Abstract:
Confocal optical device for illuminating points on a sample  807  using illuminating beams FE and for detecting beams FD coming from the illuminated points and passing through a pinholes array  806 . An exchangeable block  600  comprising a beamsplitter  602  and a redirection mirror  603  is used to superimpose the optical pathes of the illuminating beams FE and the beams to be detected FD.

Description:
FIELD OF THE INVENTION 
   The invention relates to a confocal optical device comprising a means for changing the beamsplitter that splits the illuminating beam directed towards the observed object from the beam to be detected coming from the observed object. 
   DESCRIPTION OF THE PRIOR ART 
     FIG. 1  depicts part of a confocal optical device according to the prior art. A light beam originating from a laser  308  is broadened and collimated by lenses  300 ,  301 . The illuminating beam FE having passed through the lens  301  is substantially parallel. It is then reflected by the partially reflective mirror  302  and sent back towards the lens  304  which can be the objective of the microscope or an intermediate lens. The lens  304  focuses the illuminating beam on an illuminated point  309  on the image plane  307  which can be an intermediate image plane or directly an observed object. The beam to be detected FD returning from the illuminated point passes through the lens  304  again in the reverse direction and passes through the partially transparent mirror  302 . It passes through the lens  305  and reaches a microscopic hole  306  which has a filtering action and is placed in a focal plane of the lens  305 . In the figure the partially transparent mirror  302  is in an afocal zone, that is to say the illuminating beam and the beam to be detected are substantially parallel in this zone. However, in confocal microscopy, the partially transparent mirror is not necessarily placed in an afocal zone. For example it can also be placed near the microscopic hole. The partially transparent mirror  302  can typically be a dichroic mirror separating the excitation wavelength from the emission wavelengths, in the case of a microscope operating in fluorescence mode, or a beamsplitter that is neutral as regards wavelength, for observing the light reflected by an object to be observed. 
   For the system to work, the illuminated point  309  must be conjugate with the microscopic hole  306 . But the positioning accuracy of the mirror  302  is insufficient to allow such a conjugation to be reproduced reliably when the mirror is replaced by another one or removed and then put back in place. This is because any inaccuracy in positioning the mirror modifies the direction of the illuminating beam reflected by the mirror, and consequently moves the illuminating point which ceases to be conjugate with the microscopic hole. Exchange of the mirror  302  is necessary for example, if it is a dichroic mirror, for modifying the excitation wavelength. There can also be several distinct illuminating light pathes which reach the afocal zone and are each superimposed on the beam to be detected by a partially transparent mirror. In this case, each mirror must be removable, so that an illuminating light path can be used without being interfered with by the mirror corresponding to another illuminating light path. 
     FIG. 1  does not depict the scanning device which can for example be a pair of galvanometric mirrors or a device for translational movement of the sample.  FIG. 1  can be adapted to the case of multipoint illumination by replacing the lens  300  by an array of microlenses, and replacing the microscopic hole  306  by an array of microscopic holes. 
   The problem of the loss of conjugation between the focusing point of the illuminating beam in the object, which is conjugate with the virtual focusing point of the illuminating laser, and the microscopic filtering hole, during a change of dichroic mirror, is usually solved in various ways: 
   a) by considerably magnifying the image forming in the plane  306  so as to replace the microscopic hole by a hole with larger dimensions and by placing the dichroic mirror near this hole and not in an afocal zone. This solution appreciably lengthens the optical paths and is not transposable to the case of an array of microscopic holes (multipoint illumination). This is because, in the latter case, magnifying the image implies magnifying the entire array of microscopic holes, which leads to dimensions of the array which are incompatible with the normal dimensions of a confocal device.
 
b) by providing a system for readjusting the position of the microscopic hole.
 
c) by combining the preceding solutions in order to avoid readjustments that are too large or too frequent, without excessively lengthening the optical path.
 
d) by making the illuminating beam pass through the microscopic hole, and placing the dichroic mirror before the microscopic hole on the path of the illuminating beam, and therefore after the microscopic hole on the path of the beam returning from the object. This solution simplifies the system but does not make it possible to adjust the size of the microscopic hole without also affecting the illuminating beam, nor does it make it possible to correct chromatic aberration differences between the illuminating beam and the beam to be detected returning from the observed object. It therefore results in a reduced image quality.
 
