EP0756717A1 - Systeme et technique d'illumination pour microscope optique haute definition - Google Patents

Systeme et technique d'illumination pour microscope optique haute definition

Info

Publication number
EP0756717A1
EP0756717A1 EP94914883A EP94914883A EP0756717A1 EP 0756717 A1 EP0756717 A1 EP 0756717A1 EP 94914883 A EP94914883 A EP 94914883A EP 94914883 A EP94914883 A EP 94914883A EP 0756717 A1 EP0756717 A1 EP 0756717A1
Authority
EP
European Patent Office
Prior art keywords
light
objective lens
beams
specimen
light beams
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94914883A
Other languages
German (de)
English (en)
Other versions
EP0756717A4 (fr
Inventor
Gary Greenberg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MICRO SCIENCE TECHNOLOGIES
Original Assignee
Edge Scientific Instrument Co LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Edge Scientific Instrument Co LLC filed Critical Edge Scientific Instrument Co LLC
Publication of EP0756717A1 publication Critical patent/EP0756717A1/fr
Publication of EP0756717A4 publication Critical patent/EP0756717A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/086—Condensers for transillumination only

Definitions

  • This invention relates to illumination systems for light microscopes utilizing objective lens means and more particularly to such systems that utilize illuminating light directed at an oblique angle relative to the optical axis of the objective lens.
  • patent '283 In patent '283. the location of the illuminating beam (between 15 and 17) and beam path shifting means 23 (prism) on the optical axis limits the system by permitting the use of only a single illumination beam.
  • the references cited above are typical of the prior art in that they fail to recognize the real potential of oblique lighting to enhance resolution.
  • Patent '283, in fact, does not acknowledge the resolution enhancing potential of oblique light but instead cites as a reason for its use the casting of shadows to highlight uneven areas of the specimen. It is not, therefore, necessarily an object or desideratum of patent '283 to provide a maximum oblique angle (for example, too much shadowing might obscure details).
  • one of the requirements of realizing the full potential of oblique lighting to dramatically enhance resolution is that the angle of the oblique light be maximized.
  • maximum resolution is achieved for a given condenser lens/objective lens combination by having the illumination beam exit the condenser lens' periphery so that the light illuminating the object is at a maximum oblique angle and still within the objective aperture.
  • any condenser can be used to its fullest potential.
  • a large condenser lens high numerical aperture can be used to achieve maximum oblique lighting for most objective lenses.
  • the present invention teaches that the essential requirement for realizing the maximum potential of true oblique lighting is the ability to direct two or more separate and distinct light beams onto the objective wherein each beam is at the maximum angle to the objective axis that permits the illumination to enter the objective. This, of physical necessity, requires that the beam shifting means be located off the optical axis of the objective. Also, the present invention overcomes the anistropy that is found in prior art oblique illuminating systems.
  • the present invention teaches a real time, 3-D system using multiple beams which goes far beyond what can be achieved with a single beam, such as that described in U.S. Patent 4,072,967.
  • Patent '967 teaches how to achieve a 3-D image using a microscope with a single condenser lens and a single objective lens, by placing complimentary filters across the left and right halves of the condenser lens and placing a complementary filter set in the binocular eyepieces.
  • this type of system the degree of parallax is fixed. Futhermore, there is very little disparity in parallax between the left and right images, especially at the center of the image field.
  • the left and right images are independently controlled and the degree of parallax between them can be easily adjusted to match the type of objective being employed and the type of specimen being viewed.
  • the present invention which is the ability to achieve a greater depth of field without loss of resolution, as is more fully explained below. This is a critical prerequisite for producing a sharp 3-D image.
  • the present invention resides in the illumination system for a light microscope characterized by objective lens means (which can be comprised of several lenses).
  • the objective lens means is located between the viewing means and the illuminated object or specimen.
  • the diffraction theory of microscopic vision teaches that when examining an object having very closely spaced structural details such as the markings of the diatom Amphiplewa pellucida, the image of a single point or line of detail will consist of a central beam surrounded by a number of spectra (sometimes referred to as orders of diffraction wavelets). The number and arrangement of the spectra depend on the pattern of the markings, and the wave length of light being used. The distance of the diffraction wavelets from the central beam is greater the finer the markings on the specimen (the smaller the spacing between structural details).
  • the diffraction theory further teaches that in order to obtain any image of the specimen it is necessary to collect and recombine at least one order of wavelets with the central beam. The more successive orders of wavelets recombined with the central beam the more the resolution and sharpness of the image increases.
  • oblique lighting can result in the inclusion of one or more orders of wavelets for a specimen which when illuminated by axial lighting casts all of the orders of wavelets beyond the objective.
  • the greater the angle of the oblique light the greater the number of orders of wavelets included within the objective aperture and thus the greater the resolving power of the system.
  • both the resolution as well as the sharpness of the image can be significantly increased compared to axial illumination, because the optimal oblique illumination will place the zero order wavelet near the edge of the objective aperture and thus, the objective can recombine more orders of diffraction wavelets for any given structural detail. Accordingly, it is a principal object of the present invention to provide an illumination system and method for a light microscope which produces oblique lighting thereby enhancing the microscope's resolving power and sharpness of image.
