FR2822551A1 - Confocal optical scanner for biological microscope, has optical system to focus light from object to form beam reflected by rotary mirror for scanning - Google Patents

Abstract

The confocal optical scanner for a microscope has an optical system to focus an illuminating beam (FX) from a light source to illuminate a pint on an object (1107). The system focuses the light from the object to a point (FEO) in a first image plane.. A rotary mirror (1104) reflects the light beam to scan the object. A spatial filter (1203) in the first image plane filters the light to obtain a beam to be detected.

Description

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Rapid confocal microscope Technical area
The invention relates to a fast confocal microscope, more particularly a confocal fluorescence microscope intended to operate in real time.

Prior art.

 The principle of the confocal scanning microscope is to illuminate the sample with a light beam focused on a point, and to detect only the light coming back from this point. The confocal microscope therefore comprises an illumination beam possibly filtered by a microscopic hole, and the wave coming from the illuminated object itself crosses a microscopic hole selecting the light coming from the illuminated point. The wave coming from the object is then detected.

 To generate an image of the object, it is necessary to move the illuminated point in a plane. This scanning can be carried out in various ways: i) by moving the observed sample relative to the objective. It is the simplest method but it is excessively slow. ii) -by making the microscopic holes on a disc traversed in one direction by the lighting light and in the other direction by the light coming from the object. When the disc is rapidly rotating in the image plane, the microscopic holes move and the sample is scanned. This is the Nipkow disc technique. Since the disc has many holes, this scanning technique is very fast. Detection can be done directly on a camera, and it is also possible to observe the image directly with the naked eye. On the other hand, 95% of the available light power is lost, which limits the effectiveness of this technique when the samples are weakly fluorescent. Zeiss, however, offers such a microscope (CARV) for the observation of fluorescent samples. iii) - using galvanometric mirrors to deflect the beam. The sample is then scanned point by point and a single microscopic hole is used. A photomultiplier tube allows the detection of photons having passed through this hole. This technique is the most used in confocal fluorescence microscopy because there is little loss of light energy. On the other hand, the imaging speed is limited by the saturation of the fluorescence and / or the frequency of oscillation of the galvanometric mirrors. The image cannot be observed directly and must necessarily be reconstructed by computer from the data acquired by the photomultiplier tube. The reconstitution of a horizontal plane of the sample requires knowing exactly the position of the galvanometric mirrors corresponding to the signal sampled at each instant on the photoreceptor. The good functioning of the system requires a very precise control of the galvanometric mirrors and a perfect synchronization between the movement of the galvanometric mirrors and the sampling of the signal coming from the photoreceptor.

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 To improve the imaging speed, a Nipkow disc microscope has been designed comprising a collecting disc made up of a network of microlenses, and integral with the disc carrying the microscopic holes. Each microlens is placed in front of a microscopic hole and focuses on this hole the light coming from the lighting beam. This technique is described for example in American patent number 5,162, 941 of the University of Wayne, as well as in American patent number 5,579, 157 of the company Yokogawa Electric Corporation. The corresponding microscope is marketed under the name "U1traview" by Perkin-E1mer. This technique avoids the loss of light power resulting from the use of a Nipkow disk, while retaining the high imaging speed inherent in Nipkow disk systems.

 The technique used by Yokogawa Electric Corp. poses difficult problems of mechanical production and alignment and it is expensive to implement. The disc carrying the microscopic holes and the collecting disc must be aligned with each other with great precision. The assembly constituted by these two discs must be in rapid rotation around an axis which must be perfectly fixed, perfectly orthogonal to the plane of the discs, and perfectly orthogonal to the direction of propagation of the incident light. In practice, it is extremely difficult to keep the position of the axis fixed, and this results in image defects, typically small clear or dark lines superimposed on the image. American patent number 5,579,157 to Yokogawa describes a solution making it possible to attenuate this type of defects, however this solution is not very effective. Other alignment problems exist, described for example by US patent number 5,717,519 from Yokogawa Electric Corp.

 In the Yokogawa Electric Corp. system or in Zeiss CARV, part of the lighting wave is reflected on the plate carrying the microscopic holes and then goes towards the CCD sensor. This wave being very intense compared to the wave corresponding to fluorescence, it is difficult to eliminate using dichroic filters, which results in a significant attenuation of the beam.

 In the Yokogawa Electric Corp. system or in Zeiss CARV, the same microscopic holes are successively crossed by the light beam directed towards the sample, and by the beam coming from the sample. If the diameter of these holes is reduced, the loss of intensity of the beam which results therefrom is much greater than that which results from the simple reduction in the diameter of the microscopic hole in a confocal laser scanning microscope by galvanometric mirrors. For the intensity to remain reasonably high, the microcopic holes must have a diameter close to the width of the Airy spot. This causes a sharp decrease in resolution compared to the confocal laser scanning microscope with galvanometric mirrors, in which the resolution can be increased by reducing the diameter of the microscopic hole.

 In the Yokogawa Electric Corp. system or in Zeiss CARV, the image of a microscopic hole on the CCD camera, when this image has moved a length equal to half the width of Airy's spot, covers quite the same image before moving. Therefore the resolution is limited only by the fluorescent emission wavelength, and the resolution limit

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est supérieure à ce qu'elle est dans un microscope confocal à balayage par miroirs galvanométriques, dans lequel la faible longueur d'onde d'excitation est mise à profit.

is greater than it is in a confocal scanning microscope with galvanometric mirrors, in which the short excitation wavelength is used.

Another solution for improving the imaging speed is described by American patent number 5, 351, 152 of the company Zeiss. The system described in this patent comprises a fixed network of microlenses which separates the laser beam into sub-beams which are each filtered by a microscopic hole situated in an image plane. The objective focuses in the object the beams coming from each of these microscopic holes. The beam re-emitted by the object is then redirected to a CCD sensor, each pixel of the CCD sensor being the image of a point on the object on which the beam from a corresponding microscopic hole is focused.

Scanning is performed by method (i) of moving the sample relative to the objective, although other methods are not expressly excluded. The point of the sample which is illuminated by one of the sub-beams and whose image is obtained on a corresponding point of the CCD sensor scans a reduced area of the sample. The image of the sample must be reconstructed by computer from a series of images obtained successively on the CCD sensor during the scanning operation of the sample. The scanning speed of this microscope is therefore limited by the reading speed of the CCD sensor, which must be re-read several times to obtain a single image.

