. Fig. 8 represents a detection system without wave plates that can replace the detection system described in FIG. 1. FIG. 9 shows a detection system with a camera placed in a pupillary plane. Fig. 10 serves as a support for calculating the characteristics of elliptical strips used on the mask of FIG. 7. FIG. 11 represents the mask of FIG. 7 to scale and for a particular case of realization. Fig. 12 shows a filter blade for attenuating the non-diffracted portion of the wave in the case where the mask of FIG. 7 is used. Fig. 13 shows a blade generating a phase shift of the non-diffracted portion of the illumination wave. Fig. 14 shows a camera detection system in which the acquired image can be directly displayed on a screen. Fig. 15 shows a device for direct observation using an eyepiece.

 Fig. 16 shows a side view of a set of three mirrors also shown in Figure 17. Figure 17 shows an improved embodiment, particularly suitable for direct observation by eyepieces. Figure 18 shows the electrodes of a polarization rotator used for stereoscopic observation. Figure 19 shows the frequency response of a brightfield microscope.

First embodiment.

 In the text that follows the term "lentil" can mean either a simple or compound lens, generally studied to minimize aberrations.

 Optical systems can be realized in various ways. To facilitate the design of the system and the understanding of the diagrams, use alternating space planes, designated by the letter (E) on the diagrams, and frequency planes, designated by the letter (F) on the diagrams. A space plane will be defined as an image plane such that a wave centered on a point of a pupil plane is plane in the space plane. A frequency plane will be defined as a pupil plane such that a wave centered on a point of an image plane is plane in the frequency plane. The term pupillary plane means a plane such that a plane wave in the observed sample is centered on a point of the pupillary plane. An image plane is a plane in which a point of the observed sample, on which the objective and the condenser are focused, has a point image.

 The alternations of space (E) and frequency (F) planes used in the description do not constitute a limitation of the invention and a functional system can be realized which does not include

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 no such plans. The alternation of space and frequency planes is only a particularly simple embodiment of the invention.

 Fig. 1 is an overall diagram of the first embodiment. In Figure 1 the space and frequency planes are designated respectively by the letters (E) and (F). The path of the pupil beam is in solid lines. The path of a beam from a point in the sample is shown in dashed lines on some parts of the figure.

 A beam from the laser 2000 polarized orthogonal to the plane of the figure is widened by a beam expander consisting of the lenses 2001 and 2002. It crosses the field diaphragm 2043. It then reaches the galvanometric mirror 2003 which reflects it to the fixed mirror 2004 The galvanometric mirror 2003 is rotatable about an axis passing through its center and oriented orthogonally to the plane of the figure. After reflection on 2004, the beam passes through the 2005 lens whose object focal point is in the center of the 2003 galvanometric mirror. It crosses the 2006 lens whose object focal plane coincides with the image focal plane of 2005. It is reflected by the galvanometric mirror 2007 whose center is at the focal point image of the 2006 lens. The galvanometric mirror 2007 is rotatable about an axis passing through its center and located in the plane of the figure. The beam passes through the lens 2048 whose object focal point is in the center of the galvanometric mirror 2007. It then passes through the lens 2049 whose object focal plane coincides with the image focal plane of the lens 2048. It passes through the lens 2008 whose plane focal object is confused with the focal plane image of the lens 2049. It is reflected by the mirror 2043 then by the mirror 2009 and by the partially transparent mirror 2010. It then passes through the condenser 2011. The focal plane image of the lens 2008 is in the focal plane object of the condenser 2011 so that at the outlet of the condenser the beam is parallel. The beam then passes through the observed sample 2040 which diffracts it. The whole of the wave, comprising the diffracted portion and the non-diffracted portion of the beam, then passes through the objective 2012. The wave then passes through the tube lens 2044 whose object focal plane coincides with the image focal plane of the objective. It then passes through a lens 2045 whose object focal plane coincides with the image focal plane of the lens 2044. It then passes through an optional mask 2046 placed in the image focal plane of the lens 2045. It is reflected by the mirror 2014 and passes through the 2013 lens whose object focal plane is on the optional mask 2046. It is reflected by the 2007 galvanometric mirror whose center coincides with the object focal point of the lens 2013. It is then reflected by the mirror 2015 and then passes through the 2016 lens whose object focal point is in the center of the 2007 galvanometric mirror. It crosses the 2017 lens whose object focal plane coincides with the image focal plane of the 2016 lens. It is reflected by the 2003 galvanometric mirror whose center is confused with the focal point object of the lens 2017. It crosses the lens 2018 whose object focal point is in the center of the galvanometric mirror 2003. It tra The half-wave wave plate 2019 located in the object focal plane of lens 2018 is poured. The neutral axes of this half-wave plate are oriented at 45 degrees from the plane of the figure so that the part of the wave that has passed through the half-wave plate is polarized in the plane of the figure, the portion of the wave which has passed through the hole made in this plate being polarized in the direction orthogonal to the plane of the figure. The wave then passes through an optional filter blade 2047. The wave passes through the lens 2020 whose object focal plane is on the 2019 half-wave breakthrough plate.

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 to the beam splitter 2021 which reflects one third of the light power. It then reaches the beam splitter 2026 which reflects half of the light power.

 The part of the wave which has been reflected by the beam splitter 2021 then passes through the third wave plate 2022 and the polarizer 2023 and then reaches the CCD sensor 2024 linked to the camera 2025 and located in a focal plane image of the lens 2020. A neutral axis of the third wave plate 2022 is in the plane of the figure, so that this plate induces a phase shift of 120 degrees between the part of the wave that has passed through the 2019 half-wave plate. and the part of the wave that went through the hole in that blade. The polarizer is typically 45 degrees from the plane of the figure, however different angles can be used.

 The set 2027,2028, 2029,2030 is equivalent to the set 2022,2023, 2024,2025 but the third wavelength blade is rotated 90 degrees so as to generate a phase shift of-120 degrees.

 The assembly 2031, 2032, 2033 is equivalent to the assembly 2022,2023, 2024,2025 but the third wave plate is removed.