  • an object of the invention when used with a transmitted light microscope or a reflection microscope to utilize the entirety of the source beam or beams as illumination sources for the specimen. That is to say, that the present invention, unlike so much of the prior art, does not use a mask on the condenser or between the objective and the specimen to create an oblique light beam from a small portion of the source beam.
  • the maximum potential of oblique lighting is achieved in the present invention when a plurality of independent beams are used.
  • a single beam system produces enhanced resolution, it does so predominantly along the direction of the beam axis (projected onto the specimen plane).
  • the specimen must be rotated on the stage so that the markings are oriented along the axis of the oblique illuminating beam.
  • a second oblique light beam is directed onto the objective at the opposing angle relative to the first beam, then both sides of the objective will see a beam with a greater angle than would be possible with a single central light beam.
  • multiple oblique light beams can be directed onto an objective lens at opposing angles relative to the optical axis such that the beams will combine to form an overall increase in the aperture of illumination and thus, an increase in the overall resolution of the system.
  • the final resolution of the image is dependent on the N.A. of the system.
  • the N.A. of the system will be the combination of the N.A. of objective and condenser lenses.
  • another object of the invention is to provide an illumination system and method for a light microscope utilizing a plurality of independent, separate illuminating light beams directed onto an objective lens wherein each light beam follows a path to the objective which is oblique relative to the objective's optical axis.
  • Another object of the invention is to provide an illumination system and method for a reflection light microscope wherein a plurality of independent, separate illuminating light beams are directed through an objective to illuminate a specimen wherein each light beam follows a path that is oblique relative to the objective's optical axis.
  • Another object of the invention is to provide an illumination system for a light microscope utilizing a plurality of independent separate illuminating light beams directed through an objective lens wherein the path of each light beam is independently adjustable in both its location and angle.
  • the final image is the combination of multiple images, each with extended depth of field created by an array of pre-apertured oblique illuminating beams, which have an additive effect on the overall aperture of illumination.
  • the same benefit inures to the reflection embodiment of the present invention.
  • Yet another object of the invention is to provide means for using double oblique lighting in a light microscope having an objective lens which produces enhanced resolution and real time 3-D viewing with extended depth of field.
  • each beam independently if desired, such as by interposing complementary filters and thereby produce true, real time 3-D viewing.
  • the interposition of polarizing filters in the path of one or more beams permits a variety of effects, such as selective shadow rotation, to be achieved at the same time that enhanced resolution is realized.
  • a significant part of the present invention teaches how to realize the maximum potential of oblique illumination by directing two or more separate and distinct oblique light beams onto the objective lens in a variety of configurations in order to achieve results which would not be possible with a single illuminating beam. Some of those configurations will be illustrated and their advantages discussed. However, there are other possible configurations that will not be specifically discussed but still fall within the scope of these teachings.
  • Figure 1A is a schematic diagram of microscope optical elements (including a condenser lens and an objective lens) wherein the illumination path is coincident with the axes of the lenses;
  • Figure IB is a schematic diagram of the microscope optical elements of Figure 1A wherein the illumination path is parallel to but not coincident with a condenser lens and oblique to the objective lens
  • Figure 1C is a schematic diagram of the microscope optical elements of Figure 1A wherein the illumination path is non-coincident with and oblique to the axes of both lenses;
  • Figure ID is a wave diagram illustrating the relative number of orders of wavelets that can be seen by the objective lens by the illumination arrangement of Figure 1A;
  • Figure IE is a wave diagram illustrating the relative number of orders of wavelets that can be seen by the objective lens by the illumination arrangement of Figure IB;
  • Figure IF is a wave diagram illustrating the relative number of orders of wavelets that can be seen by the objective lens by the illumination arrangement of Figure 1C;
  • Figure 2 is an optical schematic illustration of a two beam embodiment of the invention
  • Figures 2A and 2B are plan views illustrating two possible mirror arrangements for the embodiment of Fig. 2;
  • Figure 2C is a plan view illustrating a three mirror configuration
  • Figure 2D is a plan view illustrating a four mirror configuration:
  • Figure 2E is a plan view illustrating a six mirror configuration:
  • Figure 3 is an isometric, optical schematic illustration of a three beam embodiment of the invention
  • Figures 3A, 3B and 3C are plan views of the mirrors of Fig. 3 in varying arrangements;
  • Figure 4 is an isometric illustration of a beam shift means of the invention
  • Figure 5 is an optical schematic illustration of a single beam illumination system of the present invention for a reflection microscope.
  • Figure 6 is an optical schematic illustration of a dual beam illumination system of the present invention for a reflection microscope.
  • Figure 7A is a plan view illustrating a two beam illumination configuration of a reflection microscope of the present invention wherein the beams are 100° - 180° apart;
  • Figure 7B is a plan view illustrating a two beam illumination configuration of a reflection microscope of the present invention wherein the beams are 90° apart;
  • Figure 7C is a plan view illustrating a three beam illumination configuration of a reflection microscope of the present invention wherein the beams are evenly spaced about the optical axis of the objective means;