Description of the invention
The subject of the invention is a microscope in which the defects indicated above of confocal scanning microscopes with galvanometric mirrors or by Nipkow disc with or without collector are resolved. In particular, the invention allows direct observation of the image and acquisition of the image using a CCD sensor, and simplifies the problems of synchronization and alignment. This is compatible, in the present invention, with point-to-point scanning of the type used by scanning microscopes with galvanometric mirrors. The invention also makes it possible to observe the image in real time, at a speed equal to or greater than that attained by the Nipkow disc microscopes with collector, but without the difficulties of alignment and mechanical production associated with the use of these devices, at a very reduced cost, and with improved resolution by the absence of reflection on the non-transparent parts of the assembly carrying the microscopic holes.

 In a confocal scanning microscope with galvanometric mirrors of the usual type, it is possible to directly observe with an eyepiece the image plane or the beam reflected by the object reaches directly. However, the image thus observed is not a confocal image because it has not been filtered by the microscopic hole, and only the image acquired by sampling the signal passing through the microscopic hole is truly confocal. To obtain a confocal image optically, it is necessary to use a means equivalent to filtering through the microscopic hole. However, if the microscopic hole or equivalent is fixed, the image formed behind this hole does not directly constitute an image of the object: we are brought back to the system described in American patent number 5,239,178 to Zeiss. If the microscopic hole or equivalent is mobile, we are brought back to the Nipkow disc system with or without collector, and to its known defects.

According to a version of the invention, these problems are solved by a confocal scanning microscope comprising:

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 - a beam deflector A, which deflects the light beam that it reaches the observed sample, - a beam deflector B, which deflects the beam coming from the observed sample, characterized in that it comprises a beam deflector C, which deflects, by an angle proportional to the angle of deflection by said deflector B, the beam coming from the observed sample and having been deflected by said beam deflector B.

 Deflection by deflectors A and B is the technique commonly used in confocal laser scanning microscopes. The deflectors can typically be galvanometric mirrors, or acousto-optical deflectors as in the microscope marketed by the company Noran Instruments. The deflection on the deflector A makes it possible to move each illuminated point in the object. The deflection on the deflector B makes it possible to bring back to a fixed point of an image plane PB the wave coming from an illuminated point, so as to be able for example to filter this wave by a microscopic hole. Usually the wave is then detected and the confocal image is reconstructed by computer. The image formed in the plane PB is mobile in the sense that the image of a fixed point of the object moves in this plane. However, to detect this image with a camera or to observe it directly, it would be necessary for it to be fixed. The invention consists in using a deflector C which has the role of moving this image so as to compensate for its movement due to the deflector B, and to make it fixed relative to the sample. The deflectors not being placed in image planes, the deflection essentially results in changes in the direction of the beam at the exit of the deflectors, which themselves cause displacements of the image point in the image plane.

The control of the deflector C to compensate for the action of the deflector B is, in the general case, difficult. The simplest solution is to use movable mirrors or pairs of movable mirrors as deflectors satisfying the following conditions: - each movable mirror of said deflector B is coincident with a corresponding movable mirror of said deflector A, or is integral with a mirror corresponding mobile of said deflector A1, - each mobile mirror of said deflector C is merged with a corresponding mobile mirror of said deflector A, or is integral with a corresponding mobile mirror of said deflector A,
Indeed, in this case, and by means of the realization of a correctly dimensioned optical system, the movement of the mirrors of the deflector C can automatically compensate for that of the mirrors of the deflector B, so that after deflection by B and C the direction of the beam from a fixed point on the object is effectively constant. Compensation therefore does not require any particular precaution in controlling the deflectors. It requires at most an adjustment of the optical systems. The design of an appropriate optical path is facilitated if the mirrors are in afocal zones, or the wave coming from an observed point of the object is plane.

 The microscope preferably comprises at least one microscopic detection hole traversed by said beam coming from the observed sample and having been deflected by deflector B, before this beam is deflected again by said deflector C. This allows, as in a conventional confocal microscope, to filter the wave in a plane where the image of an illuminated point is fixed, that is to say does not move when the illuminated point moves in the observed sample.

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 The microscope preferably comprises a means for focusing at one or more points of an image plane the wave coming from the observed sample and having been deflected by said deflectors B and C. This focusing makes it possible to effectively form an image from the wave whose deviations have been compensated.

 The microscope preferably comprises several microscopic detection holes and means for dividing the light beam of illumination into several sub-beams, each focused at a different point on the observed sample, and each of said microscopic holes is preferably the image of 'a point on the object on which one of said sub-beams is focused. This makes it possible to scan several points in parallel, which improves the speed of the system and makes it comparable to that of a Nipkow disk microscope.

 In the case where the microscope comprises several microscopic holes, the deflector C must preferably compensate exactly for the action of the deflector B so that the direction of a beam coming from a fixed point of the object is, after deflection by said deflectors B and C, independent of the direction of this beam after passage of said deflector B. Indeed, in the contrary case, a point of the image plane would be likely to be illuminated successively by beams coming from different points of the object and crossing different microscopic holes.

 The acquisition of the confocal images on a camera involves, in the case where the excitation wavelength is notably lower than the wavelength of fluorescent emission, a loss of resolution compared to the acquisition on a sensor punctual. This is the case for example with the Nipkow confocal disc microscope, whose resolution is lower than that of the confocal laser scanning microscope. This is due to the fact that the image of a microscopic hole on the CCD camera, when this image has moved a length equal to half the width of the spot of Airy, covers the same image fairly widely before moving. According to a version of the invention, it is possible to solve this problem by reducing the size of the microscopic holes so as to attenuate the phenomenon of overlap, however this solution causes a reduction in the useful light intensity. In order to avoid this loss of light intensity, and according to a version of the invention, each of said microscopic holes is associated with a converging lens placed before this hole on the path of the wave coming from the object observed, and focusing on this hole the beam coming from a corresponding point of the object. This lens makes it possible to reduce the width of the Airy spot and therefore to use a hole of smaller diameter without loss of intensity.

