Abstract:
The fluorescence microscope comprises: a fixed-type objective lens placed between a filter set and a specimen loading portion; a partition that covers at least the specimen loading portion, the objective lens and the filter set to block extraneous light incident on the specimen loading portion; an imaging lens arranged on the outgoing surface of the absorption filter of the filter set, the imaging lens including a zoom lens capable of continuously changing the operation distance; and an imaging portion forms a fluorescent image from a fluorescence emitted from the specimen and received by the imaging lens via the absorption filter by irradiating an excitation light onto the specimen from the excitation light source via the excitation filter of the filter set and. This configuration avoids damage to the specimen or lens surface while allowing high-contrast fluorescence observation with reduced effect of extraneous light.

Description:
[0001]     The present application claims foreign priority based on Japanese Patent Application No. 2004-152548, filed May 21, 2004, the contents of which is incorporated herein by reference.  
       BACKGROUND OF THE INVENTION  
       [0002]     1. Technical Field  
         [0003]     The present invention relates to a fluorescence microscope having a function of picking up and displaying a fluorescent image of a specimen.  
         [0004]     2. Related Art  
         [0005]     Conventionally, in order to observe the microstructure of a cell and localization of a molecule, a fluorescence microscope or a laser microscope has been used. On the fluorescence microscope, a fluorescent molecule that is specifically bonded with a particular target molecule in the specimen is attached to the target molecule in order to observe distribution and behavior of the target molecule. The fluorescent molecule is also called a fluorescent probe and includes, for example, a fluorescent molecule covalently bonded with an antibody of target protein. An example of an epi microscope is described below based on  FIG. 9 . Epi illumination is an illumination method where source light is illuminated through an objective lens  950  and a fluorescence from a specimen is observed through the objective lens  950 . Illumination light (excitation light) and observation light (fluorescence) use the same optical path of the objective lens  950  that also serves as a condenser. In  FIG. 9 , in order to attain observation using epi illumination, the epi fluorescence microscope uses a dichroic mirror  914 . The dichroic mirror  914  is set in a box-shaped body generally called a dichroic cube (filter set) together with an excitation filter  912  and an absorption (barrier) filter  916 . Light having an unwanted wavelength is cut off from the illumination light of a light source by the excitation filter  912 . Only light having a wavelength that the fluorescent molecule of a fluorescent dye can absorb is transmitted through the excitation filter  912 . This attenuates the background light as an obstacle to observation. The excitation light is orthogonally reflected on the dichroic mirror  914  tilted by approximately 45 degrees with respect to an optical axis and reaches the specimen W through the objective lens  950 . The fluorescence emitted from the specimen W advances in the direction opposite to the excitation light and reaches the dichroic mirror  914  through the objective lens  950 . The dichroic mirror  914  reflects light having a wavelength below a specific wavelength and transmits the remaining light, so that a fluorescence passes through the dichroic mirror. After transmitting the fluorescence, the dichroic mirror  914  cuts the wavelength other than the target fluorescence by way of the absorption filter  916  to provide the possible darkest background and guides the transmitted light to an eye lens  9  (refer to JP-A-2000-227556).  
         [0006]     In such fluorescence observation, the intensity of a fluorescence emitted by a specimen is much smaller than that of excitation light, so that it is necessary to exclude light incident from outside other than microscope illumination. Thus, a fluorescence microscope has been placed in a darkroom for observation. The work in a darkroom is cumbersome and this approach has another problem that light emitted from an image display such as a computer and a monitor connected to the fluorescence microscope degrades the picture quality of a fluorescent image. To solve the problem, there has been developed a microscope having a lightproof section comprising a stage to be placed with a specimen, the stage surrounded by plates (refer to JP-A-2002-207177). As shown in  FIG. 10 , this microscope comprises, as a lightproof section, plate-shaped members  947   a ,  947   b ,  947   c  respectively covering the space on the stage  928  from the front face and side faces of the stage  928 , a coupling member  945  for retractably attaching the plate-shaped members  947   a ,  947   b ,  947   c  to the stage  928 , and a column for supporting the main plane of the open-state plate-shaped members  947   a ,  947   b ,  947   c  in same plane as the main plane of the stage  928 . In this way, the lightproof section  947  arranges plates around the stage  928  and blocks light incident on the stage  928  from outside. This allows use of a fluorescence microscope elsewhere than in a darkroom and prevents an observed image from being degraded even in case imaging apparatus or a computer is connected to the microscope.  