   SUMMARY OF THE INVENTION 
   The aim of the invention is to solve the problem of loss of conjugation between the focusing point  309  of the illuminating beam and the microscopic hole  306  when the beamsplitter  302  is exchanged, while avoiding the shortcomings characterising the techniques mentioned above, and in a way that is compatible with the use of multipoint illumination. 
   The invention consists of a confocal optical device for illuminating at least one illuminated point using an illuminating beam coming from an illumination source and focused on the illuminated point, and for focusing on a microscopic hole a beam to be detected coming from the illuminated point, comprising a beamsplitter passed through by a first beam and reflecting a second beam, one of the first and second beams being the illuminating beam, and the other being the beam to be detected, the beamsplitter being exchangeable, the device being characterised by the following facts: 
   it comprises a redirection mirror substantially parallel to the beamsplitter and reflecting the second beam, 
   the beamsplitter and the redirection mirror are attached to one another, so that the redirection mirror and the beamsplitter together constitute a splitter unit, which is exchanged all in one piece at the time the beamsplitter is exchanged. 
   For example, the beamsplitter can be a dichroic mirror and the redirection mirror can be a reflective-only mirror. The beamsplitter can also be a partially transparent mirror (neutral beamsplitter) or a reflective-only mirror. 
   If the beamsplitter were positioned independently of the redirection mirror, an error in positioning the beamsplitter would affect the direction of the second light beam, which would therefore not be reproducible when the beamsplitter is removed from the optical path and then put back in place. The fact that the two mirrors are attached results in the direction of the second beam at the output of the splitter unit not being affected by errors in positioning the splitter unit assembly. This is because, after reflection on two mirrors parallel to one another, a light beam retains its initial direction again exactly, irrespective of the angle between the beam and the mirrors. It is also confirmed that an error in positioning the splitter unit translationally does not affect the light beams, so that neither the position nor the direction are affected at the input and output of the splitter unit. 
   However, the presence of a redirection mirror attached to the beamsplitter and parallel to the beamsplitter is not sufficient to eliminate all sensitivity of the system to errors in positioning the splitter unit thus formed. This is because an error in positioning the splitter unit rotationally results in a corresponding translation of the second light beam, although the direction of this beam remains constant. This translation can result in a loss of optical conjugation between the illuminated point and the microscopic hole. 
   According to the invention, this problem is solved by placing said beamsplitter and redirection mirror (the splitter unit) in an afocal zone, in which the illuminating beam and the beam to be detected are substantially parallel. 
   Because the splitter unit is placed in an afocal zone, a direction of the beam at the splitter unit corresponds to a point in a plane where the beams are focused, and therefore the position of such a point (and consequently the conjugation between the focusing point of the illuminating beam and the microscopic hole) is not affected by errors in positioning the splitter unit, both rotationally and translationally. 
   The invention is adapted to both single-point systems and multipoint systems. However, in the case of multipoint systems, the existing techniques are more difficult to apply, perhaps even impossible in the case of technique (a) mentioned above. The invention is therefore particularly useful for multipoint systems, in which case the optical device according to the invention comprises means for illuminating a plurality of illuminated points using a plurality of illuminating beams, and for focusing on a plurality of microscopic holes a plurality of beams to be detected each coming from an illuminated point, said beamsplitter being passed through by a plurality of first beams, said beamsplitter and redirection mirror reflecting a plurality of second beams, said first beams being the illuminating beams and said second beams being the beams to be detected, or said first beams being the beams to be detected and said second beams being the illuminating beams. 
   The beamsplitter can be for example a dichroic mirror or a partially transparent mirror neutral as regards wavelength. The redirection mirror is preferably a totally reflective mirror. 
   In order for it to be possible to exchange the splitter unit, the device according to the invention preferably comprises a plurality of splitter units each consisting of a beamsplitter and a corresponding redirection mirror, and a means for alternately placing one or another of the splitter units on the optical path. This means can be for example a slider or a wheel turning about its axis. 
   If the beamsplitter and redirection mirror are not perfectly parallel, the direction of the second beam at the output of the splitter unit can be slightly modified with respect to its direction at the input of the splitter unit. So that several splitter units can be exchanged without loss of conjugation between the illuminated point and the microscopic hole, it is necessary that all the exchangeable units generate the same beam direction variations, with very great accuracy. This is difficult to achieve with splitter units comprising several assembled components. According to a preferred version of the invention, the beamsplitter and the redirection mirror are placed on two opposite faces of a parallel window. This window is disposed so that: 
   the optical path of the second beam comprises successively a first passing-through of the parallel window, a reflection on a first mirror, a second passing-through of the parallel window, a reflection on a second mirror, and a third passing-through of the parallel window, one of the first and second mirrors being the redirection mirror and the other being the beamsplitter, 
   and so that the optical path of the first beam comprises a passing-through of the parallel window and a passing-through of the beamsplitter. 
   The beamsplitter and the redirection mirror are for example made by depositions of thin coatings on the parallel window. The window must be sufficiently thick to allow effective splitting of the light beams. Under these conditions, good parallelism of the faces of the windows constituting several splitter units is sufficient to ensure the interchangeability of these units. This is easily achievable in an optical workshop. If the windows constituting mutually exchangeable splitter units do not have perfectly parallel faces, the angle between these faces must be the same for all the mutually exchangeable splitter units. 
   The parallel window allowing splitting of the light beams is part of the invention, in the same way as the confocal device as a whole. The invention therefore also consists of a splitter unit intended for a confocal optical device, characterised by the fact that it consists of a parallel window, 
   a first face of said window comprising a first area on which a dichroic or partially reflective mirror is made by deposition of at least one thin coating, intended to be passed through by a first light beam and to reflect a second light beam, 
   the first face of said window comprising a second non-reflective area, intended to be passed through by the second light beam, 
   a second face of said window, opposite to the first face, comprising a third area on which a redirection mirror is made by deposition of at least one thin coating, intended to reflect the second light beam, 
   the second face of said window also comprising a fourth non-reflective area, intended to be passed through by the first light beam and by the second light beam. 
   In fact such a splitter unit makes it possible to split a first light beam from a second light beam without altering the direction of these beams. It is intended as a priority to be used in a confocal microscope, but can also be used in other devices requiring the reproducible exchange of a splitter unit not altering the direction of the light beams. 