 When laser lighting is used, the microscope preferably includes means for varying the phase of each sub-beam. Indeed, in this case, the waves constituting each sub-beam are correlated with each other, which is liable to create interference in the object which can disturb the image. The correlation between sub-beams must therefore preferably be broken, which can be done by independently modifying, over time, the phase of each sub-beam. The means for varying the phase of each sub-beam is for example a transparent pane carrying extra thicknesses and rotating around an axis.

Quick description of the figures.

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 Figure 1 shows the preferred embodiment. Figure 2 shows a second embodiment. FIG. 3 represents the image on a sensor of a network of microscopic holes used in the preferred embodiment, and the trajectory of a point of this image. FIGS. 4 to 6 illustrate the command applied to a beam attenuator as a function of the position and the speed of movement of the image on the camera of a microscopic hole. FIG. 7 represents a cube integrating two networks of microscopic holes and a dichroic mirror, simplifying the second embodiment. FIG. 8 represents the image on a sensor of a network of microscopic holes used in the second embodiment, and the trajectory of a point on this image, a single galvanometric mirror being used. FIG. 9 represents an array of lenses and an array of microscopic holes used for lighting the sample. FIG. 10 represents an array of lenses and an array of microscopic holes used to detect the wave coming from the sample. Figure 11 shows a side view of three mirrors also visible in Figure 1. Figure 12 shows a third embodiment. Figure 13 shows a fourth embodiment.

Preferred embodiment.

 This embodiment is shown in Figure 1. The beam from a laser 100 passes through a beam attenuator 140 which can for example be electro-optical or acousto-optical. It then passes through a beam expander comprising for example the lenses 101 and 102. The beam then passes through a network of microlenses 103. The sub-beam from one of these microlenses has been shown in solid lines and the sub-beam in dotted lines. from another microlens. The beam from the microlens array passes through a window 106 movable in rotation about an axis 107, then an optional lens 135 which may not be used, then an array of microscopic holes 105. The array of microscopic holes 105 can consist of by depositing a reflective layer on a transparent pane by a "biographical" type method, the holes then being interruptions of the reflective layer. In this case a neutral filter placed at the laser output can be used to attenuate the effects of return of the laser light. The network of microscopic holes can also consist of a frosted metal plate in which the holes are drilled by means of a laser. This solution avoids the problems of return of the laser beam. The part of the beam which passes through a microlens of the grating 103 constitutes a sub-beam focused on a microscopic hole of the grating 105. The surface of the glass 106 is divided into a set of sub-surfaces, for example the sub-surfaces 120 and 121 Half of these sub-surfaces carry an extra thickness generating a phase shift of 180 degrees from the laser beam passing through them. The sub-surfaces carrying extra thicknesses are distributed in a pseudo-random manner throughout the sub-surfaces. Each subsurface is approximately square. The width of the side of the square is equal to the distance between two adjacent microscopic holes of the array 105. The window 106 is positioned so that each microscopic hole of the array 105 is placed below a separate subsurface. The rapid rotation of the window 106 makes it possible to generate pseudo-random phase shifts of all of the sub-beams, so that the spatial coherence of the beam is broken after crossing the glass 106. In fact, the coherence between beams is likely to slightly disturb the image. The window 106 is however of little use if the

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 microscopic holes in array 105 are sufficiently spaced, in which case it can be removed. The beam having passed through the window 106 and the network of microscopic holes 105 then passes through the dichroic mirror 104 and the tube lens 108 and is then reflected by a mirror 109. It is then reflected by a galvanometric mirror 110 which is mobile in rotation around a axis located in the plane of the figure, by a mirror 111, and by a galvanometric mirror 112 movable in rotation about an axis orthogonal to the plane of the figure. It crosses the microscope objective 113 and reaches the sample 114. The objective 113 is an objective forming an image of the observed sample ad infinitum. The network of microscopic holes 105 is in the image plane (plane where a stigmatic image of the object observed is formed). The array of microscopic holes 105 is not absolutely essential. It is useful especially to make the beam "cleaner" in the case where the microlens array 103 is of average quality. If the microlens array 103 is of very good quality, better image quality can be obtained by eliminating the array of microscopic holes 105.

 Preferably, the image focal plane of the objective 113 is in the object focal plane (in the objective direction towards lens 108) of the lens 108 and the array of holes 105 is in a focal plane of the lens 108. This makes it possible to maximize the useful opening of each sub-beam. In this case the lens 135 is not useful.

d'observation suivant laquelle est réalisée la figure 1. Le faisceau traverse ensuite la lentille 116 identique à la lentille de tube 108, et dont le plan focal objet est sur le réseau de trous microscopiques 130. Il est réfléchi par la seconde face du miroir galvanométrique 110, par le miroir 117, par la seconde face du miroir galvanométrique 112. Il traverse le filtre monochromateur 14 1. Il traverse ensuite la lentille 118 qui forme dans son plan focal image l'image du réseau 105, et donc de l'échantillon observé. Il parvient au plan focal image de la lentille 118. Un capteur CCD 119 peut être placé dans ce plan, toutefois il est également possible d'observer directement l'image formée dans ce plan. à l'aide d'un oculaire. La distance focale de la lentille 116 doit être exactement égale à la distance focale de la lentille 108, et pour permettre un ajustement The illuminated sample in return emits a non-coherent light beam. This beam passes through the objective 113, is reflected by the galvanometric mirror 112, the mirror 111, the galvanometric mirror 110, the mirror 109. It passes through the tube lens 108, is reflected by the dichroic mirror 104, then passes through the array of microlenses 131 and the array of microscopic holes 130. The pitch (distance between the centers of two adjacent holes) of the array of microscopic holes 130 is identical to the pitch of the array 105. The diameter of a microscopic hole of the array 130 may be equal to or smaller than the diameter of a microscopic hole in the array 105. Each microscopic hole in the array 130 corresponds to a microlens of the microlens array 131. The arrays 131 and 130 are positioned so that each hole in the array 130 is the image of a point of the object on which the lighting beam from a hole in the array 105 is focused. This means that each microscopic hole in the array 130 is in an image plane, by a microlent the corresponding eye, from the focal plane of the tube lens 108. The beam is then reflected by the mirror 115. It is then successively reflected by the mirrors 143, 144, 145, 146, 147 constituting the assembly 142 represented by a block in FIG. 1, which are shown in FIG. 11 in a view in the direction V indicated in FIG. 1. The assembly 142 serves to invert the angle of the beam with respect to a plane containing the optical axis and situated in the plane of FIG. . The direction P shown in Figure 11 shows the direction