 The portion of the illumination wave that passes through the partially transparent mirror 2010 reaches the CCD sensor 2041 mounted on the camera 2042 and placed in a pupil plane, on which it has a point image.

respectée :

FF FF F2008 200S-204 F2013 F2044 FcondF2049 F2045 Fobj

La lentille 2016 et la lentille 2006 sont identiques entre elles, et les lentilles 2005 et 2017 sont également

F F F F identiques entre elles. Le grossissement de l'ensemble vaut -= 20 2o] 7 20) 3 2044 F2018 2016 2045 obey

Par exemple, on peut utiliser : - un objectif planachromatique Nikon CFI60 d'ouverture numérique 1, 25 formant l'image à l'infini et corrigé indépendamment de la lentille de tube, de distance focale 2 mm. The condenser and the lens are both achromatic / aplanatic. We note Fob) the focal length of the lens and we note Fcond the focal length of the condenser. We denote by F x the focal length of the number X lens. In order that the deviations of the illumination beam and the beam passing through the sample, through the galvanometric mirrors, compensate for each other exactly, the following equality must be

respected:

FF FF F2008 200S-204 F2013 F2044 FcondF2049 F2045 Fobj

The 2016 lens and 2006 lens are identical to each other, and the 2005 and 2017 lenses are also

FFFF identical to each other. The magnification of the set is - = 20 2o] 7 20) 3 2044 F2018 2016 2045 obey

For example, it is possible to use: a Nikon CFI60 planachromatic lens with numerical aperture 1, forming the image at infinity and corrected independently of the tube lens, with a focal length of 2 mm.

 - a planachromatic Nikon condenser, with a focal length of 8 mm - a 2008 lens consisting of an optimized achromatic doublet Melles Griot, with a focal length of 800 mm - lenses 2044,2045, 2013,2016, 2017,2006, 2005, 2018, 2020 , 2048, 2049,2008 all identical to the tube lens used on Nikon microscopes, of focal length 200 mm - a red HeNe laser 2000 at 633 nm wavelength.

lenses 2001 and 2002 optimized to constitute a beam expander, dimensioned to obtain a beam of about 10 mm in diameter.

 a diaphragm 2043 approximately 8 mm in diameter.

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- CCD cameras having 512x512 usable square pixels with a pitch of 12 microns.

galvanometric mirrors having a diameter of about 10 mm.

The half-wave breakthrough plate 2019 is shown in more detail in FIG. 2. It consists of a half wave plate pierced at its center with a hole 2101 which may have been made using a power laser or by mechanical means. Hole 2101 must be on the optical axis. Its diameter is greater than the diameter of the diffraction spot formed by the beam on the blade 2019, while being sufficiently small. For example, in the particular example of dimensioning given above, its diameter may be about 50 microns. The hole 2101 may be empty, however it is preferable that it be filled with a material of index close to that of the blade. For example, the blade may be pre-pruned and pre-drilled, the hole may be filled with glass of suitable index, and the assembly may be co-polished. It is also possible to use an optical cement instead of glass. The filling of the hole with a material of index close to that of the blade makes it possible to avoid a significant phase shift of the non-diffracted portion of the wave. This is particularly useful if a laser emitting several wavelengths simultaneously is used: in the presence of a significant phase shift the images obtained for each wavelength would not be superimposed constructively.

 The system is designed in such a way that the observed sample is illuminated by a plane wave whose direction can be modified using the 2003 and 2007 galvanometric mirrors. Moreover, the system is also designed so that in the absence sample, the wave arriving at the half wave plate pierced 2019 passes through a fixed point of this blade, located on the optical axis, and coinciding with the hole 2101 practiced in this blade. So that in the absence of object the wave goes through a fixed point of the blade 2019 an adjustment of the set must be made. It is essentially to adjust the focal length of a lens, for example the 2013 lens, so that the position of the point of impact of the wave on 2019 is independent of the position of the galvanometric mirrors (in the measure where the wave actually crosses the condenser and the objective). For this purpose it is possible for example to use as lens 2013 a doublet of adjacent achromatic lenses, the distance between these lenses being adjustable. The focal length of the doublet is then modified by adjusting the distance between its two simple lenses. For the adjustment 2019 can be replaced by a CCD sensor so that the displacement of the point of impact on this sensor can be measured when the orientation of the galvanometric mirrors varies. For an appropriate adjustment of the 2013 focal length, this point of impact is fixed.

 This adjustment having been made, the pierced blade 2019 is placed in such a way that the hole 2101 coincides with the point of impact of the beam in the absence of a sample. To adjust the position of the pierced blade, it is possible, for example, to temporarily place behind this blade a mirror and a lens forming the image of the blade on an auxiliary CCD, and a polarizer set in rotation so as to strongly attenuate the part of the wave which is polarized in the plane of FIG. 1. When the hole coincides with the point of impact of the beam, the intensity reaching the auxiliary CCD is maximum. To enable this adjustment, the 2019 blade must be mounted on a 3-axis positioner.

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Since this setting has been made, it is necessary to adjust the position of the CCDs. To do this, it is possible to place in the space plane situated between the lenses 2044 and 2045 an absorbent mask having a few holes. On a computer screen, we then superimpose the images of this mask acquired using the three CCDs and we adjust the position of the CCDs to bring these images into coincidence and so that these images are clear. To enable this setting, CCD sensors must be mounted on 3-axis positioners.

The three cameras must be synchronized with each other and with the galvanometric mirrors so that their integration times coincide and also correspond to the time during which the illumination wave sweeps the object focal plane of the condenser.

1 ] =. (22032 [i!. 7]-2024 [i,7]-2029 [i, j) ±- (I2024 [ i]-2029 [ i]) 6 2-v 3

ou IX [il représente l'intensité détectée au point de coordonnées i, j du CCD numéro X. A complex elemental image is generated from the actual images detected on the three CCD cameras by performing the following calculation:

1] =. (22032 [i] 7] -2024 [i, 7] -2029 [i, j) ± - (I2024 [i] -2029 [i]) 6 2-v 3

or IX [it represents the intensity detected at the point of coordinates i, j of the CCD number X.

7T par exemple égal à-. Cette lame peut typiquement être une lame de phase du type utilisé en contraste de 16

phase, mais générant un décalage de phase plus faible. L'image de cette lame se forme sur les capteurs et

v r''i l'image complexe correspondante T, [/, ] peut être obtenue. On calcule le rapport M =' 'ou "re/'OoJ

(, V]) sont les coordonnées en pixels de l'image d'un point portant une surépaisseur, et (io, Jo) sont les coordonnées de l'image d'un point ne portant pas de surépaisseur. Ce rapport permet de normaliser l'image.

ïf 1 On utilisera ensuite l'image élémentaire normalisée Cli, jl =m ou 7 désigne la racine complexe de jm

l'unité. L'image élémentaire normalisée d'un point faiblement diffractant est réelle si ce point est uniquement absorbant et complexe si ce point est non absorbant et a un indice différent de celui du milieu dans lequel il se trouve. A reference image can be obtained by inserting, in the plane of space situated between the lenses 2044 and 2045, a blade bearing on a reduced area a slight excess thickness, causing a phase shift.

