Abstract:
A microscope for transmission viewing of a specimen, said microscope comprising a light source for producing a light beam; an objective lens positioned for focusing the light beam produced by said light source on an area of the specimen for illuminating said area; and a reflector means positioned for reflecting light transmitted through the specimen back through the illuminated area of the specimen.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    The present invention is directed to a microscope illumination device for transmission viewing of an object to be examined, with a light source for producing a light beam, an optical arrangement for focusing the light from the light source via an objective lens on an object to be examined, and a reflector mechanism, which is located on an opposite surface of the object and for reflecting the illuminated light transmitted by the object back onto the specimen.  
           [0003]    2. Description of the Prior Art  
           [0004]    “Trans-illumination” of specimen in a microscope is usually accomplished through a condenser, which directs the light from a light-source through the specimen onto the objective lens, the latter being positioned on the opposite side of the condenser. In “epi-illumination”, on the other hand, which is needed for fluorescence or reflection microscopy, illumination occurs through the objective lens, i.e., is accomplished by the same optics by which the preparation is viewed. Preferably, xenon and mercury short arc lamps are used as the light sources. If the intention is to switch back and forth quickly between incident epi- and trans-illumination, it becomes noticeable that the indicated light sources cannot be turned on and off quickly or their intensity cannot be controlled. For a microscope with automatic control of all functions, for example, by a computer, in the past mechanical closures had to be used and fast intensity control was possible within certain limits only with mercury (Hg) lamps.  
           [0005]    Another defect of existing transillumination systems concerns the fact that biological preparations often exhibit very poor contrast. As a result, many high quality transmitted light condensers are equipped for the possibility of contrast-amplifying processes, such as Zemicke phase contrast or Normarksi differential interference contrast. Both processes, however, require not only manipulation of the illumination light, i.e., the beam path in front of the specimen, but also the light after it passed through the specimen. To accomplish this, either special objective-lenses are necessary (phase contrast) or optical elements such as DIC prisms and analyzers must be placed in the beam path. Phase contrast objectives usually limit the choice of objectives and reduce light throughput to some extent, while optical elements such as analyzers requires for DIC reduce light throughput considerably. Consequently, fast switching between optimum, contrast-intensified transmitted light observation and weak-light fluorescence observation is not possible.  
           [0006]    U.S. Pat. No. 4,852,985 discloses an illumination device for a microscope, which is made as a flat array of individually triggerable light emitting diodes, which overcomes some of the described problems.  
         SUMMARY OF THE INVENTION  
         [0007]    In view of the foregoing, it is an object of the invention to overcome the aforementioned disadvantages in the prior art and to provide a microscope which enables prompt switching between incident light and transmitted light and delivers high-contrast transmitted light images for the generation of which the light beams behind the specimen need not be influenced. Preferably, illumination will be turned on and off quickly and its intensity can be changed quickly and flexibly.  
           [0008]    A second object of the present invention is to provide a microscope illumination device which allows transmission viewing of a specimen and which can be operated as easily and efficiently as possible in a function unit with epi-illumination.  
           [0009]    These and other objects are achieved in accordance with the present invention using a microscope with a light source for producing alight beam to be directed to a specimen, an optical arrangement for illuminating an objective lens, which focuses illumination light on an area of the specimen, and a reflector mechanism which is located proximate to an opposing surface of the specimen, the reflector mechanism reflecting the illumination light transmitted by the specimen back onto the illuminated area of the specimen. Such a microscope is advantageous since, in spite of the “incident light geometry” of the illumination light, i.e., the illumination light focused via the microscope onto the specimen side facing the microscope, transmission viewing of the specimen is possible. This is because the illumination light, which is focused by the microscope on the specimen and which is transmitted by the specimen, is reflected back again by the reflector mechanism onto the specimen, passes again through the specimen and can be collected by the microscope for viewing.  