observation according to which FIG. 1 is carried out. The beam then passes through the lens 116 identical to the tube lens 108, and whose object focal plane is on the network of microscopic holes 130. It is reflected by the second face of the mirror galvanometric 110, by the mirror 117, by the second face of the galvanometric mirror 112. It crosses the monochromator filter 14 1. It then crosses the lens 118 which forms in its image focal plane the image of the network 105, and therefore of the observed sample. It reaches the focal image plane of the lens 118. A CCD sensor 119 can be placed in this plane, however it is also possible to directly observe the image formed in this plane. using an eyepiece. The focal length of lens 116 must be exactly equal to the focal distance of lens 108, and to allow adjustment

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précis la lentille 116 peut être remplaçée par un doublet de lentilles, le réglage de la distance interlentilles permettant un ajustement de la distance focale de l'ensemble.

precise the lens 116 can be replaced by a pair of lenses, the adjustment of the distance between lenses allowing an adjustment of the focal distance of the assembly.

F, a = ouv. La largeur d'un trou microscopique du réseau 105 est par exemple égale à la largeur de la Flog  F tache d'Airy soit L105 = 1, 22ou F ; O8 est la distance focale de la lentille 108,/'. est la ouv FI13

distance focale de l'objectif 113, OMV est l'ouverture numérique de l'objectif 113, Â 1., est la longueur d'onde du laser. La distance D entre deux trous microscopiques adjacents est de préférence au moins 10 fois la largeur de chaque trou. Les microlentilles du réseau 103 sont des microlentilles sphériques limitées par des carrés et adjacentes les unes aux autres. La largeur de chaque microlentille (coté du carré la limitant ou distance entre les centres de deux microlentilles adjacentes) est égale à la distance D entre deux trous microscopiques adjacents. La distance focale 3 de chaque microlentille du réseau de microlentilles 103

D F DF, 1 et sa largeur D sont en outre liés par la relation ---== G ; = ûMV- -, soit F103 = ----- 2F1O3 F108 2 Fizz

Par exemple on peut avoir :

FIG. 9 shows the principle of the focusing of the laser beam on the array of microscopic holes 105, by the array of microlenses 103. The gold angle indicated in this figure is worth

F, a = open. The width of a microscopic hole of the network 105 is for example equal to the width of the Flog  F spot of Airy is L105 = 1, 22or F; O8 is the focal length of the lens 108, / '. is the open FI13

focal length of objective 113, OMV is the numerical aperture of objective 113, Â 1., is the wavelength of the laser. The distance D between two adjacent microscopic holes is preferably at least 10 times the width of each hole. The microlenses of the array 103 are spherical microlenses bounded by squares and adjacent to each other. The width of each microlens (side of the limiting square or distance between the centers of two adjacent microlenses) is equal to the distance D between two adjacent microscopic holes. The focal length 3 of each microlens of the microlens array 103

DF DF, 1 and its width D are also linked by the relation --- == G; = ûMV- -, i.e. F103 = ----- 2F1O3 F108 2 Fizz

For example we can have:

ils = 488 nm (laser argon)

they = 488 nm (argon laser)

Si la largeur des trous microscopiques du réseau 105 est égale au diamètre de la tache d'Airy,

 fi soit 1, 22 las 108, il n'y a pas de perte d'énergie mais le faisceau n'est pas gaussien, ce qui se traduit par ouv FI 13

une diminution de la résolution. Un faisceau approximativement gaussien peut être obtenu en diminuant le

 F : diamètre du trou microscopique, par exemple à 1, 22-----. Toutefois ceci entraîne une diminution 2ouv Fi13

de l'intensité utile. La meilleure solution pour obtenir un faisceau gaussien sans perdre inutilement de l'intensité utile est de ne pas utiliser le réseau de trous microscopiques, dont le rôle est surtout de pallier aux imperfections du réseau de microlentilles. Dans ce cas le faisceau est parfaitement gaussien, ce qui assure une résolution maximale. Cette solution nécessite toutefois des microlentilles de bonne qualité.

If the width of the microscopic holes of the network 105 is equal to the diameter of the Airy spot,

 fi is 1, 22 las 108, there is no loss of energy but the beam is not Gaussian, which results in Ouv FI 13

a decrease in resolution. An approximately Gaussian beam can be obtained by decreasing the

 F: diameter of the microscopic hole, for example at 1.22 -----. However, this leads to a decrease 2ouv Fi13

useful intensity. The best solution to obtain a Gaussian beam without unnecessarily losing useful intensity is not to use the network of microscopic holes, whose role is above all to compensate for the imperfections of the microlens network. In this case the beam is perfectly Gaussian, which ensures maximum resolution. This solution however requires good quality microlenses.

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jTI n 9, a = ouv F-13. On a représenté le plan focal 132 de la lentille 108. En l'abscence du réseau de F108

microlentilles 131, le réseau de trous microscopiques 130 devrait être plaçé dans ce plan, et l'angle de divergence du faisceau après passage des trous microscopiques, dans la mesure ou ces trous sont suffisamment larges, serait a. Le réseau de lentilles 131 a pour utilité d'augmenter cet angle de divergence, ce qui équivaut à diminuer le diamètre des tache d'Airy formées sur chaque trou du réseau de trous microscopiques 130. Cect permet de diminuer le diamètre des trous microscopiques du réseau 130 sans perte d'intensité lumineuse, ce qui permet ensuite une réception sur un capteur CCD sans que la résolution soit diminuée par la superposition des taches d'Airy obtenues pour des points proches. L'angle de divergence du faisceau après passage des trous microscopiques devient, compte tenu des microlentilles du réseau 131, ss = ma avec typiquement m = 1, 5 à m = 4. Les distances d2 entre le réseau de microlentilles 131 et le réseau de trous microscopiques 130, dl entre le réseau de microlentilles 131 et le plan focal 132 de la lentille 108, et la distance focale F13 1 des microlentilles du réseau 131, sont liées par l'équation des lentilles minces (qui peut éventuellement être modifiée pour prendre en compte l'épaisseur des lentilles) :