  • Figure 7D is a plan view illustrating a four beam illumination configuration of a reflection microscope of the present invention.
  • Figure 7E is a plan view illustrating a six beam illumination configuration of a reflection microscope of the present invention.
  • Figure 8 is an front view optical schematic illustration of a multiple beam illumination system of the present invention for a reflection microscope with an off-set light source;
  • Figure 9 is a top view of the embodiment of Figure 8; and Figure 10 is a side view of the embodiment of Figure 8.
  • a light beam path shift means (mirror) 11, a condenser lens means 12 having an optical axis 13, an objective lens means 14 having an optical axis 16, and a specimen support stage 17 disposed between the condenser 12 and the objective 14 and defining a specimen plane 20, are the basic components of a transmitted light microscope illumination system. While the principles of the invention are herein illustrated in connection with a transmitted light embodiment of the invention, the principles apply equally with regard to reflection microscopes as more fully described below.
  • the support stage 17 holds a specimen (not shown) to be illuminated by light beam 18 from a light beam source means (not shown).
  • the axis 13 of the condenser 12 and the axis 16 of the objective 14 are shown as being coincident which is the most common arrangement for transmitted light microscopes. Such axial coincidence is not required by the present invention, however, which is equally operative in a system where, for example, the condenser is tilted relative to the objective.
  • both the condenser means 12 and the objective means 14 are each shown diagrammatically as a single lens, it will be understood by those skilled in the art that the condenser means and the objective means may be comprised of multiple lens elements as well as other optical devices known in the art.
  • the mirror 11 When as shown in Figure 1A, the mirror 11 is positioned on the condenser axis 13 and disposed at a 45 degree angle relative to the initial path 19 of beam 18, which is normal to the condenser axis 13, the path 21a of the beam after being shifted by the mirror 11 will follow the axis 13.
  • lines indicated as representing a beam path such as 19 and 21a are schematic representations of a beam's axis. In reality, of course, a beam has an envelope which can be converging, diverging or parallel. An understanding of the present invention is best facilitated, however, by following the path of a beam's axis.
  • a beam incident a condenser such as 12 along its axis 13 will emerge from the lens along an axial path 22a.
  • the beam path 22a will pass through the specimen plane 20 at right angles thereto and include the objective lens 14 along its axis 16.
  • Figure 1A represents a typical "bright field" illumination system.
  • the shifted beam path 21b remains parallel to condenser axis 13 but is laterally displaced therefrom.
  • the effect of the beam path 21b entering condenser 12 at an off axis location is to create an angle J3 between the exit beam path 22b and the objective axis 16.
  • the exit location of the beam 22b from the condenser means 12 is not laterally displaced from the condenser optical axis 13.
  • the beam path 22b will be angled or oblique to a specimen in the specimen plane 20.
  • the important relationship is the angle $ between the exit beam path 22b from the condenser 12 and the optical axis 16 of the objective 14.
  • the advantages of the present invention do not, for example, accrue from a system that creates an oblique angle between the specimen plane and the illuminating beam path by tilting the specimen stage while at the same time allowing the illuminating beam to travel a path that is parallel to the objective axis. Such an arrangement still produces standard "bright field" illumination enhanced only by some possible shadowing.
  • mirror 11 is inclined relative to the path 19 of incident source beam 18, to be greater than 45 degrees (50 degrees for example), causing an angle of reflectance for the beam that sets the beam path 21c to the condenser means 12 at an angle ⁇ relative to the optical axis 13 of the condenser means.
  • the effect of beam path 21c entering condenser 12 at an angle ⁇ is to laterally shift the location of the exit beam path 22c from the center of the condenser lens means 12 to some location nearer the periphery.
  • the beam path shift means 11 is operative not only to control the angle of the exit path 22c but its location on the condenser 12 as well. Under these circumstances, the angle of oblique illumination is increased (angle ⁇ in Fig. IC is greater than angle $ in Fig. IB).
  • the angle of the exit beam path 22c from the condenser 12 is a function of the lateral (radial from axis 13) displacement of the incident beam path 21c, while the lateral (radial from axis 13) position of the exit beam path 22c on the condenser 12 is a function of the angle of the incident beam path 21c relative to the axis 13 of the condenser means 12.
  • One of the desiderata of the present invention is the manipulation of the beam path shift means 11 to maximize the angle of the exit beam from the condenser within the limits of the aperture of the objective. This will depend on the specification of the lenses, such as the focal length, working distance and numerical aperture.
  • the maximum oblique angle of the beam path 22c relative to the objective axis 16 within the objective aperture is achieved by having the beam path exit the condenser at or very near the edge of the condenser lens 12. This is achieved by varying the angle of mirror 11 relative to the source beam path 19 and thereby the angle ⁇ of the beam path 21c to the condenser. At the same time, i order to create true oblique lighting, all or a portion of the beam must enter the objective, requiring that for a particular objective, the exit beam path from the condenser be at a particular angle as well as location. And, as explained above, the angle ⁇ is varied as a function of the radial location of the mirror 11 and thereby the radial location of the entering beam path 21c relative to the condenser optical axis 13.
  • condensers of maximum size can be advantageously used in most systems since the beam path shift means permits the angle ⁇ of the exit beam path from the edge of the condenser to be shifted until it includes the objective. In this way, the best glass can be used and the maximum beam path angle achieved with the result of greatly enhanced resolution. Furthermore, in the present invention, unlike prior art systems, most of the optimally angled light beam can enter the objective rather than merely just an edge or small portion of the light cone, thereby creating the brightest possible image for the available light.