         [0007]     On a related art microscope, it is necessary to open the darkroom each time the magnification is changed during observation of a specimen in order to change the objective lens  951 . In this practice, extraneous light is illuminated onto the specimen so that the darkroom state is not maintained. That is, on the fluorescence microscope shown in  FIG. 10 , an objective lens is attached to the revolver to switch between a plurality of objective lenses in order to change the magnification. In a configuration where the revolver is manually rotated, an operator must insert his/her hand into a darkroom, which does not maintain the enclosed space of the darkroom. In case extraneous light is illuminated onto a specimen, the fluorescent image is disturbed and the contrast is lowered. The light causes a fast-fading specimen to start fading. To prevent this, a fluorescence microscope must be eventually placed in a darkroom. It is thus impossible to provide an easy-to-use fluorescence microscope that does nit require use of a darkroom.  
         [0008]     Another method is to automatically change an objective lens by the use of a powered revolver. In this case, changeover of an objective lens may cause the tip of the lens to come into contact with the specimen or a preparation on which the specimen is placed thus damaging it or resulting in a scratch on the lens surface of the objective lens. The greater the numerical aperture is, or the higher the magnification is, the length of an objective lens tends to become longer. The higher the magnification is, the operation distance between a specimen and the objective lens becomes shorter. For substantially high magnification, the operation distance is 1 mm or less. An attempt to change an objective lens in high-power microscopic observation is more likely to cause the tip of the objective lens to come into contact with the specimen or preparation thus damaging it or resulting in a scratch on the lens surface. Thus, an operator used to change an objective lens manually while checking that the tip of the objective lens would not come into contact with the preparation. Moreover, manual changeover requires opening of a darkroom, which causes the darkroom to disappear and the contrast of an observed image is lowered by extraneous light. Further, the distance between an objective lens and a specimen in a darkroom is hard to visually check. Thus it is difficult to check whether the objective lens is in contact with the preparation, which worsens ease-of-use. In this way, it is difficult to smoothly change the magnification while maintaining a high contrast. Thus, there has never existed a fluorescence microscope that automatically changes magnification while maintaining the darkroom state.  
       SUMMARY OF THE INVENTION  
       [0009]     This invention has been accomplished in order to solve these problems. A main object of the invention is to provide a fluorescence microscope which allows fluorescent observation without being installed in a darkroom and which is capable of changing magnification while maintaining high contrast.  
         [0010]     In order to attain the object, a fluorescence microscope according to the invention comprises: a specimen loading portion for placing a specimen as a target of observation; a filter set including a excitation filter, a dichroic mirror and an absorption filter as optical members of an optical system; a fixed-type objective lens placed between the filter set and the specimen loading portion; a partition for covering at least the specimen loading portion, the objective lens and the filter set to block extraneous light incident on the specimen loading portion; an excitation light source for emitting an excitation light onto the specimen; an imaging lens arranged on an outgoing surface of the absorption filter of the filter set; and an imaging portion for forming a fluorescent image from a fluorescence emitted from the specimen and received by the imaging lens via the absorption filter by irradiating the excitation light onto the specimen from the excitation light source via the excitation filter of the filter set. The imaging lens of the fluorescence microscope includes a zoom lens capable of continuously changing an operation distance. With this configuration, the fixed-type objective lens is provided to abolish a switching mechanism using a revolver and a slider. This avoids a situation where the tip of the objective lens comes in contact with the specimen or preparation on the specimen loading portion while the objective lens is being replaced, thus damaging the lens or scratching the lens surface. Use of a zoom lens changes magnification without changing the objective lens. In particular, the zoom lens provides continuous change in magnification, a seamless change in magnification to facilitate a search for the field of view, unlike the discrete change in magnification by changing an objective lens. A lightproof space is provided by the partition. This allows high-contrast fluorescent image observation with reduced effect of extraneous light. In particular, automatic magnification change using a zoom lens allows high-picture-quality magnification change maintaining a high contrast without the lightproof state being impaired by extraneous light at manual change of objective lenses using a revolver.  
         [0011]     Another fluorescence microscope according to the invention further comprises a display portion for displaying the fluorescent image picked up by the imaging portion. This allows a fluorescent image to be observed without providing an eye lens for visual observation. This does without a member related to an eye lens thus simplifying the overall configuration and providing a compact and low-cost fluorescence microscope.  
         [0012]     Another fluorescence microscope according to the invention is characterized in that the imaging portion is a CCD camera. It is possible to use the CCD camera to form and display a fluorescent image. In particular, use of a CCD camera that is more sensitive than human eyes allows display and observation of a fluorescent image that human eyes cannot recognize.  