FIG. 10 shows the operating principle of the microlenses of the array 131 and of the microscopic holes of the array 130. The angle indicated in this figure is worth, as in the figure

jTI n 9, a = open F-13. The focal plane 132 of the lens 108 has been shown. In the absence of the network of F108

microlenses 131, the array of microscopic holes 130 should be placed in this plane, and the angle of divergence of the beam after passage of the microscopic holes, insofar as these holes are sufficiently wide, would be a. The lens array 131 has the utility of increasing this angle of divergence, which is equivalent to reducing the diameter of the Airy spots formed on each hole of the array of microscopic holes 130. Cect makes it possible to decrease the diameter of the microscopic holes of the array 130 without loss of light intensity, which then allows reception on a CCD sensor without the resolution being reduced by the superposition of the Airy spots obtained for close points. The beam divergence angle after passage of the microscopic holes becomes, taking into account the microlenses of the array 131, ss = ma with typically m = 1.5 to m = 4. The distances d2 between the array of microlenses 131 and the array of microscopic holes 130, dl between the array of microlenses 131 and the focal plane 132 of lens 108, and the focal distance F13 1 of the microlenses of array 131, are linked by the equation of thin lenses (which can optionally be modified to take the thickness of the lenses):

et on a par ailleurs, compte tenu du coefficient m recherché :

and we also have, taking into account the coefficient m sought:

De plus pour des raisons d'encombrement on a :

In addition, for reasons of space, we have:

avec c 1. Typiquement on peut prendre c = 2.

with c 1. Typically we can take c = 2.

F; Taking into account also the expression of the angle a = open O1V, we finally obtain: F108

La largeur des trous microscopiques sur le réseau 130 peut être égale au diamètre de la tache d'Airy sur ces trous soit :

The width of the microscopic holes on the network 130 can be equal to the diameter of the Airy spot on these holes, ie:

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L130 Âfluo F, 08 m ouv F113 m ouv FI 13

ou À flua est la longueru d'onde correspondant au maximum de l'émission fluorescente.

L130 Âfluo F, 08 m open F113 m open FI 13

or À flua is the wavelength corresponding to the maximum of the fluorescent emission.

c = 2 et m = 4, et pour o = 518 nm (fluorescéine) :

Using the example of dimensioning used above for networks 103 and 105, we obtain, for

c = 2 and m = 4, and for o = 518 nm (fluorescein):

On peut améliorer la résolution en utilisant des trous microscopiques dont le diamètre est la moitié du diamètre de la tache d'Airy, ou encore inférieur, soit :

The resolution can be improved by using microscopic holes the diameter of which is half the diameter of the Airy spot, or even less, that is:

Pour une résolution maximale, avec des trous microscopiques dont le diamètre est suffisamment inférieur à la tache d'Airy, et pour permettre dans des conditions optimales une déconvolution ultérieure de l'image confocale le pas Pu 19 d'échantillonnage sur la capteur CCD (distance entre les centres de deux pixels adjacents) doit vérifier :

For maximum resolution, with microscopic holes whose diameter is sufficiently smaller than the Airy spot, and to allow under optimal conditions a subsequent deconvolution of the confocal image the sampling step Pu 19 on the CCD sensor (distance between the centers of two adjacent pixels) must check:

Âlas + Âfluo soit i8 = 4oPn9Fn3----'-Alasifluo Par exemple avec Pu 19 = 12 um et toujours dans le même exemple de dimensionnement on obtient : F, = 477 mm

Pour une résolution maximale mais sans déconvolution ultérieure de l'image confocale on peut se contenter

de : F1 l 8 =ouvXl l gFl l 3 fl soit Fl l8 = 168 mm , l -+j7Mo.luo ii8 =- ;-c'i9Fn3---,---soit Fus = 168 mm 2 2

Âlas + Âfluo ie i8 = 4oPn9Fn3 ----'- Alasifluo For example with Pu 19 = 12 um and always in the same design example we get: F, = 477 mm

For a maximum resolution but without subsequent deconvolution of the confocal image, we can be satisfied

of: F1 l 8 = ouvXl l gFl l 3 fl either Fl l8 = 168 mm, l - + j7Mo.luo ii8 = -; -c'i9Fn3 ---, --- or Fus = 168 mm 2 2

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Avec des trous microscopiques dont le diamètre est d'une tache d'airy on peut se contenter de :

With microscopic holes the diameter of which is an airy spot, we can be satisfied with:

Il est donc être utile de pouvoir modifier la distance focale de la lentille 118, ou de la remplacer par un système avec zoom ou par un système de grossissement variable avec des éléments optiques interchangeables.

It is therefore useful to be able to modify the focal distance of the lens 118, or to replace it by a system with zoom or by a system of variable magnification with interchangeable optical elements.

 The array of microlenses 131 is especially useful when the excitation wavelength is much less than the wavelength of the fluorescent emission and when the diameter of the microscopic holes is of the order of an Airy spot . If the diameter of the microscopic holes is much less than an Airy spot or if the excitation wavelength is close to the wavelength of the fluorescent emission, the array of microlenses 131 can be eliminated without great consequences . In this case, the array of microscopic holes 130 is placed directly in the image focal plane of the lens 108.

 A practical difficulty is linked to the fact that lenses of different magnifications have distinct image focal planes. Consequently, the object focal plane of the lens 108 (in the direction of the objective towards the lens 108) can only be confused with the image focal plane of the objective for one of the objectives used.