  • the single beam system described above is capable of greatly enhancing a microscope's resolution
  • the improved resolution is primarily along the direction of the axis of the illuminating beam (as projected onto the specimen plane), with the resolution along a direction 90 degrees thereto being significantly degraded.
  • Figs. ID, IE and IF illustrate the relative number of orders of wavelets that can be seen by the objective lens under the illuminating conditions shown in Figs. 1A, IB and IC, respectively.
  • Fig. IE which corresponds to the oblique illuminating conditions of Fig. IB
  • the objective lens collects and recombines more orders of diffraction wavelets 25 than shown in Fig. ID which corresponds to the axial illuminating system of Fig. 1A.
  • the increase in the order of wavelets collected in the X dimension is linked to a decrease in the order of wavelets collected in the Y dimension.
  • This increase (or decrease) in resolution relative to the resolution attainable with axial illumination is proportional to 2 times the cosine of angle 0, where angle 0 is the angle of orientation of the specimen (not shown) relative to the axis of the oblique illumination.
  • Angle 0 ranges from 0 to 90 degrees, where 0 degrees is the X dimension (or the axis of oblique illumination) and 90 degrees is the Y dimension.
  • Fig. IF which corresponds to the maximum oblique illuminating conditions, as shown in Fig. IC, the number of diffraction wavelets 25 collected and recombined by the objective is even greater than the number attainable with the oblique illuminating conditions shown by Fig. IB and IE.
  • This additional increase in resolution is proportional to the sine of the angle between the axis of the oblique illuminating beam and the optical axis 16 of the objective lens means.
  • the illumination beam shift means i.e. mirror 11
  • the condenser axis thereby permitting a plurality of such beam shift means to operate within the system simultaneously.
  • improved resolution over the entire specimen plane can be achieved by utilizing a plurality of illuminating beams positioned to have their respective axes at selected angles to one another.
  • a pair of beam path shift means in the form of mirrors 23 and 24 disposed off the optical axis 13 of condenser 12 permit the system to operate with two independent illuminating beams to the condenser lens means 12.
  • a light beam source means (lamp) 26 directs a light beam 27 along a source beam path 28 that includes the beam path shift means 23.
  • a light beam source means (lamp) 29 directs a light beam 31 along a source beam path 32 which includes beam path shift means 24.
  • Mirror 23 shifts the direction of beam path 28 to path 28a which includes condenser 12.
  • Mirror 23 is disposed a distance radially away from the condenser axis 13 and at an angle ⁇ relative to its incident light beam 27 which produces the exit beam path 33 from condenser 12 to emerge from the edge of the lens at the maximum angle which passes through the objective 14.
  • mirror 24 shifts the direction of beam path 32 to path 32a which includes condenser 12.
  • Mirror 24 operates in precisely the same way as mirror 23 to produce the desired exit beam path 34 from condenser 12.
  • the relationship of the locations of mirrors 23 and 24 relative to axis 13 is shown in Figure 2A, but can be different depending on the results desired.
  • the shift means can be disposed in essentially opposing relationship (180 degrees apart) for 3-D viewing purposes as shown in Figure 2A or at essentially right angles (90 degrees apart) as shown in Figure 2B, to achieve the best overall resolution for a two beam system.
  • Resolution over the entire specimen plane is improved by increasing the number of beams.
  • Increasing the number of beams even further to four ( Figure 2D) or to as many as six ( Figure 2E) will produce even better results. Because of the off axis placement of the beam shift means, numerous other arrangements of mirrors and spacing are possible to meet specific needs.
  • the source of light beams 27 and 28 can be from separate independent light beam sources as shown, or from a light beam source means providing a single light beam which is split by beam splitting means (not shown) which are well known in the art. More important than the source of the light, are the multiple beams 27 and 31 directed along separate, independent paths to the condenser, and the resultant exit beam paths 33 and 34 which do not fall along the optical axis 16 of the objective lens means 14.
  • mirrors provide one means of beam shifting
  • other means exist, such as prisms, and the fact that all such means are not shown does not mean that any of them are excluded from the invention.
  • the present invention in fact, encompasses an arrangement of separate micro light sources, as could be provided using fiber optics, with the beam shift means comprising mechanical or electro- mechanical means for positioning and directing these light sources.
  • the invention is manifest by separate, independent, light beams directed to the objective lens means along paths that are oblique to the optical axis of the objective lens means.
  • the beam shift means is shown to be adjustable in order to accommodate a large variety of different objective lenses.
  • the beam shift means includes such fixed systems known in the art that will direct light beams into a condenser lens at the appropriate location and angle of orientation.
  • One of the outstanding features of the multiple beam embodiment of the present invention is the intensity of light available to illuminate the specimen at the specimen plane 20.
  • the present invention does not require the use of masks or other light occluding devices.
  • the present invention makes it possible to utilize virtually all of the light from the light beam source means for illumination of the specimen.
  • the light beam source means has been shown schematically as a light bulb, it will be understood by those skilled in the art that the light beam source means may include any suitable source of radiation as well as lens means and other optical devices well known for the purpose of furnishing object illuminating light.