           [0010]    The microscope, in accordance with the present invention, can be operated very easily in a function unit with, for example, epifluorescence analysis. The same light source may be used both for trans-illumination and epi-illumination, in particular in case that this light source can be switched rapidly between different wavelengths or wavelength ranges. In general, the invention enables a completely free choice of the wavelength of the illuminating light. A further benefit is that by a corresponding design of the reflector unit, i.e. by providing for a different reflectivity in different areas thereof, an “oblique illumination” of the sample may be obtained in a simple manner, whereby the contrast of the transmission image is advantageously enhanced. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWING  
       [0011]    [0011]FIG. 1 is a sectional view of a microscope in accordance with a first embodiment of the present invention;  
         [0012]    [0012]FIG. 2 is a schematic view of an outer side of the reflector shown in FIG. 3; and  
         [0013]    [0013]FIG. 3 is a sectional view of a microscope in accordance with a second embodiment of the present invention. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0014]    [0014]FIG. 1 shows part of a microscope device having a light source (not shown) and which allows viewing of a specimen  110  in transmission, i.e., in transmitted light, or in epi-fluorescence. The microscope device preferably includes a common light source for transmission illumination and epifluorescent illumination, which allows a rapid change between different wavelengths and delivers an excitation or illumination beam  112 , which strikes and is deflected by a beam splitter  114  on an entry pupil of the microscope objective lens  116 . To excite epi-fluorescence, the beam splitter  114  is dichroic, i.e, the beam splitter  114  reflects the excitation light and transmits the emission light. The generally longer-wavelength light provided for transillumination coincides with the transmission range of the beam splitter  114 , but ordinarily enough light is reflected by the beam splitter  114  that sufficiently bright trans-illumination can be accomplished.  
         [0015]    To enable the illumination or excitation light to reach the specimen  110 , the microscope objective lens  116  is sometimes immersed in an immersion liquid  118 , which provides a medium of equal refractive index to extend from the objective lens to the biological specimen  110 , which is supported on a holder, such as a slide or cover glass  120 . The constellation makes it possible to avoid reflection of the illumination or excitation light before entering the specimen  110 . On the side of the specimen  110 , opposite the microscope objective lens  116 , i.e., underneath the object slide  120 , is a reflector means  122 , which is formed by a hemispheric transparent body, preferably glass. Preferably disposed between a flat boundary surface and the object slide  120  is a liquid medium  124  used to largely avoid the imaging errors. The liquid medium  124  is preferably water or an immersion oil, however, may be replaced by a gas or air.  
         [0016]    The reflector means  122  includes a hemispherical outer boundary surface  126 , which is completely reflective, at least in the wavelength range of the illumination light, in a first embodiment in order to reflect back all the illumination light traveling through the specimen  110  onto the area of the specimen illuminated overhead by the microscope objective lens  116 . The reflector means  122  acts in this way as replacement for a separate transmitted light source and the corresponding condenser.  
         [0017]    The illumination light reflected back by the reflecting surface  126  into the illuminated area of the specimen  110  is collected by the microscope objective lens  116  and is imaged using suitable optics onto the eye of the user or a detector. The wavelength of the light used for the illumination is preferably in the transmission range of the beam splitter  114 , so that the largest part of the light transmitted by the specimen  110  and collected by the microscope objective lens  116  is transmitted by the beam splitter  114 . At the same time, the beam splitter  114  should act by transmission for the fluorescent light of the specimen  110 , which is collected by the microscope objective lens  116 . Given the fact that much less light is required for transmitted light illumination than for fluorescence excitation, even the small fraction of light reflected by a dichroic beam splitter  114  in its wavelength-range of maximal transmission is enough to illuminate the preparation with sufficient brightness.  