It is possible to overcome this difficulty by increasing the opening of the sub-beams coming from each microlens, which decreases the available light intensity. The lens 135 overcomes this difficulty without loss of light intensity. It must be dimensioned so that a plane wave in the object has for image a plane wave between the lens 135 and the microlens array 103.

le capteur 119, pour une position de référence des miroirs galvanométriques. Le trait 301 superposé au dessin montre la trajectoire suivie par l'image d'un point 300 du réseau lorsque les miroirs galvanométriques sont commandés. Cette trajectoire est parcourue alternativement dans les deux sens. Lorsque cette trajectoire est suivie, l'image d'un trou du réseau 130 balaye une petite partie du plan image, et l'ensemble des images des trous du réseau balaye l'ensemble du plan image. Une image confocale est donc générée dans l'ensemble du plan image. Le contour 302 montre la limite de la zone utile, dans laquelle une image confocale de bonne qualité est générée. De nombreuses variantes de la trajectoire parcourue peuvent être utilisées, la contrainte essentielle étant que l'ensemble des points images des trous du réseau 130 balaye l'ensemble du plan image. The galvanometric mirrors 110 and 112 are controlled so as to move the image of the array 130 on the sensor 119, as shown in Figure 4. Figure 4 shows the image of the array 130 on

the sensor 119, for a reference position of the galvanometric mirrors. The line 301 superimposed on the drawing shows the path followed by the image of a point 300 of the network when the galvanometric mirrors are ordered. This trajectory is traveled alternately in both directions. When this trajectory is followed, the image of a hole in the network 130 scans a small part of the image plane, and the set of images of the holes in the network scans the whole of the image plane. A confocal image is therefore generated throughout the image plane. Contour 302 shows the limit of the useful area, in which a good quality confocal image is generated. Numerous variants of the trajectory covered can be used, the essential constraint being that all of the image points of the holes in the network 130 scan the entire image plane.

 The beam attenuator 140 must be controlled according to the position of the galvanometric mirrors and their speed. These parameters can be obtained in a known manner by feedback from the galvanometers, or can be obtained without feedback from the galvanometer control system, with less precision. We note Ilas the intensity of the laser beam after crossing the attenuator, and Vscan the scanning speed, that is to say the speed of movement in the image plane 119 and along the x axis of the image point d ' a microscopic hole in the array 130. This scanning speed is due only to the fastest galvanometric mirror. The position of the mirrors

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galvanométriques peut être caractérisée par la position du point image dans le plan image 119. Les figures 5

Ilas et 6 illustrent la valeur du rapport-en fonction de la position du point image d'un trou Vscan

microscopique sur l'échantillon. La figure 5 représente un ensemble de points images des trous microscopiques, dont le point 300 également représenté sur la figure 4, pour la position de référence des miroirs galvanométriques. Sur cette figure on a représenté en traits continus les arêtes de la surface

/las représentant 1a fonction - en fonction des coordonnées x, y du point 300 dans le plan image 119.

galvanometric measurements can be characterized by the position of the image point in the image plane 119. Figures 5

Ilas et 6 illustrate the value of the ratio-as a function of the position of the image point of a Vscan hole

microscopic on the sample. FIG. 5 represents a set of image points of the microscopic holes, of which the point 300 also represented in FIG. 4, for the reference position of the galvanometric mirrors. In this figure we have shown in solid lines the edges of the surface

/ las representing the function - as a function of the x, y coordinates of the point 300 in the image plane 119.

Sur la figure 6, on a représenté l'allure de cette fonction le long de la ligne 310 de la figure 5. Une valeur

constante de sur l'ensemble de la trajectoire, c'est-à-dire une commande de l'atténuateur en fonction Vscan

de la seule vitesse de balayage, permettrait en principe de supprimer les variations d'éclairage dûes aux variations de la vitesse de balayage. Toutefois, la courbe de la figure 6 permet également d'atténuer les effets d'une variation incontrôlée de l'amplitude d'oscillation des miroirs, au prix toutefois d'une perte d'intensité lumineuse. Il est également possible de faire fonctionner l'atténuateur de faisceau en mode binaire. La commande de l'intensité se fait alors conformément à la figure 7. Seule la partie centrale de la trajectoire, sur laquelle la vitesse est à peu près constante, est utilisée. Enfin, il est également possible de ne pas utiliser d'atténuateur de faisceau. L'image reste de qualité acceptable mais peut être affectée par des variations locales de l'intensité, qui peuvent être compensées par un traitement numérique ultérieur. D'une manière générale, plus le nombre de trous microscopiques est élevé et plus la surface de la trajectoire de l'image d'un point du réseau 130 dans le plan image 119 est élevée, moins les variations d'intensité d'éclairage sont notables. L'utilisation d'un réseau suffisamment dense de trous microscopiques peut donc remplacer avantageusement l'usage d'un atténuateur de faisceau. Vscan

In Figure 6, the shape of this function is shown along the line 310 in Figure 5. A value

constant of over the entire trajectory, i.e. a control of the attenuator as a function Vscan

scanning speed alone would, in principle, eliminate variations in lighting due to changes in scanning speed. However, the curve in FIG. 6 also makes it possible to attenuate the effects of an uncontrolled variation of the amplitude of oscillation of the mirrors, at the cost, however, of a loss of light intensity. It is also possible to operate the beam attenuator in binary mode. The intensity is then controlled in accordance with FIG. 7. Only the central part of the trajectory, over which the speed is approximately constant, is used. Finally, it is also possible not to use a beam attenuator. The image remains of acceptable quality but may be affected by local variations in intensity, which can be compensated for by subsequent digital processing. In general, the higher the number of microscopic holes and the greater the surface of the image trajectory of a point of the network 130 in the image plane 119, the less the variations in lighting intensity are notables. The use of a sufficiently dense network of microscopic holes can therefore advantageously replace the use of a beam attenuator.

 Each objective corresponds to an optimal dimensioning of the microscopic holes used, of the microlenses and of the lens 135. These elements can be designed so that they suit all the objectives, however the properties of the system will be sub-optimal with certain goals. It is also possible to design a series of studied objectives to give the best results with a given dimensioning of the rest of the system. However, it is preferable to be able to exchange the microscopic holes used, the microlenses and the lens 135, so as to be able to optimize them according to the objectives used, the excitation and fluorescence wavelengths, the level of fluorescence, the desired acquisition speed, the desired resolution. To this end, the arrays of microlenses 103 and 131, the arrays of microscopic holes 105 and 130, the dichroic mirror 104, and the lens 135, can be made integral with one another and constitute an exchangeable block in one piece. This saves the user from having to adjust the alignment of the different elements: alignment problems are solved when the block is manufactured. The positioning of the block in the rest of the system must simply be done with sufficient angular precision (of the order of 1 miliradian).

