Abstract:
The invention concerns a modifiable assembly of microscopic apertures comprising several plates ( 100, 110 ) that are opaque except on transparent parts ( 101, 114, 115 ), capable of moving relative to one another, to modify the size of resulting pinholes. The invention is applicable for microscopic apertures for confocal microscopy.

Description:
RELATED APPLICATIONS 
   This application is a continuation of U.S. application Ser. No. 10/474,269 filed Jul. 21, 2004, now U.S. Pat. No. 7,088,487, which was the National Stage of International Application No PCT/FR02/01222 filed Apr. 9, 2002. The entire contents of both these applications is expressly incorporated herewith by reference thereto. 

   FIELD OF THE INVENTION 
   The invention concerns a microscopic hole or a set of microscopic holes (pinholes), the number of these holes and/or their size being able to be modified easily. Such a set of pinholes is intended to be used for various applications in optics, in particular in confocal microscopy. 
   BACKGROUND 
   In confocal microscopy use is usually made of two types of pinhole:
         holes of fixed size: to modify the size of a hole, it is necessary to replace it with another. Typically several pinholes can be mounted on a wheel having a position corresponding to the use of each of these holes. The movement of the wheel must be very precise.   holes of variable size: functioning on the principle of the iris diaphragm, they require at least three blades which form a hole by crossing one another and are expensive because of the relative complexity of the mechanism.       

   Conventionally, confocal microscopy systems require the use of a single pinhole. For example, the first embodiment of French patent application number 0103860 of 22 Mar. 2001, as well as the microscope described in FIG. 3 of the U.S. Pat. No. 5,978,095 or the microscope described in the U.S. patent application No. 5,162,941. 
   Other confocal microscopy system require the use of an array of pinholes. For example, the microscopes described by FIG. 1 of U.S. Pat. No. 5,239,178 or FIG. 3 in the U.S. Pat. No. 5,978,095, or the Nipkow disk systems. 
   In certain embodiments of a microscope such as the one described in French patent number 0103860 of 22 Mar. 2001, an array of pinholes must be positioned with great precision, which is difficult using a simple technique consisting of exchanging the whole of the array. When “single” pinholes are simply exchanged, as on certain single-point confocal microscopes, their precise positioning is also difficult. In addition, the system for exchanging arrays of pinholes are necessarily bulky, since their size is the sum of the sizes of each array able to be exchanged. 
   In the case of microscopes using an array of pinholes, the size and density of the holes cannot usually be modified. However, this modification is desirable in order to adapt the size of the holes to the wavelength being studied. U.S. Pat. No. 6,002,509 affords a solution to this problem in the case of a Nipkow disk microscope. However, this solution requires the replacement of the array of holes with an array of reflective points. When the technique used consists of using reflective points produced by a multilayer treatment, each wavelength corresponds to a given size and density of the reflective points. It is then not possible to modify the size or density of the holes of the hole with a given wavelength, and the number of different sizes of holes is limited by the performance of the multilayer treatment. When the technique used consists of introducing several concentric rings on the Nipkow disk, a movement of the disk, which is not very practical, is necessary, and the size of the disk rapidly becomes excessive. The technique is difficult to adapt to systems using a fixed array of pinholes. 
   SUMMARY OF THE INVENTION 
   The object of the invention is a a plurality of pinholes of variable size, the changes to which are obtained by a simplified method which is precise and inexpensive. In particular, one object of the invention is to produce pinholes which can be modified without problems in positioning and which are of reduced bulk. “Holes” means holes in the optical sense of the term, that is to say small areas through which light can pass, not necessarily void. A “hole” can for example be an interruption in an opaque layer deposited on glass. 
   To this end, the object of the invention is a modifiable array comprising a plurality of microscopic apertures and adapted to filter a light beam in a confocal microscope,
         comprising a plurality of plates each of which comprises a plurality of intermediate apertures,   wherein each microscopic aperture results from the superimposition of intermediate apertures in each of said plates,   wherein each of said intermediate apertures contributes to the formation of at most one microscopic aperture,   at least one of said plate being adapted to move, to switch from a first configuration to a second configuration,   wherein the size of the microscopic apertures in said second configuration differs from the size of the microscopic apertures in said first configuration, and   wherein each microscopic aperture is made up of the superimposition of the same intermediate apertures in the second configuration as in the first configuration.       