For example 7T. This blade can typically be a phase plate of the type used in contrast of 16

phase, but generating a lower phase shift. The image of this blade is formed on the sensors and

the corresponding complex image T, [/,] can be obtained. We calculate the ratio M = '' or 're /' OoJ

(, V]) are the pixel coordinates of the image of a point bearing an excess thickness, and (io, Jo) are the coordinates of the image of a point not bearing excess thickness. This report is used to standardize the image.

We will then use the normalized elementary image Cli, jl = m or 7 denotes the complex root of jm.

unit. The normalized elementary image of a weakly diffracting point is real if this point is only absorbing and complex if this point is non-absorbing and has an index different from that of the medium in which it is located.

These formulas are similar to those used in WO99 / 53355. The non-diffracted portion of the wave, which passes through the hole 2101, is used as the reference wave and phase offsets are applied to the diffracted part of the wave by means of the third wave plates. The polarizers can be oriented at 45 degrees from the plane of FIG. 1, but it is possible by modifying their orientation to modify the relative intensity of the reference wave and of the diffracted wave. The three polarizers must however be oriented in the same way, so that the reference wave has the same amplitude on the three corresponding sensors.

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This microscope has several modes of use: Mode 1) Generation of cuts of the observed object. In this imaging mode the mask 2046 and the filter blade 2047 are not used. To generate sections of the observed object, the galvanometric mirrors are controlled so that the point of impact of the illumination wave in the object focal plane of the condenser moves in such a way that the light power received in a point of the focal plane during the integration time of the camera is independent of the position of this point within the limits defined by the aperture diaphragm. For example, the point of impact of the illumination wave in the object focal plane of the condenser can traverse a trajectory of the type shown in FIG. 3, the speed of displacement of the point being approximately constant in the rectilinear parts of this trajectory, and the entire trajectory being traversed during the integration time of the camera. In FIG. 3 shows the diaphragm opening 2111 of the condenser and the path 2112 of the point of impact of the illumination wave.

l'échantillon, avec )/, J= CJ/, y] ou C [/, j estl'image élémentaire normalisée obtenue pour la position caractérisée par l'indice k. Ce tableau peut être amélioré par une déconvolution permettant de compenser la point spread function ou réponse impulsionnelle du système. Le filtre de déconvolution peut être obtenu par des considérations théoriques ou par mesure à l'aide d'un échantillon ponctuel, par exemple une bille du type utilisé pour calibrer les microscopes confocaux. La trajectoire du point d'impact de l'onde dans le plan focal du condenseur peut être contrôlée à l'aide du capteur CCD 2041. La partie réelle de l'image obtenue correspond, pour des échantillons faiblement diffractants, à l'absorptivité. La partie imaginaire correspond, pour des échantillons faiblement diffractants, à l'indice de réfraction. Le filtre de déconvolution est le même que celui utilisé par exemple dans l'article Reconstructing 3D lightmicroscopic images by digital image processing , par A. Erhardt & al. applied optics vol. 24 no 2,1985. Such a trajectory can typically be obtained by using a resonant galvanometric mirror and a slower second galvanometric mirror, according to a method commonly used in confocal microscopy. The complex image C [i, it obtained from the real images detected on the three sensors is a section of the observed object. However this cut is imperfect and can be improved by taking several successive cuts, the position of the sample along the optical axis being incremented by a constant value between each cut. These sections are indexed by an index k. We thus obtain a complex three-dimensional array H [i, j, k] in which each element of the array corresponds to a point of

the sample, with) /, J = CJ /, y] or C [/, j is the normalized elementary image obtained for the position characterized by the index k. This table can be improved by a deconvolution to compensate the point spread function or impulse response of the system. The deconvolution filter can be obtained by theoretical considerations or by measurement with the aid of a point sample, for example a ball of the type used to calibrate confocal microscopes. The trajectory of the point of impact of the wave in the focal plane of the condenser can be controlled by means of the CCD 2041. The real part of the image obtained corresponds, for weakly diffracting samples, to the absorptivity. The imaginary part corresponds, for weakly diffracting samples, to the refractive index. The deconvolution filter is the same as that used for example in the article Reconstructing 3D lightmicroscopic images by digital image processing, by A. Erhardt & al. applied optics vol. 24 No. 2.1855.

Mode 2) Use in tomographic mode.

In this imaging mode the mask 2046 and the optional filter blade 2047 are not used.

The use in tomographic mode consists in using a method of the type described in patent WO99 / 53355. However, there is no random phase shift to compensate. Moreover, the frequency representation parts whose superposition generates the frequency representation of the object are obtained in a slightly different manner.

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J2Z ('P + q) We use the Fourier transform [p, q] = Y C [i, ile'-of the normalized elementary image I, J Cl!, Y obtained for a given position of the galvanometric mirrors. N x Np, X is the dimension of N N the useful area of the CCD sensor, and the indices vary from - to -1.

C[p, q] est la projection sur un plan horizontal (suivant l'indice 1 déterminant la direction verticale d'une partie sphérique de la représentation fréquentielle tridimensionnelle de l'objet Ffp, q,/]. La Fig. 4 montre en coupe verticale suivant q, l la partie sphérique 2120 de la représentation F[p,q,l], ainsi que le vecteur d'onde Je de l'onde d'éclairage ramené à l'échelle de cette représentation, et le support bidimensionnel 2121 de Cfp, q]. L'image détectée sur le CCD 2041 permet l'obtention du vecteur d'onde Je et donc la détermination de la position de la portion de sphère 2120. Il est alors possible de projeter E[p, q] sur cette portion de sphère, suivant la direction verticale 2122, pour obtenir une portion de la représentation fréquentielle de l'objet. La représentation tridimensionnelle F[p, q, l] est alors obtenue, comme dans le brevet W099/53355, par superposition d'un ensemble de telles portions de sphères obtenues pour une série d'ondes d'éclairage différant entre elles par leur direction et obtenues par déplacement des miroirs galvanométriques. La représentation spatiale est obtenue par inversion de la transformation de Fourier. 2 2

C [p, q] is the projection on a horizontal plane (according to index 1 determining the vertical direction of a spherical part of the three-dimensional frequency representation of the object Ffp, q, /]. vertical section along q, l the spherical portion 2120 of the representation F [p, q, l], and the wave vector I of the lighting wave brought to the scale of this representation, and the two-dimensional support 2121 of Cfp, q] The image detected on the CCD 2041 makes it possible to obtain the wave vector I and thus the determination of the position of the sphere portion 2120. It is then possible to project E [p, q on this portion of sphere, in the vertical direction 2122, to obtain a portion of the frequency representation of the object, the three-dimensional representation F [p, q, 1] is then obtained, as in patent WO99 / 53355, by superposition of a set of such portions of spheres obtained for a series of illumination waves differing in their direction and obtained by displacement of the galvanometric mirrors. The spatial representation is obtained by inverting the Fourier transform.