   
     BRIEF DESCRIPTION OF THE FIGURES 
       FIG. 1  shows a confocal optical device according to the prior art. 
       FIG. 2  shows a confocal optical device according to the invention. 
       FIG. 3  shows in perspective a splitter unit according to the invention. 
       FIG. 4  shows the same splitter unit in cross-section. 
       FIG. 5  shows several splitter units associated within a mirror-changing slider. 
       FIG. 6  shows in cross-section another type of splitter unit. 
       FIG. 7  shows a slider associating several splitter units of the type depicted in  FIG. 6 . 
       FIG. 8  shows a preferred type of splitter unit. 
       FIG. 9  shows the association in a slider of several splitter units of the type depicted in  FIG. 8 . 
       FIG. 10  shows a single-point confocal device according to the invention using the splitter unit of  FIG. 8 . 
       FIG. 11  shows a multipoint confocal device according to the invention using the splitter unit of  FIG. 8 . 
       FIG. 12  shows a rotating wheel turning about an axis. 
   

   DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     FIG. 2  depicts a simple embodiment of the device according to the invention. The system is identical to that of  FIG. 1 , but the beamsplitter  302  has been replaced by the splitter unit consisting of the beamsplitter  321  and the redirection mirror  320  and situated in an afocal zone. 
     FIGS. 3 and 4  show one particular embodiment of the splitter unit. This comprises a glass component  403  comprising a surface  401  on which the beamsplitter is made, and a surface  400  on which the redirection mirror is made. It is necessary to use a second glass component  402  so as not to disrupt the beam that passes through the beamsplitter. The optical paths of the illuminating beam FE and detection beam FD have been drawn as dotted lines. As shown in  FIG. 5 , several independent splitter units  410 ,  411 ,  412  can be associated in a slider  414  allowing them to be brought into the optical path one after another. 
   As a general rule, it is desirable to have precise parallelism between the faces  400  and  401  and between the faces  404  and  406  so that two distinct splitter units direct the beam in the same direction. The faces  404 ,  406 ,  405  must also be perfectly parallel to one another. 
     FIG. 6  shows another type of splitter unit according to the invention. This comprises a support  500  with holes in it for letting the light beam pass through, on which a beamsplitter  501  and a redirection mirror  502  are positioned. The illuminating beam passes through the hole  503 , is reflected by the redirection mirror  502 , is reflected by the beamsplitter  501 , and leaves the device via the hole  504 . The mirrors  501  and  502  are made by deposition of a reflective coating on glass windows. They are held resting on the surface of the support  500  by spring steel elements, for example  505  and  506 , which apply pressure on the periphery of the mirrors. They can also be fixed by a thin layer of adhesive. If the support  500  is itself made of glass, “molecular bonding” is also possible. Several splitter units can be associated in a single slider. In this case, for these assemblies to be easily interchangeable, the surfaces on which the redirecting mirror  502  and the beamsplitter  501  are respectively positioned must be highly parallel. This constraint can be lessened by making in a single component several supports of the type shown by  FIG. 6 . For example  FIG. 7  shows a multiple support  520 , comprising partially transparent mirrors  511  to  514  corresponding to the mirror  501  of  FIG. 6 , a hole  510 , and holes  521  to  525  corresponding to the hole  503  of  FIG. 6 . Good flatness of the surfaces of the multiple support thus made is in fact sufficient to obtain good reproducibility of the direction of the beam, even when several partially transparent mirrors are used successively and when there is a slight parallelism error between the surfaces of the two mirrors  501 ,  502 . However, it is difficult to put the beamsplitter and redirection mirror in position on their resting surfaces with the necessary accuracy. 
   The sliders can be motorised. However, it is also possible to mount several splitter units  901 ,  902 ,  903 ,  904  on a wheel  900  turning about a spindle  905  as shown on  FIG. 12 , which makes it possible to reduce the friction compared with a slider system and therefore facilitate motorisation. 