         [0013]     Another fluorescence microscope according to the invention is characterized in that the fluorescence microscope is an inverted fluorescence microscope. While it is difficult to observe a specimen alive on an upright microscope, it is possible to observe a specimen alive on an inverted microscope. In general, for the inverted fluorescence microscope, a fluorescence obtained via an objective lens arranged below a specimen in an inverted way needs to be polarized up to the eyes of the observer in upward direction. This introduces a polarization mirror to provide a U-shaped optical path, thus resulting in a larger-size, more complicated and higher-cost system. This disadvantage is offset by abolishing an eye lens for visual observation and members for the eye les and a mirror used to change the optical path, thereby providing a more compact inverted fluorescence microscope.  
         [0014]     Another fluorescence microscope according to the invention is characterized in that the partition has a rectangular shape covering the optical path of the fluorescence microscope. This allows members of the optical path to be arranged in the rectangular partition in order to maintain the lightproof state without the interference with the optical path by extraneous light.  
         [0015]     Another fluorescence microscope according to the invention is characterized in that part of the partition comprises an aperture for insertion or retrieval of the specimen. This allows the aperture to be opened to place a specimen on the specimen loading portion or replace the specimen. Once the specimen is set, the aperture is blocked so that the inside of the partition will be maintained as a lightproof space thus allowing a specimen to be observed while magnification is being changed at high contrast.  
         [0016]     Another fluorescence microscope according to the invention is characterized by comprising a microscope illumination system for irradiating illumination light onto the specimen and the excitation light source emits excitation light onto the specimen on the specimen loading portion and.  
         [0017]     According to the fluorescence microscope of the invention, it is possible to observe a fluorescent image at high contrast by shielding light to a specimen from outside, without placing the specimen in a darkroom. This further prevents excessive fading. Further, a related art fluorescence microscope requires manual switching between objective lenses using a revolver or a slider to change magnification and the lightproof space is impaired each time the magnification is changed thus degrading the contrast as well as change in magnification is cumbersome. Further, extreme care must be exercised in switching between objective lenses so as not to damage a specimen with the objective lens. With the fluorescence microscope of the present invention, use of a zoom lens has enabled automatic magnification change. Once a specimen is set and a lightproof space provided, magnification change is made easy while maintaining the lightproof state. This provides an excellent advantage that safe and high-picture-quality observation is possible while maintaining high contrast and without the contact of the objective lens with the specimen in changing modification, due to the fixed-type objective lens. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0018]      FIG. 1  is a block diagram showing a fluorescence microscope according to an embodiment of the invention;  
         [0019]      FIGS. 2A-2C  are perspective views of the partition of the fluorescence microscope shown in  FIG. 1 ;  
         [0020]      FIG. 3  shows a list of representative fluorescence pigments and the corresponding excitation filters and absorption filters.  
         [0021]      FIG. 4  is a block diagram showing a fluorescence microscope system according to another embodiment of the invention;  
         [0022]      FIG. 5  shows a screen layout of an exemplary display screen of the display portion;  
         [0023]      FIG. 6  is a block diagram showing a fluorescence microscope according to another embodiment of the invention;  
         [0024]      FIG. 7 a  block diagram showing a fluorescence microscope according to still another embodiment of the invention;  
         [0025]      FIG. 8  is a block diagram showing the control system of a fluorescence microscope according to another embodiment of the invention;  
         [0026]      FIG. 9  is a block diagram showing a related art epi fluorescence microscope; and  
         [0027]      FIG. 10  is a front view showing another related art fluorescence microscope. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0028]     Embodiments of the invention are described below based on drawings. Note that the embodiments described below are intended to illustrate a fluorescence microscope to embody the technical philosophy behind the invention and do not limit the invention. In particular the specification does not limit the members defined in the claims to those in the embodiments. The size or alignment of members in the drawings may be exaggerated for the purpose of illustration. In the following description, a same name or sign designates a same or homogenized member and detailed description is omitted as required. Each member of the invention may be such that the same member comprises a plurality of elements in order to let a single member serve as a plurality of elements. Conversely, a plurality of members may share the function of a single member.  