  • Another important feature of the multiple beam embodiment is that it is able to overcome the anistropy that is inherent in all oblique illuminating systems known in the prior art.
  • the anistropy of resolution and sharpness has been discussed above.
  • Another effect of the anistropy associated with prior art systems is the obvious uneven illumination of the image field. That is, one side of the field of view appears bright while the opposite side appears dark.
  • the introduction in the present invention of multiple beams makes it possible to produce an evenly illuminated field of view.
  • a plurality of light beams following different paths to the condenser makes it possible to individually manipulate those beams for a variety of possible results in addition to enhanced resolution.
  • real time 3-D is achieved by interposing complimentary polarizing filters 36 and 37 in beam paths 28 and 32, respectively together with providing similar eye piece polarizing filters 38 and 39 in binocular eye piece 41 having a pair of viewing lenses 42 and 43.
  • the filters 36 and 37 are denoted by positive and negative symbols to indicate that they could be complementary in a variety of different ways known in the art. They may be plane polarizers oriented with their polarizing axes mutually at right angles.
  • the filters may be circular polarizers, one of the pair producing left-hand polarization, the other producing right-hand polarization.
  • the filters may be complementary color filters (such as red and green) of either the absorption or dichroic type.
  • the eye piece filters 38 and 39 interact with filters 36 and 37 to selectively limit the light from only one of the light sources 26 and 29 so that the image produced by the light along beam path 33 does not exit the viewing lens 43, and the image produced by the light along beam path 34 does not exit the viewing lens 42.
  • the present invention goes far beyond what can be achieved with a single beam, real time, 3-D system in which the degree of parallax is fixed.and there is very little disparity in parallax between the left and right images, especially at the center of the image field.
  • the left and right images are independently controlled and the degree of parallax between them can be easily adjusted to match the type of objective being employed and the type of specimen being viewed.
  • there is another and possibly even more important advantage with the present invention which is the ability to achieve a greater depth of field without loss of resolution. This is a critical prerequisite for producing a sharp 3-D image.
  • a microscope utilizing the illumination system of the present invention can use any of the many light beam manipulation devices known in microscopy, such as polarizing filters, aperture stops, collimator, etc. In multiple beam systems of the present invention these devices can be used to provide beams having different characteristics or those having the same characteristics.
  • mirror surfaces 45, 46 and 47 are supported on beam shift means 48, 49 and 50, respectively.
  • Each mirror surface is disposed in one of the source beam paths 51, 52 and 53, of light beams 54, 56, and 57, respectively, emanating from light beam sources means 58, 59 and 61.
  • the shift means 48, 49 and 50 as best seen with reference to Figures 3A and 3B, are movable along paths 55 that are radial relative to the optical axis 13 of the condenser means 12 (see Figs. 2 and 3).
  • the beam shift means are positioned at locations on their paths 55 that place the beam reflecting mirror surfaces 45, 46 and 47 radially outward from the axis 13. As fully described above, varying the location of a mirror (45 for example) along its radial path 55 varies the angle of the beam exit path 66 (see Fig. 3) from condenser lens means 12.
  • "bright field” illumination is available in the present system by locating one of the mirror surfaces (47 for example) over the optical axis 13 and in a position in which it creates a beam path that travels along the condenser lens means axis 13.
  • the other mirror surfaces can be deployed to provide oblique lighting at the same time or disabled (mirrors moved out of range of the condenser means or associated light beam source means turned off) for standard "bright field" illumination.
  • Positioning of the shift means 48, 49 and 50 can also result in "dark field” illumination.
  • "dark field” illumination is made possible.
  • each mirror surface 45, 46 and 47 is also angularly tiltable relative to its associated source means beam so as to vary the angle of reflectance of its mirror surface.
  • the angle of the beam path from the shift means to the condenser means 12 is varied and in turn the location of the exit beam path from the condenser means is varied.
  • the source means beams 54, 56 and 57 follow source beam paths 51, 52 and 53 to the light path shift means 48, 49 and 50 that are generally normal to the axis 13 of condenser means 12 and evenly angularly spaced about the axis 13 of the condenser 12 and the axis 16 of the objective lens means 14 which axes are shown as being coincident (see Fig. 2).
  • the mirrors 45,46 and 47 are positionable radially and angularly to establish the direction of the beam paths 62, 63 and 64 to the condenser lens means 12, and thereby control the location and direction of the exit paths 66, 67 and 68 from the condenser means to the objective means..
  • a large field lens 71 acts as a pre-condenser lens means permitting the gathering of all of the light from the incident beams and the accurate direction of those light beams onto the condenser lens 12.
  • the raising or lowering of the field lens 71 relative to the condenser lens 12 has the effect of sizing the beam on the specimen plane 20 to accommodate low power as well as high power systems.
  • a field lens aperture (iris stop) 72 can be used to control .depth of field and contrast, provided the condenser means 12 is slightly under focused.
  • Prior art systems reduce the condenser lens aperture to increase depth of field but in doing so reduce resolution due to a concomitant reduction in the numerical aperture of the light beam exiting the condenser.
  • the condenser aperture 69 remains fully open while the field lens aperture 72 can be reduced to increase depth of field without a concomitant loss in resolution. This is because the aperture of each illuminating beam is reduced while the overall aperture of illumination that exits the condenser lens is not significantly reduced. The multiple beams illuminate the full condenser aperture and no loss of resolution is experienced.