         [0018]    As shown in FIG. 3, in order to obtain an image with contrast as high as possible during illumination and transmission viewing of the specimen  110 , the reflector means  122  can be made such that it does not reflect with uniform intensity over the entire hemisphere of the illumination light, but rather acts as a mirror only in one certain range for the wavelength(s) of the illumination light in order to achieve “oblique illumination” of the specimen  110 . In a particular embodiment only less than one quarter  128  of the hemispheric surface  126  is reflective, while the remaining area  130  does not act as a mirror for the wavelengths of the illumination light.  
         [0019]    The arrangement shown in FIG. 2 forms an extremely versatile and flexible overall system for combined transmission and epi-fluorescence microscopy. It does not require additional transmission illumination and can be selectively operated as a transmission microscope or as an epi-fluorescence microscope with a single light source by quickly changing the wavelength of the light source between the illumination light and the excitation light as. A particularly suitable light source, which can be switched quickly between different wavelengths, is described in the German Patent DE 42 28 366, where white light is guided via a parabolic mirror onto a holographic diffraction grating, which is turned by way of a scanner and diffracts light with a spectral composition, which depends on the angle of rotation of the reflection grating, onto the parabolic mirror, from where it is supplied via an optical fiber to the excitation beam path of the microscope. Similar embodiments of a light-source corresponding to DE  42   28   366 , but with direct coupling to the microscope without the use of fiberscan also be imagined.  
         [0020]    Instead of being equipped with an immersion objective, the microscope can also be made as an inverse microscope, and it can then be advantageous to immerse the reflector means into the preparation of the specimen.  
         [0021]    An arrangement according to FIG. 1 can be used especially advantageously for Two-photon microscopy (TPM) because the reflector surface  126  can be used to increase the collecting efficiency for fluorescence photons. To accomplish this, the surface  126  must be made reflective such that the visible emission light is completely reflected, while the light for the oblique illumination is only partially reflected. An example is shown in FIG. 2, where only a quadrant  128  is made reflective, whereas a large fraction of the surface ( 130 ) is transmitting. In this way, the collection efficiency can be increased by a factor of two with a single optical component and at the same time oblique illumination can be accomplished.  
         [0022]    Furthermore, it is possible to provide the reflection means  122  on the side facing the top surface of the specimen  110  with a suitable opening whereby the action of a pulsed laser beam focused by the microscope objective lens  116  on the specimen  110  causes particles from the specimen  110  to be propelled outward from the specimen  110  where they are subsequently collected. The particles are cut free or prepared before being flung outward via the highly focused, pulsed laser beam from the sample  110  and then are flung outward from the specimen  110  via a second laser blast in order to be captured by a receiving vessel such as the reflection means  122 . In particular, it is possible to specifically control and capture individual cells discharged from the specimen  110 . In this embodiment of the invention the reflection means  122  is preferably made as an economical, disposable article, which can be replaced after capturing a cell for a new capture process. Alternatively, in order to capture several different particles or cells from a specimen  110 , there can be a series of reflection means  122  formed in an array with several identical or different reflection bodies. The array is displaced inbetween “shots” in such a way that each cut out cell or part thereof ends up in a different vessel, i.e. reflection means.  
         [0023]    [0023]FIG. 3 shows an exemplary embodiment of the invention in which essentially the same optics described in FIG. 2 are used, but in a form optimized for another operating mode, for example, “total internal reflection fluorescence” (TIRF) microscopy. TIRF microscopy is a process in which a laser beam  150  is coupled from the outside into the interior of the reflection means  122  by the nonreflecting area  130  of the reflection surface  126  and is totally reflected on the boundary surface between the object slide  120  and the specimen  110  or the preparation medium  118 . In this way, the laser beam  150  illuminates the specimen  110  only by near field action at the total reflection point. Since the angle of incidence of the exciting laser beam  150 , and thus, the penetration depth of the laser light into the specimen  110  can be varied, the arrangement can be optimally adapted for special circumstances. The emission light emitted in the part of the specimen  110  illuminated by the laser beam  150  is collected by the microscope objective lens  116  and sent for detection.