Mode 3) obtaining projections of an object.

 Projections of an object can be obtained by means of an opaque mask 2046 of the type shown in FIG. 7, having an opening 2161 in the form of an elliptical strip, and placed in a pupillary plane.

d'obtention des caractéristiques d'une ellipse limitant la bande elliptique 2161. Sur cette figure, le

F paramètre R vaut R = nFobJ 2045 ou n est l'indice de l'huile optique pour laquelle l'objectif est conçu. Fig. 10 specifies the calculation intermediaries of this elliptical band and details the mode

for obtaining the characteristics of an ellipse limiting the elliptical band 2161. In this figure, the

F parameter R is R = nFobJ 2045 where n is the index of the optical oil for which the objective is designed.

Le diamètre DO du cercle 2160 limite la partie utile du plan pupillaire, compte tenu de l'ouverture de

zu l'objectif. Ii vaut DO = 20MV-7,.--. L'angle entre la direction de projection et l'axe optique est F2O44

e. Les autres paramètres se déduisent de la figure :

Le rapport petit axe sur grand axe est -= cos (0). La position du centre Cl de l'ellipse par rapport au D2

centre optique C0 s'obtient par : D1 = H-d F2O44

The diameter OD of the circle 2160 limits the useful part of the pupillary plane, taking into account the opening of

zu the goal. Ii is DO = 20MV-7, .--. The angle between the projection direction and the optical axis is F2O44

e. The other parameters are deduced from the figure:

The small axis ratio on the major axis is - = cos (0). The position of the center Cl of the ellipse with respect to the D2

optical center C0 is obtained by: D1 = Hd

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H = RsinO d=hsin D2 = R2 - (R-hf 2 2

d'ou finalement :

D1= DO sinS 20MV DO 7ï7

Par exemple, dans l'exemple particulier de dimensionnement donné plus haut, on a D0=5mm et une ellipse limitant un masque utilisable pour 0 = 8 degrés a par exemple les caractéristiques suivantes, obtenues à partir des équations indiquées précédemment :

H = RsinO d = hsin D2 = R2 - (R-hf 2 2

of or finally:

D1 = OD sinS 20MV DO 7I7

For example, in the particular example of dimensioning given above, D0 = 5 mm and an ellipse limiting a usable mask for 0 = 8 degrees have for example the following characteristics, obtained from the equations indicated above:

<Tb>
<tb> parameter <SEP> value
<tb> Dl <SEP> 0, <SEP> 30
<tb> D2 <SEP> 4, <SEP> 25
<tb> D3 <SEP> 4, <SEP> 21
<Tb>

La largeur de la bande elliptique doit être supérieure au diamètre de la tache de diffraction formée dans le plan pupillaire. Dans le cas présent elle peut valoir par exemple 20 microns. Ce masque à bande elliptique a été représenté sur la figure 11 pour une largeur de la bande elliptique égale à 20 microns. Plus la largeur de la bande est élevée, plus la profondeur de champ de l'image obtenue est faible, et plus la luminosité est élevée. Il est donc préférable d'utiliser une largeur de bande faible dans la mesure ou cette largeur de bande reste compatible avec la luminosité nécessaire.

The width of the elliptical band must be greater than the diameter of the diffraction spot formed in the pupillary plane. In the present case it may be for example 20 microns. This elliptical band mask has been shown in FIG. 11 for an elliptical band width of 20 microns. The higher the width of the band, the lower the depth of field of the image obtained, and the higher the brightness. It is therefore preferable to use a low bandwidth as long as this bandwidth remains compatible with the necessary brightness.

If a projection along the optical axis is sought, the band is annular and centered on the optical axis. If a more inclined projection direction is sought, the ellipticity becomes more pronounced and the band is more eccentric to the optical axis.

It is also possible to place the elliptical band mask in another pupillary plane, for example a pupillary plane where the phase plate is usually placed in a phase contrast lens.

The galvanometric mirrors must then be controlled so that the point of impact of the illumination beam on this mask travels the elliptical band during the integration time of the sensors. The image Cry, I obtained is then a projection in the vertical direction. This image can be improved by a two-dimensional deconvolution whose characteristics can be obtained either by measurement of the impulse response or by theoretical considerations.

In this imaging mode most of the diffracted wave is stopped by the mask 2046.

On the other hand, the non-diffracted portion of the wave passes entirely through the mask. In the absence of special precautions, the diffracted wave becomes negligible compared to the undiffracted wave and can therefore hardly be detected. To remedy this defect, it is necessary to use a filter blade 2047.

This blade is shown in FIG. 12. It consists, for example, of a transparent pane comprising in addition an absorbent element 2201 which may be, for example, of glass or stained plastic and

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 have a diameter, in the particular example of dimensioning given above, of about 50 microns. This absorbent element 2201 must be placed just above the hole made in the pierced blade 2019. It is also possible to remove the glass 2047 and to directly pour a plastic absorbent element into the hole 2101 made in the pierced blade. The absorbent element serves to attenuate the undiffracted wave to facilitate the detection of the diffracted wave. The filter blade 2047 may also be used to perform more sophisticated filtering of the image, and may for example have a decreasing absorptivity of its center 2201 towards its edges.

 By means of beam splitters and fast switches, for example based on ferroelectric liquid crystals, it is also possible to separate two paths on which two different masks can be used. By using these two paths alternately, two projections forming a stereoscopic image are generated. The mask may also consist of a liquid crystal space modulator, which allows to change the direction of observation at will.

 Different modes of use, intermediate between the section generation, projection, and tomographic modes, can be used. In general, the tomographic mode is the one that allows the best image quality, and the other two modes are used for real-time observation.

 Variations of this embodiment may be used. In particular, it is possible to use several lasers at different wavelengths, with a switching system between these lasers or a superposition system of the lasers, to obtain a color effect.