  • the interposition of the field lens 71 and iris stop 72 does not interfere with the operation of the present invention since adjustment of the mirror surfaces 45, 46, and 47 continues to control the direction and location of the exit paths of the beams from the condenser lens 12.
  • the interposition in the source beam paths 54, 56 and 57 of such devices as lamp condensers 73, zoom lens 74 (to adjust beam size), and polarizing filters 76 does not interfere with the operation of the present invention and in fact highlights one of its major advantages.
  • the use of such light manipulating devices on the light beams either separately (between the light source and the shift means) or together, such as by the field lens 71, the field lens aperture (iris) 72 or a polarizing filter 77 ( between the shift means and an eye piece 78), does not reduce the system's resolution.
  • light sources 58, 59 and 61 are independent (as opposed to a single source split by optical means) they can be varied in intensity to add yet a further investigatory variation.
  • the present invention does not limit the use of well known optical devices for light manipulation nor does it result in operation at low light levels relative to the light provided by the light source means.
  • the illumination system of the present invention enhances resolution and at the same time makes it possible to create illumination conditions that can satisfy a wide variety of investigation needs.
  • a multi- beam system of the present invention enjoys enhanced resolution both from an increase in the oblique orientation of the illuminating beams relative to the objective lens means optical axis (increase in orders of wavelets recombined) as well as from an increase in the overall aperture of illumination of the condenser lens due to the additive effect of the multiple light beams that exit the condenser from around its periphery.
  • polarizing filters 76 in the source beam paths 54, 56 and 57 from the light source means to the beam shift means are complementary, rotation of polarizing filter 77 in the combined beam between the objective lens means 14 and the eyepiece lens 78 permits rotation of the shadow effect of the oblique lighting on the specimen by effectively attenuating the illumination from one or two of the beams while looking at the effects of the other.
  • the present invention is independent of any particular mechanical or electrical system for positioning and directing the illuminating beams. This includes systems that may be adjustable or pre-adjusted and fixed, and may utilize mirrors, prisms, fiber optics or other known or unknown devices. Such mechanical systems can take any number of forms known to those skilled in the art. By way of example, such a mechanical arrangement for positioning the mirrors of the shift means is described with reference to Figure 4.
  • a shift means 80 includes a mirror 81 affixed to a tilt arm 82 which is rotatably connected by hinge 83 to an "L" shaped mount member 84 which is secured to a car 86 that runs in tracks 87.
  • a cable 88 attached to a tab 89 formed on the end of mount member 84 provides the means for positioning the car 86 on the track 87 and thus the radial position of the mirror 81 relative to an optical axis.
  • a pivot arm 91 is pivo tally attached at one of its ends to the tilt arm 82 and at its other end to a slide 92 that runs in a groove 93 in the mount member.
  • a cable 94 affixed to a tab 96 formed in the end of slide 92 positions the slide in its groove and in doing so adjusts the tilt of the tilt arm 82 and the angle of the mirror 81.
  • the use of micrometers (not shown) attached to the ends of the cables 94 and 98 to operate the cables makes it possible to achieve the degree of precision necessary for the invention.
  • a number of shift means 80 in the same system can be mechanically inter-connected (by means well within the skill of the art) so that their positions will be inter-dependent. That is, the movement of one shift means to a new radial location or the tilting of a mirror to a different angular position will cause corresponding movement in the other shift means.
  • This arrangement sets a fixed relationship between the mirrors and makes it possible to easily assure that all of the beams are substantially identical in their paths through the system other than their circumferential position relative to the objective axis 16.
  • the positioning of the shift means is most advantageously mechanically independent.
  • the mirror members are selectively mechanically inter-connected for unified movement and mechanically unconnected for independent movement. Such a system is capable of satisfying the needs of a wide variety of microscope uses.
  • the method of the present invention for increasing resolution, sharpness and depth of field in a transmitted light microscope having a condenser lens means with an optical axis, and an objective lens means with an optical axis constitutes the steps of directing a plurality of independent light beams onto the condenser lens means along paths that are not coincident with the condenser lens means optical axis; and fixing the location and direction of the paths of the light beams to the condenser lens means so that the light beams that exit the condenser lens means are directed along paths that include the objective lens means and are oblique relative the optical axis of the objective lens means. Further, the directions of the paths of the light beams onto the condenser lens means are selected to produce exit paths from the condenser lens that are at the optimal angle relative to the optical axis of the objective lens means that includes the objective lens means.
  • the number of beams When the number of beams is two and they are directed along paths that are in opposition to one another (essentially 180 degrees apart) they provide illumination for real-time 3-D viewing. When they are at right angles (90 degrees to one an other) they provide the best overall resolution using just two beams. When the number of beams is three or more they are preferably radially positioned and spaced about the optical axis of the condenser lens means for the best overall resolution at the specimen plane.
  • the primary teaching of the invention - multiple oblique beams from a specimen to the objective - is applicable to reflection microscopes as well as transmitted light microscopes where the objective lens acts as a condenser lens to direct illumination onto the specimen from different angles.