   An iris diaphragm also comprises plates sliding with respect to each other. The invention is distinguished in this by the fact that these plates carry pinholes and by the fact that one of the plates carries at least two pinholes. This particular arrangement simplifies the design of the system and makes it possible to produce arrays of modifiable pinholes, whilst iris diaphragms are designed only for a single modifiable pinhole. 
   A modifiable set of pinholes can also be obtained by a system physically exchanging two sets of pinholes produced on different plates. This solution is used in certain single-point confocal microscopes. The present invention is distinguished from this simple technical solution by the use of several superimposed plates, which makes it possible to modify the array of pinholes by means of movements which are also microscopic, rather than macroscopic as is the case in the state of the art. This simplifies the positioning problems. 
   Various techniques for producing plates can be employed. For example, and according to one characteristic of the invention, two of said plates can be transparent windows on which said pinholes are produced by the deposition of an opaque layer by a lithographic method. The opaque layers on these two plates can then be turned towards each other, so that the space separating them is as small as possible. The advantage of this technique is that the window have good rigidity (deform little). 
   According to one characteristic of the invention, at least one of the plates is a fine opaque sheet in which said pinholes are obtained by piercing. This solution is necessary when more than two plates are used. This is because, if only glass plates are used, their thickness does not make it possible to produce an array of holes correctly. 
   According to one characteristic of the invention, the plates consisting of fine opaque sheets are placed against each other and held between two thick plates, in order to prevent any deformation of said plates consisting of fine opaque sheets. This is because one difficulty in the manufacturing of the device is the tendency to the deformation of the fine sheets, which do not have the necessary rigidity and must therefore be placed between thicker supports. 
   According to one characteristic of the invention, the plates are separated from each other by layers of a transparent lubricating liquid. This is because, in the contrary case, friction between the plates make correct functioning difficult. Another solution is to use plates which do not touch each other, but this solution is difficult since it requires excellent surface evenness of the plates. 
   Sliding of one plate with respect to another can in general take place along two axes. However, the system is simplified, according to one characteristic of the invention, if this sliding takes place along only one axis. In this case, it is possible to use a guide rail to help maintain correct relative positioning of the plates. However, such a rail is expensive and poses problems of positioning. In order to facilitate the relative positioning of the plates, and according to one characteristic of the invention, two adjacent plates sliding with respect to each other along one axis are positioned with respect to each other by microscopic guide rails. A microscopic rail being fragile, it is preferable, according to one characteristic of the invention, to use several microscopic guide rails. These rails can for example be produced by lithography. 
   Various solutions can be used for the arrangement of the plates and the distribution of the holes on the plates. 
   According to one characteristic of the invention, an appropriate solution consists of continuously moving the plates with respect to each other so that this continuous movement results in a continuous modification of the surface of each pinhole in the modifiable set. It is possible for example to use several identical plates and one reference position for which the pinholes in said several plates are exactly superimposed on each other. So that the reduction in size of the pinholes takes place regularly, it is preferable to generate a translation movement of the i th  plate in a direction oriented at 
             2   ⁢           ⁢   π     N         
radians in a reference frame common to the N plates constituting the set of pinholes. In the case where N plates are used, the pinholes are preferably polygons with 2N sides, although they can also have other shapes, for example circular. The directions of the translation movement of the plates with respect to each other are then preferably directed along midperpendiculars of the polygons.
 
   According to one characteristic of the invention, the sliding of the plates with respect to each other is obtained by means of an iris diaphragm mechanism. This solution is well adapted to the previous case, where it makes it possible to coordinate the continuous movement of several plates. This iris diaphragm mechanism can, in the case where a rotation of the set of pinholes must be avoided, be supplemented by a supplementary rotation device for compensating for the rotation caused by an iris diaphragm mechanism with a single movable element. 
   According to one characteristic of the invention, one of the plates is moved by means of a linear positioner along an axis. This solution is preferred when a technique based on discrete movements is used. It may also be necessary to move one of the plates by means of a two-axis positioner. This solution is one which allows the maximum flexibility. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  depicts a plate used in a first embodiment. 