 It is also possible to use a light source described in FIG. 5. In this figure of light is produced at the focus 2130 of a high intensity lamp, for example a mercury vapor lamp. This light is collected by a collector 2131 and then focused by a lens 2132 to a hole 2133. If this hole is sufficiently small, it constitutes a point source in a pupillary plane. The light from this microscopic hole passes through a lens 2134 and then the field diaphragm 2135. The object focal plane of the lens 2134 is on the microscopic hole 2133. The image focal plane of the lens 2134 is on the diaphragm 2135. The light emerges Field diaphragm 2135 can replace light from the laser and its beam expander. The major advantage of this light source is that it is inexpensive and polychromatic, which allows by means of a monochromator filter to select various wavelengths. By cons, even optimizing the system, most of the light from the source is lost and the useful intensity is quite reduced. This problem can be solved by simultaneously widening the microscopic hole 2133 and the hole 2101 made in the blade 2019.

However this introduces approximations that can decrease the quality of the image. Indeed, if the illuminated area in the object focal plane of the condenser is too large: - the width of the microscopic hole 2101 must be sufficient to cover the image of this area in the plane of the blade 2019. The diffracted part of the wave also crossing this zone, a significant part of this diffracted wave will be modified in the same way as the undiffracted wave.

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 - the width of the ellipse used in "get projections of an object" mode (extended depth of field) must remain greater than the width of the image of the illuminated area in the plane of the mask 2046. This limits the depth field that can be obtained.

 similarly, in "tomographic" mode, the extension along the vertical axis of the three-dimensional image that can be obtained is limited.

If such a source is used, a sufficiently sensitive camera should be used. In addition, the half wave and third wave blades must be achromatic, which is also the case when several lasers are used.

 It is not essential to use wave plates to generate the phase offsets. Fig. 8 shows a detection device that can be substituted for that of FIG. 1. After passing through the lens 2018, the wave is divided in three by the beam splitters 2170 and 2175. The portion of the wave that is reflected by 2170 passes through then a blade 2171 placed in a pupillary plane and carrying an extra thickness at the point of impact of the non-diffracted portion of the illumination wave. The extra thickness must be such that it generates a phase shift of 120 degrees between the wave passing through it and the wave that does not pass through it. The wave then passes through the lens 2172 whose object focal plane is on the slide 2171 and reaches the CCD 2173 located in the image focal plane of 2171 and attached to the camera 2174. The set 2176.2177, 2178.2179 is equivalent 2171,2172, 2173,2174 but the extra thickness generates a phase shift of 240 degrees. The assembly 2180, 2182, 2183 is also equivalent but does not include a blade with extra thickness (it may possibly include a blade without extra thickness).

trois décalages de phase doivent être prises successivement. Les trois caméras sont par exemple remplaçées par le dispositif de la figure 6. Sur cette figure, le faisceau issu de la lentille 2018 après avoir traversé la partie du dispositif de la Fig. 1 qui se trouve avant cette lentille, parvient à un séparateur de faisceau 2140. It is also possible to use a single camera. In this case, the images corresponding to

three phase shifts must be taken successively. The three cameras are for example replaced by the device of Figure 6. In this figure, the beam from the lens 2018 after passing through the portion of the device of FIG. 1 which lies before this lens, reaches a beam splitter 2140.

The portion of the beam which is reflected by 2140 is then reflected by the piezoelectric mirror 2141 and reaches the microscopic hole 2142 which coincides with the point of impact of the wave in the absence of observed sample. The portion of the wave that passes through this microscopic hole essentially comprises the non-diffracted portion of the wave and will be used as the reference wave. It then passes through the beam splitter 2144.

 The portion of the wave which passes through the beam splitter 2140 is reflected by the mirror 2143 and the semi-transparent mirror 2144. The two superimposed waves then pass through the lens 2145 and reach the CCD 2146 sensor integrated in the camera 2147.

 The orientation of the mirror 2143 must be adjusted to obtain the flat tint on the camera in the absence of observed object.

caméras est remplaçée par l'acquisition successive d'images correspondant à des décalages de phase de 0, 120, 240 degrés. Les décalages de phase sont réalisés au moyen du miroir piézoélectrique 2141. Lors des The operating mode is similar to the previous one but the simultaneous acquisition on all three

cameras is replaced by the successive acquisition of images corresponding to phase shifts of 0, 120, 240 degrees. The phase offsets are made by means of the piezoelectric mirror 2141.

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 three successive acquisitions necessary to obtain a complex image, the galvanometric mirrors must be ordered in exactly the same way.

 It is possible to add in the pupillary plane situated between 2140 and 2143 a slide bearing an absorbing point at the point of impact of the non-diffracted part of the wave, in order to keep on this path only the diffracted wave.

piézoélectrique 2195. Mais la valeur complexe obtenue au pixel de coordonnées i, j représente maintenant Cj7, y] au lieu de C[i, j] et une transformée de Fourier inverse doit donc être effectuée pour retrouver l'image de l'objet. Des systèmes de détection utilisant trois caméras dans des plans pupillaires et une séparation du front d'onde par polarisation peuvent également être conçus. Il est possible de rajouter un plan pupillaire intermédiaire avant la caméra, dans lequel on place une lame portant un point absorbant situé au point d'impact direct de l'onde non diffractée. Ceci permet d'éviter la saturation de la caméra en ce point. The cameras are not necessarily in an image plane. Fig. 9 shows a camera detection system placed in a pupil plane. The wave having passed through the lens 2018 passes through the beam splitters 2190 and 2191 before reaching the CCD 2192 located in a frequency plane for this wave. The portion of the wave that is reflected by the beam splitter 2190 passes through the microscopic hole 2194 which coincides with the fixed point of impact of the non-diffracted portion of the wave. The part of the wave that passed through 2194 constitutes the reference wave and the microscopic hole 2194 can be considered as being in a space plane for this wave. The wave coming from 2194 is reflected on the piezoelectric mirror 2195 and on the mirror 2196. It passes through the lens 2197, is reflected by the beam splitter 2191 and reaches the CCD 2192 attached to the camera 2193. The mode of use is similar to that of the device described in FIG. 6, phase shifts now being made using the mirror

piezoelectric 2195. But the complex value obtained at the coordinate pixel i, j now represents Cj7, y] instead of C [i, j] and an inverse Fourier transform must therefore be performed to find the image of the object. Detection systems using three cameras in pupillary planes and a polarization wavefront separation can also be designed. It is possible to add an intermediate pupillary plane before the camera, in which is placed a slide bearing an absorbing point located at the point of direct impact of the undiffracted wave. This avoids saturation of the camera at this point.