  • an objective lens means 101 having an optical axis 102 is optically disposed between a specimen 104 at a specimen support stage 106 and a viewing means 107.
  • the specimen 104 is illuminated by a light beam source means 108 which provides a light beam 109 that travels to the specimen 104 via initial beam path 111 which is shifted by a light beam shift means 112, including a partially silvered mirror 115, to a path 113 that is oblique to the optical axis 102 of the objective lens means 101 and which includes the objective lens means 101.
  • the objective lens means 101 directs the beam 109 onto the specimen 104 along path 114 which is also oblique to the optical axis 102 of the objective lens means 101.
  • the beam 109 reflects off of the specimen 104 along reflection path 116 which is at the same angle relative to the optical axis 102 as the incident beam path 114, and passes through the objective lens means 101 after which it follows path 117 to the viewing means 107 through the partially silvered mirror 115.
  • the angle and location of the beam path 113 is adjustable by movement of the shift means 112 both angularly 112A and transversely 112T to permit different size objective lens means 101 to be used and the optimal oblique angle achieved for each.
  • a lamp condensing lens 122, and/or an iris 123 can be disposed in the beam path 111 as can other optical devices commonly used in microscopy and shown in Figure 3.
  • the reflected light from the specimen passes through the objective lens means 101 at an oblique angle relative to the optical axis 102 of the objective lens means 101 and, thereby, has all of the advantages described above in connection with the transmitted light embodiment of Figures IB and IC.
  • the performance of the reflected light embodiment improves when multiple, independent, oblique light beams are used.
  • the specimen 104 is illuminated by a second light beam source means 126 which provides a light beam 127 that travels to the specimen 104 via initial beam path 128 which is shifted by a light beam shift means 129 including a half silvered mirror 130 to a path 131 that is oblique to the optical axis 102 of the objective lens means 101 and which includes the objective lens means 101.
  • the objective lens means 101 directs the beam 127 onto the specimen 104 along path 132 which is also oblique to the optical axis 102 of the objective lens means 101.
  • the beam 127 reflects off of the specimen 104 along path 133 which is at the same angle relative to the optical axis 102 as the incident beam path 132, and thus passes through the objective lens means 101 and follows a path 134 to the viewing means 107 through partially silvered mirrors 115 and 130.
  • the angle and location of the beam path 131 is adjustable by movement of the shift means mirror 129 both angularly 129A and transversely 129T to permit different size objective lens means to be used and the optimal oblique angle achieved for each.
  • a condensing lens 141, and/or an iris 142 can be disposed in the beam path 128 as can be other optical devices commonly used in microscopy, such as filters.
  • the specimen 104 is illuminated by multiple, independent light beams that are oblique to the optical axis of the objective lens means and which follow reflection paths to the viewing means that includes the objective lens means and which are oblique to the optical axis of the objective lens means. All of the advantages described above attributable to multiple oblique transmitted lighting pertain equally to the multiple oblique reflected lighting described above.
  • the incident path (113, and 114) of beam 109 may be the reflected path (133 and 134) of beam 127, and the incident path of beam 127 (131 and 132) may be the reflected path (116 and
  • a two beam system can be arranged with the beams at various angles relative to one another. At right angles (see Figure 2B) the best overall resolution is achieved for two beams. At between 100° and 180° (see Figure 7A) the best 3-D is achieved.
  • a housing 141 extending perpendicular to the axis 142 of a reflection microscope 143 supports a beam shift means 144 having a plurality of adjustable mirrors 146.
  • Each mirror 146 is aligned to receive a beam from a light source means 147 and reflect that beam onto a main beam shift means 149 which includes a partially silvered mirror 151. All beams reflected onto mirror 151 are directed through the objective lens means 148 of microscope 143 onto a specimen 152. The beams reflected off of the specimen and into the viewing means 153 pass through only a single beam splitting device - the partially silvered mirror 151.
  • the shift means 144 directs the beams onto the main shift means 149 at an angle that causes all of the beams reflected onto the specimen to be oblique to the axis 148 (which is also the axis of the objective lens means) both as specimen illumination beams and reflected beams. While only a dual light beam system with a single main light shift means has been illustrated in detail, a system of three, four, six or more light beams would essentially look the same except that the number of mirrors 144 and number of light sources 147 would increase.
  • Figures 8, 9 and and 10 permits multiple oblique light beams to be used in a reflection microscope to illuminate a specimen without compromising light intensity by having beams pass through multiple beam splitters.
  • a pellicle as the main shift means 151, any problems that might arise due to a beam shifting as it passes through a beam splitter in the form of a partially silvered mirror, for example, are avoided.
  • Superior 3-D viewing is possible, when more than one beam is used.
  • the use of the 3-D viewing head described in my co ⁇ pending patent application, serial number 07/957286, filed October 6, 1992, for IMPROVEMENTS IN STEREOSCOPIC MICROSCOPES permits real time viewing or photographing without the use of, filters or choppers.
  • the present invention goes far beyond what can be achieved with a conventional single beam reflection microscope, by offering a real time, 3-D system.
  • the left and right images are independently controlled and the degree of parallax between them can be easily adjusted to match the type of objective being employed and the type of specimen being viewed.
  • there is another and possibly even more important advantage with the present invention which is the ability to increase contrast and achieve a greater depth of field without loss of resolution.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