       FIG. 2  depicts a device used for preventing leakages of the liquid separating the plates, the device being filled under vacuum. 
       FIG. 3  depicts a similar device but comprising an overflow having a breather. 
       FIG. 4  depicts a plate used in a second embodiment. 
       FIG. 5  illustrates the movement of 3 plates in this embodiment. 
       FIG. 6  shows a supplementary plate used in this embodiment. 
       FIG. 7  shows in front view the device driving 3 identical plates on the principle of  FIG. 5 . 
       FIG. 8  shows this device in section, supplemented by a fourth plate. 
       FIG. 9  depicts in section the assembly of two plates produced on metallic sheets. 
   

   DETAILED DESCRIPTION OF THE INVENTION 
   First Embodiment 
   This first embodiment makes it possible to obtain square holes of continuously variable size. It uses two identical plates depicted for example by  FIG. 1 . When the hole  401  in the first plate is exactly superimposed on the hole  401  in the second plate a set of square holes equivalent to the first plate alone is obtained. When the two plates are moved with respect to each other along the axis  402 , the size of the square holes resulting from the superimposition of the plates is decreased. 
   The two plates moving with respect to each other can be separated by a layer of optical liquid. The system can be made impermeable as indicated in  FIG. 2  by means of a flexible closure  500 , for example made from plastic, which closes the whole of the system. The liquid can then be injected under vacuum between the two plates and into the area included inside the flexible closure  500 . This device makes it possible to reconcile the movement of the plates  110  and  100  with the absence of leakages of liquid. One alternative to filling under vacuum is the overflow system depicted in  FIG. 3 . A tube  105  leads into a reservoir  502  provided with a breather and raised up and ensures the maintenance of a level of optical liquid in the area included between the plates. 
   Second Embodiment 
   This second embodiment is particularly adapted to the case where a high density of pinholes is sought. Although more complicated than the first embodiment, it allows for hexagonal pinholes, which is better than square pinholes. In a basic version, it requires the use of 3 plates which move continuously with respect to each other. They are driven by means of a diaphragm device with modified iris in order to compensate for the rotation of the whole. The pinholes are hexagonal. 
   The three plates carry arrays of identical holes which, in a reference position, are superimposed on each other.  FIG. 4  shows a plate  1000  comprising pinholes, for example  1001 . The broken lines, for example  1002 , delimit hexagonal locations not carrying any hole. In the reference position, the appearance of the modifiable set of pinholes is the same as the appearance of each of the plates and is therefore depicted by  FIG. 4 . 
   The size of the holes in the modifiable set of pinholes is modified by moving the plates with respect to each other in the manner indicated by  FIG. 5 . This figure depicts part of the array of pinholes. In the figure, the broken lines depict the limits of the pinholes in each of the three plates and the intersection of these holes, which constitutes the effective hole of the modifiable array, has been depicted in white. The arrows show the direction of movement of the plates from the reference position. By moving the plates in the direction of the arrows the width of the holes is decreased continuously. The shape of the holes of the modifiable set of pinholes is not modified when their width decreases. This is due to the fact that there are 3 plates, that the holes have 2×3=6 sides, and that the directions of the movement are along the midperpendiculars of the hexagons. 
   In a version also making it possible to modify the density of pinholes, a fourth plate is necessary. This plate is depicted in  FIG. 6 . The set of modifiable pinholes obtained by means of three plates and depicted in  FIG. 4  in the reference position constitutes a first intermediate set. The plate in  FIG. 5  constitutes a second intermediate set. When the hole  1010  in the plate in  FIG. 6  is superimposed on the hole  1001  in the first intermediate set in  FIG. 4 , the density of pinholes is at a maximum. When the hole  1013  in the plate in  FIG. 6  is superimposed on the hole  1001  in the first intermediate set in  FIG. 4 , the number of pinholes per unit surface area is divided by 4. When the hole  1012  in the plate in  FIG. 6  is superimposed on the hole  1001  in the first intermediate set in  FIG. 4 , the number of pinholes per unit surface area is divided by 9. When the hole  1011  in the plate in  FIG. 6  is superimposed on the hole  1001  in the first intermediate set in  FIG. 13 , the number of pinholes per unit surface area is divided by 16. 