         [0000]     (Inverted Fluorescence Microscope)  
         [0029]      FIG. 1  shows a block diagram of a fluorescence microscope according to an embodiment of the invention. The fluorescence microscope shown in  FIG. 1  is an inverted fluorescence microscope. The following example pertains to a case where a plurality of fluorescent dyes (fluorescent pigments) are introduced into a specimen W (also called a test specimen or a sample) in order to dye the specimen and cause the specimen to develop in multiple colors for multicolor fluorescent observation. A fluorescence microscope  100  shown in  FIG. 1  comprises an excitation light source  48 , a collector lens  54 , a filter set  1 , an objective lens  50 , an imaging lens  52 , and an imaging portion  22 . These members are arranged on a certain optical path. The excitation light source  48  emits excitation light to excite a fluorescent dye. For example, a high-pressure mercury lamp or a high-pressure xenon lamp is used. These lamps irradiate light having a wide wavelength. The excitation light source  48  may be a light-emitting diode that features low power consumption, compact design and high efficiency. A member of the excitation light source may be provided in a unit, and such units may be assembled to form a fluorescence microscope. These units facilitate installation, transportation and maintenance of a fluorescence microscope system. The illumination light from the excitation light source  48  is formed into pencils of light substantially parallel to each other by the collector lens  54 . The pencils of light are introduced as excitation light into the filter set  1 . The collector lens  54  may be a fluorescence epi illumination lens in the case of epi illumination. While it is assumed that epi illumination is used to illuminate the specimen in the following example, the invention is also applicable to other illumination methods such as transmissive illumination and total reflection illumination.  
         [0030]     The filter set  1  is a combination of a single-pass filter that selectively transmits light having a wavelength fit for observation of a specific fluorescent dye and a mirror. As shown in  FIG. 1 , the filter set  1  comprises an excitation filter  12 , an absorption filter  16  and a dichroic mirror  14 . The filter set  1  comprises a plurality of types that are changeable. each filter set  1  comprises a combination of the excitation filter  12 , absorption filter  16  and dichroic mirror  14 . By changing the filters or mirrors, different monochrome images may be picked up. A plurality of filter sets  1  are set to a filter holder  56  and changed in a filter switch portion  18 . The excitation light selected in the excitation filter  12  of the filter set  1  is reflected on the dichroic mirror  14 , passes through the objective lens  50  and projected onto the specimen W.  
         [0000]     (Objective Lens  50 )  
         [0031]     The objective lens  50  also serves as a condenser lens. The objective lens  50  is a fixed objective lens of a single type, instead of a plurality of objective lenses one of which is selected using a revolver. While the objective lens  50  is fixed with fixing means such as a screw or a stop piece, the objective lens  50  may be detached with the fixing means released. So that it is possible to replace the objective lens  50  with another depending on the purpose of observation. For example, the objective lens  50  may be replaced with an objective lens dedicated to observation of phase difference, differential interference, a bright field, or a dark field. It is possible to mount an appropriate objective lens such as an oil-immersed lens, a water-immersed lens, a dry lens a lens for a cover slide sample, or a lens for a non-cover slide sample, depending on the purpose of observation or application. In the example shown, the objective lens  50  is fixed with a screw. The screw may be loosened to remove the objective lens  50  and replaced with another objective lens  50  for use in a variety of applications.  
         [0000]     (Lightproof Space  46 )  
         [0032]     The specimen W is placed on the specimen loading portion  28 . In general, observation of a low-light specimen such as a fluorescent specimen is made in a darkroom because it is necessary to exclude extraneous light. A fluorescence microscope  100  according to this embodiment arranges a specimen loading portion  28 , an objective lens  50  and a filter set  1  in a lightproof space shielded from extraneous light, the lightproof space  46  serving as a darkroom, thereby providing an easier-to-use fluorescence microscope capable of fluorescence observation without using an additional darkroom. The specimen loading portion  28  may use an XY stage to allow traveling in X-axis and Y-axis directions. The specimen loading portion  28  may be designed to travel in vertical direction (Z-axis direction) so as to change the relative distance to an optical system  10  thus allowing focusing.  