1 [ ; surépaisseur 2411 générant par exemple un décalage de phase de-. La surépaisseur 2411 doit être placée 2 au point d'impact fixe de la partie non diffractée de l'onde d'éclairage. Pour une image en fond clair la lame 2403 n'est pas indispensable, et dans le cas d'une image en mode obtention de projections la lame 2047 doit également être utilisée. It is also possible to delete the cameras and observe the image directly using an eyepiece, or use only one camera but on which a single image is acquired and retransmitted on the screen. a computer without prior treatment. In this case the part of the system of FIG. 1 situated after the lens 2018 is replaced by that represented in FIG. 14. The wave issuing from 2018 passes through a plate 2403 located in the image focal plane of the lens 2018, then passes through the 2404 lens whose object focal plane is on the slide 2403, and reaches the CCD 2405 sensor attached to the camera 2406. The sensor and the camera can optionally be replaced by a 2407 eyepiece forming the image on the retina of the eye 2408 as shown in Fig. 15. Blade 2403 is shown in Fig. 13 in the case where a phase contrast image is sought. It consists of a window with a center window

1 [; overthickness 2411 generating for example a phase shift of. The excess thickness 2411 must be placed 2 at the point of fixed impact of the non-diffracted portion of the illumination wave. For a brightfield image the blade 2403 is not essential, and in the case of a projection mode image the blade 2047 must also be used.

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 The diagrams of FIGS. 1, 6, 8, 9, 14, 15 can be usefully complemented by neutral filters, possibly adjustable, to adjust the light intensity in the different parts of the device.

Second embodiment (preferred mode)
This second embodiment is shown in FIG. 17. It differs from the first embodiment in that the wave which has passed through the pierced blade is then again redirected by the galvanometric mirrors. This solution has the advantage that: - it is not essential to place the galvanometric mirrors exactly in space planes, - the system is less sensitive to the long-term position drifts of these mirrors - the non-diffracted part of the beam Lighting has a variable direction at the output of the system, which allows direct observation without the danger associated with the use of coherent light.

 The linearly polarized beam from the laser 1000 passes through the beam expander consisting of the lenses 1001 and 1002, then the diaphragm 1003. It passes through the polarizing beam splitter 1004, is reflected by the galvanometric mirror 1005, the mirror 1006 and the Galvanometric mirror 1007. It then passes through the polarizing beam splitter 1021, then the lens 1009. It is reflected by the mirrors 1010 and 1011, then by the mirror 1012. It passes through the condenser 1013, the object observed 1014, the objective 1015, the tube lens 1016, the lens 1017. It passes through the polarizer 1050 which selects only the main polarization direction (direction of polarization of the non-diffracted part of the wave) so as to obtain a perfectly linear polarized beam. It passes through the polarization rotator 1019 whose electrodes are shown in FIG. 18 and which makes it possible to pass the entire beam or the beam passing through the frequency plane on one or more elliptical strips as desired. It then passes through the orthogonally polarized polarizer 1051 to the polarizer 1050. It is reflected by the mirror 1018 and passes through the lens 1020. It is then reflected by the polarizing beam splitter 1021. It is then reflected by the galvanometer mirror 1007, by the mirror 1006, by the galvanometric mirror 1005, and by the polarizing beam splitter 1004. It is reflected by the mirrors 1022, 1023, 1024 shown in Figure 16 in side view. It passes through the lens 1025, the pierced blade 1026, the filtering blade 1027. It is reflected by the mirrors 1028 and 1029, passes through the retardation plate 1030, the steerable polarizer 1031, the lens 1032. It is reflected by the second mirror face 1005, by the mirror 1033, by the second face of the galvanometric mirror 1007. It passes through the lens 1047, is reflected by the mirror 1046, passes through the lens 1045. It is then separated in two by the semi-transparent mirror 1034. part of the beam is reflected by the mirrors 1039, 1040 through the shutter 1041 composed of a polarization rotator liquid crystal and a polarizer. This part of the beam passes through the eyepiece 1042 and reaches the eye 1043. The other part of the beam is reflected by the mirror 1035, passes through the shutter 1036, the eyepiece 1037, and reaches the eye 1038.

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 The laser 1000 may be a laser emitting several wavelengths simultaneously, or a combination of several lasers whose outputs are superimposed by dichroic mirrors, so as to obtain a color image.

est confondu avec le plan focal objet du condenseur 1013. Le plan focal objet de la lentille 1016 est confondu avec le plan focal image de l'objectif 1015. Le plan focal image de la lentille 1016 est confondu avec le plan focal objet de la lentille 1017. Le rotateur de polarisation 1019 est dans le plan focal image de la lentille 1017 et dans le plan focal objetde la lentille 1020. Le plan focal image de la lentille 1020 est confondu avec le plan focal objet de la lentille 1009. Le plan focal objet de la lentille 1025 est confondu avec le plan focal image de la lentille 1020. La lame demi onde percée 1026 est dans le plan focal image de la lentille 1025. Le plan focal objet de la lentille 1032 est confondu avec le plan focal image de la lentille 1025. La distance entre 1033 et 1005 soit égale à la distance entre 1006 et 1005. Le plan focal image de la lentille 1032 est sur le miroir 1033. Le plan focal objet de la lentille 1047 est sur le miroir 1033. Le plan focal image de la lentille 1047 est confondu avec le plan focal objet de la lentille 1045. Le plan focal image de la lentille 1045 est le plan image 1048 observé à l'aide des oculaires 1042 et 1037. The focal plane object of the lens 1009 is on the mirror 1006. The focal plane image of the lens 1009

is coincident with the object focal plane of the condenser 1013. The object focal plane of the lens 1016 is coincident with the image focal plane of the objective 1015. The image focal plane of the lens 1016 coincides with the object focal plane of the lens 1017. The polarization rotator 1019 is in the image focal plane of the lens 1017 and in the object focal plane of the lens 1020. The image focal plane of the lens 1020 coincides with the object focal plane of the lens 1009. The focal plane object of the lens 1025 is coincident with the image focal plane of the lens 1020. The half wave wave pierced 1026 is in the focal plane image of the lens 1025. The object focal plane of the lens 1032 is coincident with the image focal plane of the lens 1025. The distance between 1033 and 1005 is equal to the distance between 1006 and 1005. The image focal plane of the lens 1032 is on the mirror 1033. The object focal plane of the lens 1047 is on the mirror 1033. The plane focal image of the lenti 1047 is coincident with the object focal plane of the lens 1045. The image focal plane of the lens 1045 is the 1048 image plane observed with the eyepieces 1042 and 1037.