   The three identical plates depicted in  FIG. 4  can be driven by means of an iris diaphragm drive system depicted in  FIGS. 7 and 8 . The plates in  FIG. 4  are the plates  1040 ,  1041 ,  1042  depicted in  FIG. 8 . These plates are metallic sheets tensioned over circular holding rings  1022 ,  1021 ,  1020  and carrying holes produced for example by laser piercing. These three fixing rings are connected to two control rings  1023 .  1024 . For example, the internal ring  1020  is connected to the control ring  1023  by a bar  1028  turning freely about an axis  1029  fixed in the control ring  1023 . The internal ring  1020  is connected to the control ring  1024  by a bar  1026  turning freely about an axis  1027  fixed in the control ring  1024 . The housing  1031  of the axis  1027  is oversized so as to be able to combine a rotation and translation with respect to the axis  1027 . A tie rod  1030  is used for keeping the housing  1031  in abutment on the axis  1027 . The other two holding rings are connected in a similar manner to the control rings. When the two control rings turn simultaneously in opposite directions and by an equal angle, the three plates move in translation with an angle of 120 degrees between the directions of each movement axis, as indicted in  FIG. 5 . 
     FIG. 8  also depicts the fourth plate  1051  which is a sheet tensioned on a holding ring  1050 . The holding ring  1050  is mounted on a two-axis positioner. 
   The plates  1051  and  1042  are themselves pressed on thick plates of glass  1052  and  1043 . When the whole of the system is in position the two glass plates  1052  and  1043  prevent deformations of the sheets (plates)  1042 ,  1041 ,  1040 ,  1051  carrying pinholes. 
   Method of Guiding and Positioning the Plates 
   The plates carrying pinholes move in translation with respect to each other along a single axis. For example, in the first embodiment, and also in the second embodiment with regard to the three plates moving by means of an iris diaphragm mechanism. This solution simplifies the system in that each plate moves with respect to another along a single axis. As indicated above a guide rail can be used for guiding the plates However, a macroscopic guide rail is difficult to produce with the required precision. In order to obtain good positioning of the plates it is possible to replace such a guide rail with a set of microscopic guide rails. General principles of a guidance method descrbied in U.S. patent application No. 7,088,487 column 6 lines 1 to 44. 
   This guidance method can be adapted to the case where the plates are metallic sheets, as in the second embodiment. In this case male or female rails can be produced on each side of each sheet. The glass plates are then replaced by the metallic sheets. The diagram in  FIG. 9  is equivalent to that in  FIG. 14  of U.S. Pat. No. 7,088,487 but illustrates the case of plates formed by fine metallic sheets. The plate  1134  carries the male rail  1132  separated from the plate by a layer of protective resin  1133  used for producing the rail  1132  by lithography. The plate  1137  carries the female rail  1138  produced by lithography in a metallic layer  1135  separated from the plate by a resin  1139 . The space between the two plates is filled with a lubricanting liquid. A guide rail has been shown only on one side of each plate but it is possible to produce one on each side of each plate. In the case of the second embodiment, the guide rails which separate each metallic sheet guided by two adjacent sheets participate in the maintenance of the shape of the metallic sheets and in the prevention of deformations. Naturally sheet number  1051  in  FIG. 8  can be guided by this method only if its movement is restricted to a single direction. 
   INDUSTRIAL APPLICATIONS 
   The present set of pinholes can be used in a confocal microscope with multipoint illumination. For example, if a set of pinholes of the type described in the first embodiment replaces the set of pinholes used in the system described by FIG. 1 of U.S. Pat. No. 5,239,178 it becomes possible to modify the size of these pinholes. Likewise, the array of pinholes in the first embodiment of the present invention can replace, with the same effect, the array of pinholes used in FIG. 3 of the U.S. Pat. No. 5,978,095. By using a modifiable array of pinholes according to the present invention in the microscope described by one of the first two embodiments of French patent application number 0103860 of 22 Mar. 2001, it is possible to easily modify the diameter of the pinholes, which affects the speed/resolution or speed/penetration depth compromise in the sample.