         [0000]     (Partition  47 )  
         [0033]     A partition  47  that constitutes a lightproof space has a shape of a box as shown in  FIG. 2 . As illustrated, it is possible to shield extraneous light reliably by covering the specimen loading portion  28 , the objective lens  50  and the filter set  1  with the box-shaped partition  47 . While the partition shown in  FIG. 2A  is designed to cover the entirety of the microscope, it may be enclosed with plates so that it will cover only the part of the specimen loading portion  28 , the objective lens  50  and the filter set. A retractable aperture  47   b  is formed in part of the partition  47  for attachment and replacement of a specimen. As shown in  FIGS. 2   b  and  2   c , the aperture  47   b  rotates part of the lid in a crank-like motion so that it will move from the forward end position to the lowermost position. This allows a preparation with a specimen set to be placed on the specimen loading portion  28  for replacement. Besides crank-like rotation of the lid, the aperture  47   b  may be such that the lid is slid open in vertical direction, the lid is slid open in horizontal direction (sliding door), the door is opened/closed by way of a hinge provided on the side face of the aperture  47   b , or a panel to block the opening is removed. While the aperture  47   b  is provided in part of the front side wall of the partition  47  in the example of  FIG. 2   b , the while face may be opened/closed. The shape of the partition may be a hollow rectangular parallelepiped, a cylinder, a half cylinder, or a prism such as a triangular prism. The shape may be chamfered as required.  
         [0034]     In this way, when a specimen is set on the specimen loading portion or replaced, the aperture  47   b  may be opened. Once the specimen is set, the aperture  47   b  may be blocked to maintain the internal of the partition  47  as a light-shielded lightproof space. This allows fluorescence observation at high contrast without a fluorescence microscope being installed in a darkroom. The fluorescence is not affected by the light from a computer or a monitor connected to the fluorescence microscope. Fading of a specimen is prevented. Further, work in a darkroom is made unnecessary. Thus, freedom of installation is enhanced and the operation area is bright enough for easy operation or work. Moreover, as mentioned later, use of a zoom lens ensures an excellent operation environment where change in magnification is allowed with the darkroom state maintained and adjustment to a desired magnification is made possible in the observation of a high-picture-quality fluorescent image.  
         [0035]     Among the fluorescent dyes included in the specimen W, a fluorescent dye corresponding to the irradiated excitation light emits a fluorescence, which passes through the objective lens  50  and is incident on the filter set  1 , then passes through the dichroic mirror  14 . In this way, the dichroic mirror  14  reflects illumination light and transmits a fluorescence. The fluorescence is transmitted and the optical components other than a fluorescence such as illumination light are selectively absorbed by the absorption filter  16 . The absorption filter  16 , also called a barrier filter, is arranged closer to the face where a fluorescent image is formed than the dichroic mirror  14 . The light that has exited the filter set  1  passes through an imaging lens  52  and is incident on an imaging portion  22 . The imaging portion  22  is arranged in a position conjugate with the focal face of the objective lens  50 . The imaging portion  22  converts a fluorescence to an electric signal. Based on the signal, an image is formed and displayed on a display portion  24 . Thus, the imaging portion  22  is composed of an image pickup device. A semiconductor image pickup device such as a CCD camera is preferably used. The CCD camera, arranged in a two-dimensional plane, simultaneously picks up a single screen without sequentially scanning the screen as in a laser microscope. The noise characteristic of a CCD camera is improved when it is cooled. Thus, a CCD camera using a Peltier device or liquid nitrogen for cooling may be used. As mentioned above, the fluorescence microscope allows automatic change of single-pass filter set  1  by way of the filter switch portion  18 , and is capable of simultaneously displaying a monochrome image picked up by each filter set  1  and an superposed image where such monochrome images are superposed one on another.  
         [0000]     (Filter Set  1 )  
         [0036]     The filter set  1  includes a set of an excitation filter  12 , an absorption filter  16  and a dichroic mirror  14  in a box-shaped body generally called a dichroic cube. A combination of a excitation filter  12 , an absorption filter  16  and a dichroic mirror  14  of the filter set  1  is determined depending on the fluorescent dye introduced into the specimen W. A combination of single-pass bandpass filters is determined so that only the light having a desired wavelength component will be extracted and the remaining wavelength components rejected in order to allow correct observation of a color developing with a fluorescent dye. Thus, the filter set  1  used is determined depending on the fluorescent dye used. In general, the filter set  1  of different fluorescent colors is used. For example, a color combination such as RGB and CMY corresponding to fluorescence pigments may be used as required. A list of combinations of representative fluorescence pigments and the corresponding excitation filters and absorption filters is shown in  FIG. 3 . In  FIG. 3 , the reagent name (common name), the wavelength of its excitation light and the wavelength of the absorption light are shown in major peak values in the band. The plurality of filter sets  1  may be changeable by the filter switch portion  18 . The plurality of filter sets  1  are set to a filter holder  56  and any one of the plurality of filter sets  1  is set on the optical path by the filter switch portion  18 . The filter switch portion  18  may use a turret to change the filter set  1  in a motor-driven rotary fashion or sliding fashion. Control of the filter set  1  is set by the switching set portion  20 . It is possible use the filer set  1  to change at a time a necessary set of an excitation filter  12 , an absorption filter  16  and a dichroic mirror  14 . Change operation may be made at a single section to facilitate high-speed operation and maintenance. Individual change means for individually changing a plurality of excitation filter, absorption filters and dichroic mirrors may be provided instead of using a filter set including a combination of an excitation filter, an absorption filter  16  and a dichroic mirror. Based on this configuration, respective change means may be controlled in an interlocked fashion to arrange a predetermined set of an excitation filter, an absorption filter and a dichroic mirror on the optical path. Further, it is possible to change an optical path by using a mirror thereby substantially change the filters for later image pickup.  