La présente invention a pour objet un système et une technique d'illumination permettant d'améliorer la résolution, le contraste, la profondeur de champ et la perception de la profondeur obtenus avec un microscope optique doté d'objectif (14) caractérisé par un axe optique (16), et un ensemble de miroirs réfléchissants (23, 24) conçus pour réfléchir un ou plusieurs faisceaux lumineux d'illumination (28, 32) qui sont ensuite focalisés sur l'échantillon selon des trajets (33, 34) traversant l'objectif sans être en coïncidence avec l'axe optique de ce dernier.
EP94914883A 1994-04-21 1994-04-21 Systeme et technique d'illumination pour microscope optique haute definition Withdrawn EP0756717A4 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1994/004438 WO1995029419A1 (fr) 1994-04-21 1994-04-21 Systeme et technique d'illumination pour microscope optique haute definition

Publications (2)

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EP0756717A1 true EP0756717A1 (fr) 1997-02-05
EP0756717A4 EP0756717A4 (fr) 1997-11-26

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EP (1) EP0756717A4 (fr)
JP (1) JPH10502742A (fr)
AU (1) AU6711294A (fr)
WO (1) WO1995029419A1 (fr)

Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2020141497A1 (fr) * 2019-01-04 2020-07-09 Ho Jun Eom Microscope dentaire/chirurgical pouvant être monté

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3544914B2 (ja) 2000-03-17 2004-07-21 住友化学工業株式会社 光学顕微鏡装置および顕微鏡観察方法。
DE10208594A1 (de) 2002-02-27 2003-09-04 Leica Microsystems Beleuchtungseinkoppelung für eine optische Betrachtungseinrichtung
JP5633706B2 (ja) * 2011-12-07 2014-12-03 横河電機株式会社 共焦点光スキャナおよび共焦点顕微鏡
CN112424668A (zh) * 2018-06-04 2021-02-26 业纳光学系统有限公司 用于捕捉显微图像的显微镜和方法以及平面反射器的用途

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JPS63191114A (ja) * 1987-02-03 1988-08-08 Canon Inc 眼科手術用顕微鏡
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Publication number Priority date Publication date Assignee Title
WO2020141497A1 (fr) * 2019-01-04 2020-07-09 Ho Jun Eom Microscope dentaire/chirurgical pouvant être monté

Also Published As

Publication number Publication date
AU6711294A (en) 1995-11-16
WO1995029419A1 (fr) 1995-11-02
JPH10502742A (ja) 1998-03-10
EP0756717A4 (fr) 1997-11-26

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