 As previously FN is noted the focal length of the lens number N, opens the aperture of the lens and the condenser, Fond the focal length of the condenser, Fobj the focal length of the lens.

unes les autres, les relations suivantes doivent être vérifiées :

Fcond F1016 1020 1 Fl1009Fobj F1017 F1032 = F1025-

Les électrodes du rotateur de polarisation 1019 sont représentées sur la figure 18. Elles forment deux bandes elliptiques pratiquement annulaires, chacune étant du type représenté sur la figure 11. In order for the deflections of the beam on the various parts of its trajectory to effectively compensate for the

the others, the following relationships must be verified:

Fcond F1016 1020 1 Fl1009Fobj F1017 F1032 = F1025-

The electrodes of the polarization rotator 1019 are shown in FIG. 18. They form two substantially annular elliptical bands, each of the type shown in FIG.

The set of these two bands is composed of the electrodes 1111 to 1115. The rest of the surface of the rotator is composed of the electrodes 1116 to 1119 which are all connected to the same electrical potential.

 To obtain a sectional image the galvanometric mirrors are controlled so that the point of impact of the non-diffracted portion of the illumination wave sweeps the portion of the ploarization rotator 1019 which is accessible taking into account the opening of the lens, or equivalently so that the point of impact of the light wave sweeps the entire object focal plane of the condenser 1013.

polariseur 1051. Les interrupteurs 1041 et 1036 sont ouverts. The set of rotator electrodes are then controlled so that the light can pass through the

1051. The switches 1041 and 1036 are open.

 To obtain a stereoscopic image, the electrodes of the polarization rotator 1019, the galvanometer mirrors 1007 and 1005, and the shutters 1041 and 1036, must be controlled synchronously. Formation of a stereoscopic image is composed of a left image forming phase, and a right image forming phase. These two phases alternate quickly enough that the eye can not distinguish them.

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During left image formation phase: - the shutter 1041 is open.

shutter 1036 is closed.

the electrodes 1110, 1111, 1112, 1113 are controlled in such a way that the light passing through them has its modified polarization and passes through the polarizer 1051.

the other electrodes of the polarization rotator 1019 are controlled so as not to modify the polarization of the light passing through them, so that this light is stopped by the polarizer 1051.

The galvanometric mirrors 1005, 1007 are controlled so that the point of impact of the illumination wave has an elliptical trajectory, moving on the ellipse formed by the electrodes 1110, 1111, 1112, 1113.

 During the right image forming phase: the shutter 1041 is closed.

shutter 1036 is open.

the electrodes 1110, 1111, 1114, 1115 are controlled in such a way that the light passing through them has its modified polarization and passes through the polarizer 1051.

the other electrodes of the polarization rotator 1019 are controlled so as not to modify the polarization of the light passing through them, so that this light is stopped by the polarizer 1051.

The galvanometric mirrors 1005, 1007 are controlled so that the point of impact of the illumination wave has an elliptical trajectory, moving on the ellipse formed by the electrodes 1110, 1111, 1114, 1115.

 The polarizer 1051 is not absolutely essential in that the polarizing beam splitter 1021 is sufficient to select a suitable polarization.

 The half-wave half-wave plate 1026 is of the same type as the half-wave half-wave plate 2019 of the previous embodiment. The filtering blade 1027 is of the same type as the filtering blade 2047. The delaying blade 1030 can optionally be replaced by a variable phase shift assembly of the type described in: P. Hariharan, "Achromatic phase shifting for polarization interferometry", Journal of modem optics vol. 43 no 6 pp. 1305-1306, 1996.

 By rotating the polarizer 1031 and the moving wave plate of the variable phase shift assembly replacing the retarding plate 1030, a bright background with high contrast and phase contrast can be switched continuously from the conventional bright background. By exchanging the filter blade 1027 it is possible to modify the type of filtering applied and to obtain a black-field image.

 To compensate for the relatively slow galvanometric mirrors, it is possible to complete the system with an accoutero-optical deflector placed for example between the laser 1000 and the lens 1001. This deflector can be used to quickly deflect the beam. The modification of the direction of the beam in the object is then partly due to the galvanometric mirrors and partly to the accousto-optical deflector.

The deflection due to the acousto-optical deflector must remain much smaller in amplitude than that due to the galvanometric mirrors, so that the point of impact of the non-diffracted part of the wave on the half-wave plate pierced moves in a small direction. area around its average position. It is then sufficient to enlarge slightly the hole of the pierced blade so that the non-diffracted part of the wave passes through it whatever the state

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 the accousto-optical deflector. If the accousto-optical deflector only deflects the beam in one direction, it is possible to give the hole of the pierced blade an elongated shape.

When the electrodes of the polarization rotator 1019 are all passing and in the absence of the matching plate 1027, the frequency response of the microscope (Fourier transform of the two-dimensional point-to-point response function, for a point of the object located in the plane of focus), has the appearance shown in Figure 19, or the module of the two-dimensional spatial frequency is represented on the abscissa, and or the amplitude of the frequency representation is ordinate. This frequency response is the same as for a conventional brightfield microscope. The amplitude decreases for the high spatial frequencies, which results in a decrease in the resolution as perceived by the observer. This resolution can be improved by correcting the frequency response by means of a suitable filter blade 1027, the frequency response of the corrected system then being approximately constant up to the maximum spatial frequency reached.

r c* lame filtrante 1027, est proportlOnelle a rmax - r avec rmax = ouv'Fobj --, et ou r est la F1016 F1020 lame filtran

distance à l'axe optique. Pour compenser cette réponse, on peut utiliser une lame filtrante ayant une transmissivité T (r) dépendant de la distance r à l'axe optique et valant :

2 ré-r - si : r () =i rmax-r

ou rl. est une valeur limite comprise entre 0 et rm O. La résolution effective obtenue vaut alors à peu près, au sens de Rayleigh :

1, 2 1 2 2rma 1 i max 40MV+

Par exemple on peut utiliser rhm = 0, 75 et on a alors une limite de résolution de 0, 57 fois la limite de résolution usuellement obtenue avec un microscope en fond clair (limite de Rayleigh). Dans ce cas la valeur minimale atteinte par r (r) est T (0) = 0,0625. The frequency representation of FIG. 19, brought back to the Fourier plane in which the

rc * 1027 filter blade, is proportlocal to rmax - r with rmax = open Fobj -, and or r is the F1016 F1020 filter blade

distance to the optical axis. To compensate for this response, it is possible to use a filter blade having a transmissivity T (r) depending on the distance r to the optical axis and being:

2 re-r - si: r () = i rmax-r

or rl. is a limit value between 0 and rm O. The effective resolution obtained is then worth approximately, in the Rayleigh sense:

1, 2 1 2 2rma 1 i max 40MV +

For example, it is possible to use rhm = 0.75 and then there is a resolution limit of 0.57 times the resolution limit usually obtained with a brightfield microscope (Rayleigh limit). In this case the minimum value reached by r (r) is T (0) = 0.0625.