         [0037]     Another method for performing multicolor fluorescence observation is the use of a dual or triple bandpass filter capable of simultaneously observing a plurality of fluorescence pigments with an image pickup device such as a CCD camera attached to the fluorescence microscope, or the use of an appropriate single-pass (monochrome) filter set corresponding to each of the fluorescent pigments to be used.  
         [0000]     (Display Portion  24 )  
         [0038]     The display portion  24  is a display for displaying an image picked up by the optical system  10 . The display constituting the display portion  24  is a monitor that can display the image at a high resolution and may be a CRT or a liquid crystal display panel. The display portion  24  may be integrated into a fluorescence microscope or an externally connected monitor. Or, an external connection device  58  connected to a fluorescence microscope  200  may be used as a display portion as shown in  FIG. 4 . For example, in case a computer  58 A is used as an external connection device  58 , the monitor  24 A of the computer  58 A may provide the function of the display portion. A plurality of display portions may be used for each of the fluorescence microscope  200  and the external connection device  58 .  
         [0039]     Next, an example of the display screen of the display portion  24  is shown in  FIG. 5 .  FIG. 5  shows an exemplary GUI representation of a fluorescence microscope image display program. As illustrated, the display portion  24  comprises a plurality of image display areas G. The display portion  24  simultaneously displays different images in the image display areas G for comparison. In particular, in this embodiment, one of the image display areas G is used as an superposed image display area O for displaying an superposed image where images observed using the filter sets are superposed one on another. This allows comparison of an image observed using a filter set and such an superposed image on the same screen. As mentioned later, in this embodiment, the images can be displayed virtually in real time, so that it is possible to readily observe the state of a specimen under various conditions. In the example of  FIG. 5 , total four image display areas G are provided, one of which is used as an superposed image display area O. The number of image display areas may be three or less or five or more. Preferably, the number of image display areas is the number of filters sets in the filter holder plus the number of superposed image area so that all the filter sets and an superposed image thereof can be observed on a single screen. It is of course possible to enlarge any selected screen or toggle between enlarges screens for filter sets or select an superposed image. It is not necessary to display all images on one screen. Images may be displayed in separate windows or all-image display may be selected. In this way, image display practices may be changed as required in accordance with the number of filters, purpose of observation, and user&#39;s taste. In the example of  FIG. 5 , three filter sets corresponding to the fluorescent dyes  1  through  3  are loaded into the filter holder  56 , and monochrome images picked up while toggling between these sets and an superposed image where the monochrome images are superposed one on another are displayed.  
         [0000]     (Image Adjustment Portion  40 )  
         [0040]     The setting screen shown in  FIG. 5  comprises an image adjustment portion  40 . In the example of setting screen shown in  FIG. 5 , switches for adjusting image adjustment parameters including the position, height, magnification, brightness and contrast for movement of field view are provided as the image adjustment portion  40  to the right of the image display area G. The image adjustment parameters may be adjusted by the user or automatically set to optimum values. For example, the “One-touch Auto” button to automatically adjust the exposure time and the “One-Touch Focus” button to automatically adjust the focus are provided. Among the image adjustment parameters, the position is adjusted interns of travel in X and Y directions, allowing a shift of the eyepoint of an image being displayed. For example, in the screen of the image display area G, dragging an arbitrary position with a mouse and shifting the position in predetermined direction moves the position or eyepoint. The specimen loading portion  28  travels in X and Y directions in accordance with the travel amount of the mouse. The up/down and right/left button as well as the crosshair button may be used to move the specimen loading portion  28 . A guide may be provided for displaying the section where the currently displayed eyepoint is located in the display area.  