 The present embodiment can be combined with all the imaging modes known in confocal microscopy. In particular, the object-holder can be provided with a vertical displacement system

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 piezoelectric or by stepper motor, so as to be able to generate three-dimensional images by modifying the focusing plane. Systems based on acoustic-optical attenuators can be used to switch several lasers and excite different fluorophores, so as to generate images rich in information by superimposition. The system is also compatible with the use of the two-photon method, the number of microscopic holes then having to be adjusted so that sufficient intensity remains available at each focal point of the beam. Filter wheels (filter 141) can be used to modify the detected wavelengths. The dichroic mirror 104 can be replaced by a semi-transparent mirror so as to be able to change excitation and fluorescence wavelengths by switching a laser and changing the filter 141, without having to exchange the dichroic mirror 104. replacing the dichroic mirror 104 with a semi-transparent mirror also makes it possible to use the apparatus for observing in reflection non-fluorescent diffractive samples.

 It is of course possible to use only one microscopic hole, in which case the speed characteristics are those of a confocal scanning microscope by galvanometric mirrors of the current type. However, the advantage of the system remains to be able to view the image directly and to be able to record it on a camera. Embodiments 3 and 4 illustrate in more detail embodiments with a single microscopic hole or equivalent.

Second embodiment:
The second embodiment differs from the first in that non-coherent lighting is used.

It is shown in Figure 2. The lighting is provided for example by the glowing arc 150 of a mercury vapor lamp. It passes through a collector 151, a field diaphragm 152, a monochromator filter 153 selecting the excitation wavelength of the fluorescence. It passes through the lens 154, the aperture diaphragm 155, the lens 156, and reaches the array of microscopic holes 105 directly.

The assembly made up of elements 150 to 156 constitutes Köhler lighting and can be replaced by any other Köhler lighting system. The rest of the system is identical to the previous embodiment.

 A large part of the available light intensity is reflected by the array of microscopic holes 105, which limits the clarity of the image formed in the plane 119. So that the acquisition time and the clarity of the image remain within within reasonable limits, it is preferable to use an array 105 consisting of microscopic holes very close to each other. While in the previous embodiment, the distance between two microscopic holes can typically be about 20 times the width of each hole, in the present embodiment it is preferable to limit this distance, which can for example be 2 at 4 times the width of the microscopic hole. This generates disturbances at low spatial frequency on the image, but the high spatial frequencies, which are the most information carriers, continue to benefit from the confocal effect.

 To allow the exchange of the two networks of microscopic holes without this exchange requiring complex adjustment, and to reduce the manufacturing cost, the network of lenses 131 can be eliminated and the networks of microscopic holes 105,130 and the dichroic mirror 104 can be integrated into a single easily achievable element, represented in FIG. 7. The dichroic mirror 400 is integrated into a cube

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 transparent 401. Windows 402, 403 are mounted on the sides of the cube, of which they are secured by suitable parts, for example 404. Empty spaces 405,406 are left between the windows and the cube 401. The side of the windows 402,404 which looks towards the cube is covered with a thin metallic layer and a thin layer of photosensitive resin. The microscopic holes are made by exposure to the photosensitive resin using a white light projector. The appearance of the light beam from this projector has been symbolically represented in dotted lines. The projector is focused for example on the plane formed by the face of the window 402 which carries the thin layers. Each illuminated point of the window 402 will become a microscopic hole. Due to the configuration of the system, the points of the glass 403 which are illuminated are correctly placed without any additional adjustment being necessary, and will become the microscopic holes corresponding to the holes in the glass 402. After exposure, an appropriate liquid is introduced in the empty spaces 405.406 so as to remove the resin at the exposed locations, then an acid is used to remove the metal at these same points. The microscopic holes were then obtained. A solvent can be used to remove the residual layer of resin, and the assembly can finally be cleaned. Finally, it may be useful to introduce an optical liquid or a transparent plastic into the empty space, preferably with the same index as the blades 402, 403. This avoids unnecessary reflections on the contact surfaces. If a material of the same index as the blades 402,403 is introduced into the empty spaces, then it is possible to use to make all the panes 402,403 frosted on their face which looks towards the cube. This allows a better dispersion of the incident light by the reflecting parts. Monochromators 153 and 141 can also be integrated into this cube, which makes it possible to modify the imaging mode by exchanging a single component.

 In this configuration, it is also possible to delete one of the galvanometric mirrors 110 or 112, provided that the axis of rotation of the remaining mirror is slightly modified. FIG. 8, produced according to the same conventions as FIG. 3, shows the trajectory 500 of the image of a microscopic hole in the plane 119, as well as the area 502 in which a good quality image is obtained, the position of the images of the microscopic holes in the reference position being as indicated in the figure, limited by the contour 501. Because the microscopic holes are numerous and close to each other, a slightly oblique trajectory makes it possible to scan the whole of the plan without using a second galvanometric mirror.

 This second embodiment has the advantage of being less expensive than the first insofar as it does not require a laser beam. It also has the advantage of allowing easy modification of the excitation and emission wavelengths of fluorescence throughout the visible and UV range. On the other hand, the weak lighting hardly allows real-time imaging.

 This second embodiment can be combined with the first in the same device, shutters that can be used to switch from one lighting mode to another.

Third embodiment:
FIG. 12 shows a confocal laser scanning fluorescence microscope according to a third embodiment of the invention. A beam from a laser 1100 passes through a beam expander or

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collimator formed of the lenses 1101, 1102 then is reflected by the dichroic mirror 1103 which reflects the wavelength of the laser 1100 and lets pass the wavelength re-emitted by fluorescence. The beam is then reflected on the galvanometric mirror 1104 mobile in rotation about an axis located in the plane of the figure, then on the galvanometric mirror 1105 mobile in rotation around an axis orthogonal to the plane of the figure. It then crosses the polarizing beam splitter 1204 and then the microscope objective 1106. The beam re-emitted by fluorescence from this point is collected by the objective 1106, crosses the polarizing beam separator
1204, is reflected successively by the two galvanometric mirrors 1105 and 1104, passes through the dichroic mirror 1103, and is focused by the lens 1108 on a reflecting point 1203 situated on the rear face of a quarter-wave plate 1202. The part of the beam passing through the quarter-wave plate then reaches an absorbent cavity. The reflecting point can for example be produced by optical litography.