     FIG. 8  shows a preferred embodiment of the splitter unit making it easier to manufacture an independent and easily interchangeable splitter unit. In fact, the embodiments described previously are difficult to implement with sufficient accuracy for distinct splitter units to be interchangeable without disrupting the point-to-point conjugation relationships between the different image planes of the device of the invention. The splitter unit depicted in  FIG. 8  solves this problem. It consists of a sufficiently thick parallel window  600 , on which the beamsplitter  602  is made by deposition of a thin coating (for example a multi-coating deposit in the case of a dichroic mirror) and the redirection mirror  603  is also made by deposition of a thin coating (typically a metallic coating or a multi-coating deposit). The illuminating beam FE enters the parallel window via an area  604  which can be antireflection coated, passes through it and reaches the redirection mirror  603  which reflects it. It again passes through the parallel window and is reflected by the beamsplitter  602 . It then passes through the parallel window which it leaves via the area  601  which can be antireflection coated. The beam to be detected FD enters the window via the area  601 , passes through it, reaches the mirror  602  and passes through it. As shown in  FIG. 9 , several splitter units  701 ,  702 ,  703 ,  704  of the type depicted in  FIG. 8  can be associated in a slider  700  making it possible to exchange one unit for another. The parallel window can typically be made of glass and the making of two perfectly parallel faces on a glass window does not pose any technological difficulties. The dimensions of the parallel window depend on the width of the beams and can typically be 15 mm (thickness separating the faces bearing the beamsplitter  602  and the mirror  603 )×15 mm (width)×45 mm (length). This solution therefore makes it possible to obtain at an acceptable cost easily interchangeable splitter units intended to be mounted on wheels or sliders (for example). 
   This device generates a lateral shift of the light beams which can be compensated for by a corresponding shift of the lenses of  FIG. 2 .  FIG. 10  is a modification of  FIG. 2  for the use of the parallel window  600  described in  FIG. 8 . The same numbering as in  FIG. 2  is used, adding the numbers  602  and  603  used in  FIG. 8  and corresponding respectively to the mirrors  321  and  320  of  FIG. 2 .  FIG. 11  depicts a preferred embodiment of the invention in the case of multipoint illumination and use of the splitter unit described in  FIG. 8 . A collimated laser beam  800  is split by the array of microlenses  801  into a plurality of illuminating beams FE. The figure depicts one of these beams in solid lines and another in dotted lines. The illuminating beams then pass through the lens  802  after which each illuminating beam is substantially parallel. They are reflected by the mirror  803 . The illuminating beams then reach the splitter unit consisting of the parallel window  600 . They enter the window, are reflected by mirrors  603  and  602 , and then leave the window. They pass through the objective  804  and are focused on illuminated points of the object  807 . The beams to be detected FD coming from the illuminated points then pass through the objective  804 , pass through the parallel window  600  and the mirror  602 , pass through the lens  805  and are focused on the holes of the array of microscopic holes  806 . The scanning device, which can typically be a galvanometric mirror placed between the parallel window and the objective  804 , has not been depicted. 
   In the figures, the beamsplitter is passed through by the beam to be detected. The beamsplitter may also reflect the beam to be detected and be passed through by the illuminating beam, which does not alter the nature of the invention. 
   INDUSTRIAL APPLICATIONS 
   The device of the present invention allows rapid and reliable exchange of the dichroic mirror in confocal microscopes, in particular multipoint ones. This exchange is for example necessary during the observation of cells marked with several fluorescent markers, in order to successively obtain images corresponding to each marker.