         [0041]     Adjustment of height is made by determining the relative distance in Z direction, that is, between the specimen W and the optical system  10  (objective lens  50 ). This adjusts the focus of the image. Through adjustment of height on the height specification section, the specimen loading portion  28  is vertically moved. Adjustment of height or magnification may be made continuously and visually by using a slider, a level meter, or a scale. The example in  FIG. 5  uses a slider  32 A as a height specification section. In any case, the specimen loading portion  28  is moved for adjustment, the same result is obtained by moving the optical system  10 .  
         [0000]     (Zoom Lens)  
         [0042]     Adjustment of magnification is made using an imaging lens  52 . The imaging lens  52  is a lens arranged between the filter set  1  and the imaging portion  22  for imaging a fluorescence from the absorption filter  16  of the filter set  1 . In this example, the imaging lens is a zoom scaling lens. A single imaging lens  52  may be used to continuously change the magnification for example from 10 times to 100 times. The magnification, number of apertures and operation distance of each of the objective lens and the imaging lens are set to appropriate values depending on the purpose of observation and conditions. The fluorescent image formed by the imaging lens  52  is projected onto the imaging portion  22 , converted to an electric signal and displayed on an externally connected display portion  24 . The image data of the fluorescent image is captures into an external connection device  58  such as a computer  58  for later processing. Processing conditions and processing results are displayed on the display of the computer  58 A. The computer  58 A may also use with the display portion. The observer can observe a fluorescent image on the display portion and operate the computer  58 A to apply desired processing to the fluorescent image and observe the processing results on the display portion or display.  
         [0000]     (Abolishment of Eye Lens)  
         [0043]     As shown in  FIG. 1 , an inverted fluorescence microscope according to the invention does without an eye lens for visual observation to simplify the configuration of the inverted fluorescence microscope. A related art inverted fluorescence microscope polarizes upward the optical path of a fluorescence obtained via an objective lens or a filter set provided below a specimen loading portion so that it arranges an eye lens at the height of the eyes of the user. The optical path is bent and extended so that the structure of the microscope is complicated and the resulting larger system design was a cause of high costs. Such an eye lens is abolished and the display portion for displaying an image picked up by the imaging portion is externally connected in order to simplify the system and attain the compact design of the apparatus.  
         [0044]     On a related art fluorescence microscope, the maximum field of view of the objective lens where the imaging portion can pick up an image is small. In case an objective lens of an appropriate magnification and high NA is selected, a field of view with large NA is secured. In this system, in order to attain magnification of 10 times to 60 times that is frequently used, a 20× apochromat objective lens (NA=0.75) is selected. The zoom lens is set to magnification of 0.5 times (zoom out) to three times (zoom in) to attain performance approximately equivalent to NA of standard specification 10 times to 60 times.  
       Other Embodiments  
       [0045]      FIG. 6  shows a block diagram of an inverted fluorescence microscope according to another embodiment of the invention. The same member names as  FIG. 1  uses substantially identical members so that the corresponding description is omitted. Major difference between the configuration in  FIG. 6  and that in  FIG. 1  is polarization of the optical axis of the imaging lens  52 B. In the configuration shown in  FIG. 1 , a fluorescence is arrange approximately in vertical direction with respect to the excitation light irradiated approximately in horizontal direction. The configuration is extended both in horizontal and vertical directions, which results in a larger geometry of the fluorescence microscope apparatus. In contrast, for the configuration shown in  FIG. 6 , the imaging lens  52 B that receives a fluorescence is arranged in horizontal direction and a CCD camera  22 C as an imaging portion is arranged ahead of the imaging lens  52 B. This limits protrusion in vertical direction, thereby reducing the overall size of the fluorescence microscope apparatus. The apparatus tends to be inherently long in horizontal direction since the path of the excitation light is arranged in horizontal direction. By arranging a path for extracting a fluorescence in horizontal direction also, the apparatus is long in horizontal direction alone with protrusion in vertical direction suppressed. Note that the horizontal direction and the vertical direction may be exchanged. When the paths of excitation light and a fluorescence are arranged in vertical direction, compact fluorescence microscope apparatus is obtained that is long in vertical direction and whose protrusion in horizontal direction is suppressed. The paths of excitation light and a fluorescence may be arranged in oblique direction. The path of excitation light and that of a fluorescence is preferably not parallel but in skew relation without crossing. In case the path are arranged in parallel, the excitation light and the fluorescence interferes with each other and produces a crosstalk, which could result in improper imaging. The paths are arranged in skew relation and in close proximity to each other, not apart from each other, in order to prevent possible interference. This contributes to downsizing of the apparatus.  