 The part of the beam which is reflected by the point 1203 crosses the quarter-wave plate 1202, the lens 1108, the dichroic mirror 1103, is reflected by the galvanometric mirrors 1104,1105, is reflected by the polarizing beam splitter 1204, then is focused by the lens 1205 in the plane of a CCD sensor 1206 fixed on the camera 1207.

The polarization of the laser 1100 is chosen so that the wave coming from this laser crosses the beam splitter 1204. The quarter-wave plate 1202 has its neutral axis oriented at 45 degrees from the axis passing through the polarizer
1201 and is quarter wave for the wave re-emitted by fluorescence. Its role is to rotate the direction of polarization by 90 degrees, so that only the wave reflected by the reflecting point is then reflected by the polarizing beam splitter 1204, excluding the wave resulting from parasitic reflections on The lens
1204.

 Under these conditions, the confocal image of the object is formed directly on the CCD sensor 1206 when the object is scanned by means of the galvanometric mirrors. An imperfect control of the galvanometric mirrors results at worst in dark areas on the image, but in no case by a displacement of the points of the object or any geometric imprecision. The object must be scanned during the integration time of the sensor. The images can then be transferred from the CCD sensor to a sampler and a computer.

 The sensor can also be replaced by an eyepiece, possibly a binocular assembly, allowing direct observation of the image formed in the plane or on the diagram the CCD 1206.

In this case, the scanning must be done quickly enough not to be noticeable by the eye.

 This embodiment can be adapted to a multibeam system of the type used in the first embodiment. Its main advantage is that it does not require any precise adjustment apart from the matching of the laser focal point with the reflecting point. Its major drawback is the loss of light intensity which results from the use of semi-transparent or polarizing mirrors. to allow the use of a single side of the mirrors for all of the optical paths.

 You can replace the quarter-wave plate 1202 with a transparent window and replace the polarizing beam splitter 1204 with a non-polarizing beam splitter. The system remains perfectly functional under these conditions, the noise due to parasitic reflections being simply a little higher. We

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 can then also replace one of the galvanometric mirrors with an acousto-optic deflector, which makes it possible to speed up the imaging procedure.

Fourth embodiment:
This embodiment is shown in FIG. 13, and differs from the previous embodiment in that each galvanometric mirror has two usable faces, which are actually used.

 The beam from the laser 1300 passes through the beam expander or collimator formed by the lenses 1301,1302 then is reflected by the dichroic mirror 1303. It is then reflected by the galvanometric mirror 1304 which is mobile in rotation about an axis located in the plane of the figure, then by the fixed mirror 1305 and by the galvanometric mirror 1306 movable in rotation about an axis orthogonal to the plane of the figure. It then crosses the objective 1307 infinitely forming an image of the sample, and is focused at a point of the observed sample 1308. The light re-emitted by fluorescence from this point crosses the objective 1307 in the opposite direction, is reflected by the galvanometric mirror 1306, the fixed mirror 1305, and the galvanometric mirror 1304.

It passes through the dichroic mirror 1303, and is focused by the lens 1309 and the fixed mirror 1310 on the microscopic hole 1311. The light having passed through the microscopic hole 1310 is reflected by the mirror 1312, collimated by the lens 1313, reflected by the mirror 1314, then by the second face of the galvanometric mirror 1304. It is then reflected by the mirror 1315 then by the second face of the galvanometric mirror 1306. It is then focused by the lens 1316 on a point of the image plane in which the sensor is located CCD 1317 attached to camera 1318. As before, the CCD sensor can be replaced by an eyepiece.

 The operation of the assembly is the same as in the first embodiment.

Industrial applications
The present microscope can be used for real-time imaging of biological objects.

Claims (9)

Claims (1/2)  1-confocal scanning microscope, comprising: - a beam deflector A, which deflects the light beam as it reaches the observed sample, - a beam deflector B, which deflects the beam coming from the observed sample , characterized in that it comprises a beam deflector C, which deflects by an angle proportional to the angle of deflection by said deflector B, the beam coming from the observed sample and having been deflected by said beam deflector B. 2-microscope according to claim 1, characterized in that: - said beam deflectors are mobile mirrors or pairs of mobile mirrors, - each mobile mirror of said deflector B is merged with a corresponding mobile mirror of said deflector A, or is secured to a corresponding movable mirror of said deflector A1, - each movable mirror of said deflector C is merged with a corresponding movable mirror of said deflector A, or is secured to a corresponding movable mirror of said deflector A, 3-microscope according to claim 2 , characterized in that - said movable mirrors of said deflector A are each merged with a corresponding movable mirror of said deflector B, - said movable mirrors of deflector C are each made on the opposite face of a corresponding movable mirror of said deflector A,  4-microscope according to one of claims 1 to 3, characterized in that said beam deflectors are placed in afocal planes. 5-microscope according to one of claims 1 to 4, characterized in that it comprises several microscopic holes crossed by said beam coming from the observed sample and having been deflected by said deflector B. 6-microscope according to one of claims 1 to 5, characterized in that it comprises means for focusing at one or more points of an image plane the wave coming from corresponding points of the observed sample and having been deflected by said deflectors B and C. <Desc / Clms Page number 18>

 Claims (2/2)

 7-microscope according to one of claims 5 or 6, characterized in that it comprises a means for dividing the light beam of illumination into several sub-beams each focused at a different point of the observed sample, and by the fact that each of said microscopic holes is the image of a point on the object on which one of said sub-beams is focused. 8-microscope according to one of claims 1 to 7, characterized in that said deflector C deflects the beam coming from a given point of the object so that the direction of this beam, after deflection by deflectors B and C , or independent of the position of said deflectors B and C. 9-microscope according to one of claims 1 to 8 and according to claim 7, characterized in that it comprises means for varying the phase of each of said sub-beam. 10-microscope according to claim 9, characterized in that said means for varying the phase of each of said sub-beams is a transparent pane carrying extra thicknesses and rotating about an axis.