 If only an increase in resolution in brightfield is sought, one can delete the pierced blade 1026, the wave plate 1030 and the polarizer 1031.

 This method of increasing the resolution can also be used in the other embodiments.

 As shown in Fig. 17, an inexpensive system can be obtained by replacing the laser and the beam expander with the system shown in Fig. 5 and already described in the first embodiment. The wave issuing from the emitting zone 2130 of an arc lamp passes through the collecting lens 2130 and is then refocused by a lens 2132 on a hole 2133 situated in a frequency plane. This hole is placed in the object focal plane of the lens 2134, the field diaphragm 1003 being placed in the image focal plane of

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F2134 rlim-rmax F1009 r max 1009 rmax

Pour compenser la perte de luminosité qui, comparativement à un miroscope classique, découle de la faible largeur du trou 2133 et de la faible valeur de T (0), il est préférable d'utiliser une source lumineuse 2130 de très forte luminosité, par exemple une lampe à arc. this lens. For the brightness to be maximum while remaining compatible with the method of increasing the resolution described here, the diameter of the hole 2133 of FIG. 5 can be for example:

F2134 rlim-rmax F1009 r max 1009 r max

To compensate for the loss of luminosity which, compared to a conventional miroscope, results from the small width of the hole 2133 and the low value of T (0), it is preferable to use a very bright light source 2130, for example an arc lamp.

. For example, it is possible to use in this case: a Nikon CFI60 planachromatic lens of numerical aperture 1.25 forming the image at infinity and corrected independently of the tube lens, with a focal length of 2 mm.

 a planachromatic Nikon condenser with aperture 1.4 diaphragm at 1.25, with a focal length of 8 mm.

a lens 1009 consisting of a Melles Griot optimized achromatic doublet, with a focal length of 800 mm.

- lenses 2134, 1016, 1017, 1020, 1025, 1032, 1047, 1045 all identical to the tube lens used on Nikon microscopes, with a focal length of 200 mm - a mercury arc lamp.

galvanometric mirrors having a diameter of about 20 mm; a hole 2133 of about 0.625 mm in diameter.

a filter blade defined as above with rmax = 2.5 mm and rlim = 1.875 mm
However, this dimensioning requires galvanometric mirrors of large size to not reduce the field. It is advantageous to modify the focal length of the lenses in the following manner: the lenses 1020, 1025, 1032, 1047, 2134 have a focal length of 50 mm.

 the lens 1009 has a focal length of 200 mm. the other lenses being as before. This solution makes it possible to use smaller (about 6 mm) and faster galvanometric mirrors without reducing the size of the field. However, lenses with a focal length of 50 mm are more difficult to optimize by avoiding aberrations.

 In the indicated configuration the position of the lens 1032 and the mirror 1033 must be adjusted with sufficient precision so that the image plane is fixed after reflection on the galvanometric mirrors. This can be slightly simplified by using a single galvanometric mirror movable around two axes, the other galvanometric mirror being replaced by a fixed mirror. In this case the position setting of the mirror 1033 becomes unnecessary.

 A change of lens in the device of FIG. 17 necessitates modifications in the rest of the optical system, which make more expensive a system adapted to a series of different objectives, in which various lenses must be exchanged at the same time as the goal. However, the increase in resolution made possible by the present invention is especially useful with high resolution objectives.

To limit the cost and in the case where the essential objective of the device is an increase in resolution in brightfield, it is advantageous to combine the present method, used for example with the objective x100 oil or dry, with a method conventional lightfield microscopy used with other lenses.

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 This can be done by sizing the system for the x100 objective, and using with this lens the described method, which involves a proper control of the galvanometric mirrors allowing the scanning of the focal plane object of the condenser by the beam of illumination With the others objectives, the hole 2133 is replaced by a diaphragm opening sufficiently open, a fixed position of the galvanometric mirrors is used, the blades 1026, 1027, 1030 and the polarizer 1031 are suppressed, and the polarization rotator is controlled so as to completely pass, which allows to obtain a classic brightfield image. It is also possible, but more expensive, to use a partially distinct optical path for the conventional bright background and for the system with resolution increase.

Industrial applications:
This microscope can replace microscopes in brightfield, phase contrast, or DIC. It offers a very superior image quality, as well as the possibility of obtaining either cuts or projections of the object.

Claims (10)

Claims (1/2)  1. Microscope operating in transmission, in which the illumination beam is not focused on a point of the observed object, characterized in that it comprises: a beam deflector A placed between the illumination source and the condenser, and making it possible to modify the direction of the illumination wave in the sample, - a beam deflector B placed between the objective and the detector, and making it possible to modify the direction of the wave coming from the sample and having crossed the objective, 2. Microscope according to claim 1, characterized in that it comprises a differentiating device for applying, in an intermediate plane reached by the light after deflection by said deflector B, a mean phase and / or attenuation modification and / or polarization different to the non-diffracted part of the illumination wave and to the wave diffracted by the observed object. 3. Microscope according to one of claims 1 or 2, characterized in that it comprises a filtering plate placed in an intermediate plane reached by the light after deflection by said deflector B, and whose transmissivity depends on the distance to the axis optical. 4. Microscope according to one of claims 2 to 3, characterized in that said beam deflectors A and B are controlled or arranged so that the direction of the light wave in the sample is variable and so the non-diffracted part of the illumination wave arrives at a fixed point of said intermediate plane. 5. Microscope according to one of claims 1 to 4, characterized in that said beam deflectors A and B are movable mirrors or pairs of movable mirrors. 6. Microscope according to one of claims 1 to 5, characterized in that each movable mirror of said deflector B coincides with a corresponding movable mirror of said deflector A, or is integral with a corresponding movable mirror of said deflector A. 7. Microscope according to one of claims 1 to 6, characterized in that it comprises a deflector C for changing the direction of the wave from the sample after it has been deflected by said deflector B. <Desc / Clms Page number 24>  8-microscope according to claim 7, characterized in that: - said deflector C is a movable mirror or a pair of movable mirrors, - each movable mirror of said deflector C coincides with a corresponding movable mirror of said deflector B, or is integral a corresponding mobile mirror of said deflector B.

 Claims (2/2)

9-microscope according to claim 8, characterized in that each movable mirror of said deflector C is formed on the opposite face of a corresponding movable mirror of said deflector B. 10-microscope according to one of claims 1 to 9, characterized in that it comprises a mask placed in the image focal plane of the objective, or in an image plane of this focal plane which is placed in the path of the front beam deflection by said deflector B or after deflection by said deflector C, which is absorbing everywhere except on an elliptical shaped strip.