         [0000]     (Upright Fluorescence Microscope)  
         [0046]     While the inverted fluorescence microscope is described in the above example, the invention is also applicable to an upright fluorescence microscope.  FIG. 7  shows an upright fluorescence microscope as a fluorescence microscope according to another embodiment of the invention. As shown in  FIG. 7 , the upright fluorescence microscope arranges an objective lens  50  and a filter set  1  above the specimen loading portion. This fluorescence microscope includes a lightproof space where all optical paths of an optical system  10  are enclosed by a partition  47  and the partition  47  is a box-shaped case covering the entire fluorescence microscope. This completely shields extraneous light elsewhere than the microscope illumination system, thus allowing high-quality fluorescent image observation.  
         [0000]     (Control System  64 )  
         [0047]      FIG. 8  shows an exemplary block diagram of a control system  64  of the fluorescence microscope. In  FIG. 8 , the details of the optical system  10  are not shown. As shown in  FIG. 8 , the fluorescence microscope comprises as an imaging system  66 : a stage  28 A as a form of a specimen loading portion  28  on which a specimen is placed; a stage elevator  30 A for moving the stage  28 A; an optical system  10  for exciting a fluorescent dye with excitation light irradiated onto a specimen W placed on the stage  28 A and forming a fluorescence on an imaging portion  22 ; a CCD  22 A as a form of the imaging portion  22  for electrically reading, per two-dimensionally arranged pixel, a fluorescence incident via the optical system  10  from the specimen W fixed to the stage  28 A; and a CCD control circuit  22 B for performing driving control of the CCD  22 A. The stage elevator  30 A is a form of a height adjustment portion  30  and includes, for example, a stepping motor  30   a  and a motor control portion  30   b  for controlling up-and-down movements of the stepping motor  30   a . The stepping motor  30   a  moves the stage  28 A in the z axis direction as an optical axis direction and in X and Y directions as a plane perpendicular to the optical axis direction.  
         [0048]     The fluorescence microscope comprises as a control system  64  for controlling the imaging system  66 : a I/F portion  68 ; a memory portion  34 ; a display portion  24 ; an operation portion  70 ; and a control portion  26 . The I/F portion  68  communicates an electric signal carrying data with the imaging system  66  by way of communications means. The memory portion  34  retains image data electrically read by the imaging portion  22 . The display portion  24  displays a picked-up image, a synthesized image, or various setting. The operation portion  70  performs operation such as input and setting based on the screen displayed on the display portion  24 . The control portion  26  controls the imaging system  66  in accordance with the conditions set on the operation portion  70  to perform imaging as well as synthesizes acquired image data to generate a 3D image or performing various processing such as image processing. The imaging system  66  and the control system  64  may be included to complete operation in the fluorescence microscope alone. Or, as shown in  FIG. 4 , an external connection device  58  such as a computer  58 A may be connected to a fluorescence microscope  200  and the fluorescence microscope  200  may operate the imaging system while the external connection device  58  may operate the control system. Members of the imaging system and those of the control system are not strictly distinguished; for example the memory portion may be included in the imaging system, or the CCD control circuit or motor control portion may be included in the control system.  
         [0049]     The operation portion  70  is connected, either wiredly or wirelessly, to a fluorescence microscope or a computer of a fluorescence microscope system, or is fixed to the computer. Examples of a general operation portion includes a mouse, a keyboard, and various pointing devices such as a slide pad, TrackPoint, a tablet, a joystick, a console, a jog dial, a digitizer, a light pen, a ken-key pad, a touch pad, and Acupoint. Such operation portions may be used for operation of a fluorescence microscope image display program as well as operation of a fluorescence microscope and its peripherals. A display for representing an interface screen may include a touch screen or a touch panel for the user to directly touch the screen with his/her finger for data input or system operation. Voice input means or other existing input means may be used instead or in combination with the above means. In the example of  FIG. 8 , the operation portion  70  includes a mouse (refer to  FIG. 4 ). Use of the mouse allows operation of a slider  32 A as a height specification section  32  or focusing on an image and other operations. In this way, by displaying an operation menu together with an image on the display portion  24  and selecting an operation item and operating a function on the screen, it is possible for the user to correctly grasp the operation details and states without operation errors, thus providing a tactile and easy operation system.  
         [0050]     A fluorescence microscope according to the invention is applicable to for example a fluorescence antibody test where the serum and cell nucleus of a patient is caused to react with each other, then a fluorescence indicator is added and an antinuclear antibody is observed on a fluorescence microscope, and whether the antibody is positive or negative is determined based on the fluorescence of the antinuclear antibody.